COMPOSITIONS AND METHODS TO SELECT FOR REDUCED GRAIN MOISTURE IN MAIZE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/602,188, filed Feb. 23, 2012, the entire content of which is herein incorporated by reference.

[0002] The present disclosure relates to compositions and methods useful in selecting maize plants with reduced grain moisture.

[0003] Grain moisture is of primary importance for maize (Zea mays L.) production and breeding. Low (or reduced) grain moisture decreases the economic impact of artificial drying and allows earlier harvesting, which permits the grower to obtain a higher price for the crop at an earlier date and reduces exposure of the crop to adverse weather and field conditions that hinder the harvest operation (Sweeney et al (1994) Crop Sci 34:391-396). Warmer temperatures and lower humidity promote rapid field drying of corn grain. However, in mid- to short-season environments, harvest time is often characterized by decreases in temperature and increases in humidity. Thus, hybrids with superior yielding ability that dry at a more rapid rate are highly desirable.

[0004] Historically, selection of inbred lines to produce hybrids with reduced grain moisture has been performed by measuring grain moisture directly. However, some studies have suggested that grain moisture can also be selected for indirectly by measuring traits such as field grain drying rate (FDR) (Sala et al (2006) Theor Appl Genet 112:462-471) or husk senescence (Sweeney et al (1994) Crop Sci 34:391-396). Silking date and black layer formation were also found to be significantly correlated with grain moisture in some studies; however, other studies have shown a limited association (Sweeney et al (1994) Crop Sci 34:391-396). The use of any phenotypic selection method can require extensive resources and is somewhat imprecise, with environmental factors and complex associations between different agronomic traits complicating selection.

[0005] Selection through the use of molecular markers associated with reduced grain moisture allows selections based solely on the genetic composition of the progeny. As a result, plant breeding can occur more rapidly, thereby generating commercially acceptable maize plants with reduced grain moisture in a relatively short amount of time. Thus, it is desirable to provide compositions and methods for identifying and selecting maize plants with reduced grain moisture. These plants can be used in breeding programs to generate high-yielding hybrids that dry at a more rapid rate.

[0006] QTL for grain moisture have been reported on chromosome 9 (Frascaroli et al. (2007) Genetics 176:625-644; Sala et al. (2005) Theor Appl Genet 112:462-471; Blanc et al. (2006) Theor Appl Genet 113:206-224; Moreau et al. (2004) Theor Appl Genet 110:92-105; Lu et al. (2003) Theor Appl Genet 107:494-502; Austin et al. (2000) Crop Sci. 40:30-39).

[0007] Compositions and methods for selecting maize plants for grain moisture are provided herein, including compositions and methods for identifying and selecting maize plants with reduced grain moisture.

[0008] In one embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a marker allele is detected in the maize plant. The marker is located on chromosome 9 at 72-79 cM on a single meiosis map or at 200.4-230.6 cM on an IBM2 map; and an allele of the marker is associated with reduced grain moisture. Maize plants that have one or more marker alleles associated with reduced grain moisture are then identified and/or selected. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0009] In one aspect, the marker is PHM18206, PHM9929, PHM6726, PHM4910, PHM6667, PHM229, PHM6567, PHM5208, PHM15758, PHM14370, PHM3159, PHM17246, or PHM4691.

[0010] In another aspect, the marker detects a SNP polymorphism. The SNP polymorphism can be PHM18206-5, PHM9929-8, PHM6726-10, PHM4910-5, PHM6667-11, PHM229-15, PHM6567-10, PHM5208-11, PHM15758-6, PHM14370-9, PHM3159-14, PHM17246-16, or PHM4691-9.

[0011] In another aspect, the allele is a “T” at PHM18206-5, a “T” at PHM9929-8, an “A” at PHM6726-10, a “G” at PHM4910-5, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, a “T” at PHM17246-16, or a “T” at PHM4691-9.

[0012] In another embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a marker allele is detected in the maize plant. The marker is located in a chromosomal interval defined by and including PHM18206 and PHM4910; and an allele of the marker is associated with reduced grain moisture. Maize plants that have one or more marker alleles associated with reduced grain moisture are then identified and/or selected. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0013] In one aspect, the marker is PHM18206, PHM9929, PHM6726, PHM4910, PHM6667, PHM229, PHM6567, PHM5208, PHM15758, PHM14370, PHM3159, PHM17246, or PHM4691.

[0014] In another aspect, the marker detects a SNP polymorphism. The SNP polymorphism can be PHM18206-5, PHM9929-8, PHM6726-10, PHM4910-5, PHM6667-11, PHM229-15, PHM6567-10, PHM5208-11, PHM15758-6, PHM14370-9, PHM3159-14, PHM17246-16, or PHM4691-9.

[0015] In another aspect, the allele is a “T” at PHM18206-5, a “T” at PHM9929-8, an “A” at PHM6726-10, a “G” at PHM4910-5, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, a “T” at PHM17246-16, or a “T” at PHM4691-9.

[0016] In another embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a marker allele is detected in the maize plant. The marker is located on chromosome 9 at 93-98 cM on a single meiosis map or at 317-323 cM on an IBM2 map; and an allele of the marker is associated with reduced grain moisture. Maize plants that have one or more marker alleles associated with reduced grain moisture are then identified and/or selected. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group, or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0017] In one aspect, the marker is PHM10270, PHM4598, PHM14092, PHM18292, PHM3507, PHM3004, or PHM14122.

[0018] In another aspect, the marker detects a SNP polymorphism. The SNP polymorphism can be PHM10270-13, PHM4598-22, PHM14092-77, PHM18292-9, PHM3507-13, PHM3004-13, or PHM14122-22.

[0019] In another aspect, the allele is a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, or a “G” at PHM14122-22.

[0020] In another embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a marker allele is detected in the maize plant. The marker is located on in a chromosomal interval defined by and including PHM4598 and PHM14092; and an allele of the marker is associated with reduced grain moisture. Maize plants that have one or more marker alleles associated with reduced grain moisture are then identified and/or selected. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group, or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0021] In one embodiment, the marker is PHM10270, PHM4598, PHM14092, PHM18292, PHM3507, PHM3004, or PHM14122.

[0022] In another aspect, the marker detects a SNP polymorphism. The SNP polymorphism can be PHM10270-13, PHM4598-22, PHM14092-77, PHM18292-9, PHM3507-13, PHM3004-13, or PHM14122-22.

[0023] In another aspect, the allele is a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, or a “G” at PHM14122-22.

[0024] In another embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a haplotype is detected in the maize plant. The haplotype can comprise: a “T” at PHM9929-8, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, and a “T” at PHM17246-16. Maize plants that have the haplotype are identified and/or selected.

[0025] In another embodiment, methods for identifying and/or selecting a maize plant with reduced grain moisture are presented in which a haplotype is detected in the maize plant. The haplotype can comprise: a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, and a “G” at PHM14122-22. Maize plants that have the haplotype are identified and/or selected. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group, or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0026] In another embodiment, maize plants identified and/or selected by any of the methods presented above are included.

[0027] In another embodiment, a maize plant comprising within its genome a haplotype consisting of: a “T” at PHM9929-8, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, and a “T” at PHM17246-16; that exhibits reduced grain moisture when compared to a maize plant or progeny plant that does not have the haplotype, is presented. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group, or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0028] In another embodiment, a maize plant comprising within its genome a haplotype consisting of: a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, and a “G” at PHM14122-22; that exhibits reduced grain moisture when compared to a maize plant or progeny plant that does not have the haplotype, is presented. The maize plant may be an inbred in the Stiff Stalk Synthetic heterotic group, or may be a progeny plant derived from an inbred in the Stiff Stalk Synthetic heterotic group.

[0029] In another embodiment, methods of introgressing a reduced grain moisture QTL allele into a maize plant are provided. The methods include: screening a population with at least one marker to determine if one or more maize plants from the population comprise(s) an allele of a marker associated with a reduced grain moisture QTL allele, wherein the reduced grain moisture QTL allele comprises a “T” at PHM18206-5, a “T” at PHM9929-8, an “A” at PHM6726-10, a “G” at PHM4910-5, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, a “T” at PHM17246-16, and a “T” at PHM4691-9; and selecting from said population at least one maize plant comprising an allele of said marker locus associated with a reduced grain moisture QTL allele. A marker of interest can be located within 5 cM, 2 cM, or 1 cM of the grain moisture QTL.

[0030] In another embodiment, methods of introgressing a reduced grain moisture QTL allele into a maize plant are provided. The methods include: screening a population with at least one marker to determine if one or more maize plants from the population comprise(s) an allele of said marker associated with a reduced grain moisture QTL allele, wherein the reduced grain moisture QTL comprises: a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, and a “G” at PHM14122-22; and selecting from said population at least one maize plant comprising an allele of said marker locus associated with a reduced grain moisture QTL allele. A marker of interest can be located within 5 cM, 2 cM, or 1 cM of the grain moisture QTL.

[0035] SEQ ID NO:1 is the reference sequence for marker PHM18206.

[0036] SEQ ID NO:2 is the reference sequence for marker PHM6567.

[0037] SEQ ID NO:3 is the reference sequence for marker PHM4691.

[0038] SEQ ID NO:4 is the reference sequence for marker PHM229.

[0039] SEQ ID NO:5 is the reference sequence for marker PHM9929.

[0040] SEQ ID NO:6 is the reference sequence for marker PHM3159.

[0041] SEQ ID NO:7 is the reference sequence for marker PHM15758.

[0042] SEQ ID NO:8 is the reference sequence for marker PHM5208.

[0043] SEQ ID NO:9 is the reference sequence for marker PHM6667.

[0044] SEQ ID NO:10 is the reference sequence for marker PHM17246.

[0045] SEQ ID NO:11 is the reference sequence for marker PHM14370.

[0046] SEQ ID NO:12 is the reference sequence for marker PHM6726.

[0047] SEQ ID NO:13 is the reference sequence for marker PHM4910.

[0048] SEQ ID NO:14 is the reference sequence for marker PHM4598.

[0049] SEQ ID NO:15 is the reference sequence for marker PHM3507.

[0050] SEQ ID NO:16 is the reference sequence for marker PHM18292.

[0051] SEQ ID NO:17 is the reference sequence for marker PHM3004.

[0052] SEQ ID NO:18 is the reference sequence for marker PHM14122.

[0053] SEQ ID NO:19 is the reference sequence for marker PHM10270

[0054] SEQ ID NO:20 is the reference sequence for marker PHM14092.

[0055] SEQ ID NO:21 is the PHM18206 forward external primer.

[0056] SEQ ID NO:22 is the PHM18206 forward internal primer.

[0057] SEQ ID NO:23 is the PHM18206 reverse internal primer.

[0058] SEQ ID NO:24 is the PHM18206 reverse external primer.

[0059] SEQ ID NO:25 is the PHM6567 forward external primer.

[0060] SEQ ID NO:26 is the PHM6567 forward internal primer.

[0061] SEQ ID NO:27 is the PHM6567 reverse internal primer.

[0062] SEQ ID NO:28 is the PHM6567 reverse external primer.

[0063] SEQ ID NO:29 is the PHM4691 forward external primer.

[0064] SEQ ID NO:30 is the PHM4691 forward internal primer.

[0065] SEQ ID NO:31 is the PHM4691 reverse internal primer.

[0066] SEQ ID NO:32 is the PHM4691 reverse external primer.

[0067] SEQ ID NO:33 is the PHM229 forward external primer.

[0068] SEQ ID NO:34 is the PHM229 forward internal primer.

[0069] SEQ ID NO:35 is the PHM229 reverse internal primer.

[0070] SEQ ID NO:36 is the PHM229 reverse external primer.

[0071] SEQ ID NO:37 is the PHM9929 forward external primer.

[0072] SEQ ID NO:38 is the PHM9929 forward internal primer.

[0073] SEQ ID NO:39 is the PHM9929 reverse internal primer.

[0074] SEQ ID NO:40 is the PHM9929 reverse external primer.

[0075] SEQ ID NO:41 is the PHM3159 forward external primer.

[0076] SEQ ID NO:42 is the PHM3159 forward internal primer.

[0077] SEQ ID NO:43 is the PHM3159 reverse internal primer.

[0078] SEQ ID NO:44 is the PHM3159 reverse external primer.

[0079] SEQ ID NO:45 is the PHM15758 forward external primer.

[0080] SEQ ID NO:46 is the PHM15758 forward internal primer.

[0081] SEQ ID NO:47 is the PHM15758 reverse internal primer.

[0082] SEQ ID NO:48 is the PHM15758 reverse external primer.

[0083] SEQ ID NO:49 is the PHM5208 forward external primer.

[0084] SEQ ID NO:50 is the PHM5208 forward internal primer.

[0085] SEQ ID NO:51 is the PHM5208 reverse internal primer.

[0086] SEQ ID NO:52 is the PHM5208 reverse external primer.

[0087] SEQ ID NO:53 is the PHM6667 forward external primer.

[0088] SEQ ID NO:54 is the PHM6667 forward internal primer.

[0089] SEQ ID NO:55 is the PHM6667 reverse internal primer.

[0090] SEQ ID NO:56 is the PHM6667 reverse external primer.

[0091] SEQ ID NO:57 is the PHM17246 forward external primer.

[0092] SEQ ID NO:58 is the PHM17246 forward internal primer.

[0093] SEQ ID NO:59 is the PHM17246 reverse internal primer.

[0094] SEQ ID NO:60 is the PHM17246 reverse external primer.

[0095] SEQ ID NO:61 is the PHM14370 forward external primer.

[0096] SEQ ID NO:62 is the PHM14370 forward internal primer.

[0097] SEQ ID NO:63 is the PHM14370 reverse internal primer.

[0098] SEQ ID NO:64 is the PHM14370 reverse external primer.

[0099] SEQ ID NO:65 is the PHM6726 forward external primer.

[0100] SEQ ID NO:66 is the PHM6726 forward internal primer.

[0101] SEQ ID NO:67 is the PHM6726 reverse internal primer.

[0102] SEQ ID NO:68 is the PHM6726 reverse external primer.

[0103] SEQ ID NO:69 is the PHM4910 forward external primer.

[0104] SEQ ID NO:70 is the PHM4910 forward internal primer.

[0105] SEQ ID NO:71 is the PHM4910 reverse internal primer.

[0106] SEQ ID NO:72 is the PHM4910 reverse external primer.

[0107] SEQ ID NO:73 is the PHM4598 forward external primer.

[0108] SEQ ID NO:74 is the PHM4598 forward internal primer.

[0109] SEQ ID NO:75 is the PHM4598 reverse internal primer.

[0110] SEQ ID NO:76 is the PHM4598 reverse external primer.

[0111] SEQ ID NO:77 is the PHM3507 forward external primer.

[0112] SEQ ID NO:78 is the PHM3507 forward internal primer.

[0113] SEQ ID NO:79 is the PHM3507 reverse internal primer.

[0114] SEQ ID NO:80 is the PHM3507 reverse external primer.

[0115] SEQ ID NO:81 is the PHM18292 forward external primer.

[0116] SEQ ID NO:82 is the PHM18292 forward internal primer.

[0117] SEQ ID NO:83 is the PHM18292 reverse internal primer.

[0118] SEQ ID NO:84 is the PHM18292 reverse external primer.

[0119] SEQ ID NO:85 is the PHM3004 forward external primer.

[0120] SEQ ID NO:86 is the PHM3004 forward internal primer.

[0121] SEQ ID NO:87 is the PHM3004 reverse internal primer.

[0122] SEQ ID NO:88 is the PHM3004 reverse external primer.

[0123] SEQ ID NO:89 is the PHM14122 forward external primer.

[0124] SEQ ID NO:90 is the PHM14122 forward internal primer.

[0125] SEQ ID NO:91 is the PHM14122 reverse internal primer.

[0126] SEQ ID NO:92 is the PHM14122 reverse external primer.

[0127] SEQ ID NO:93 is the PHM10270 forward external primer.

[0128] SEQ ID NO:94 is the PHM10270 forward internal primer.

[0129] SEQ ID NO:95 is the PHM10270 reverse internal primer.

[0130] SEQ ID NO:96 is the PHM10270 reverse external primer.

[0131] SEQ ID NO:97 is the PHM14092 forward external primer.

[0132] SEQ ID NO:98 is the PHM14092 forward internal primer.

[0133] SEQ ID NO:99 is the PHM14092 reverse internal primer.

[0134] SEQ ID NO:100 is the PHM14092 reverse external primer.

[0135] SEQ ID NO:101 is the reference sequence for marker PHM10028.

[0136] SEQ ID NO:102 is the reference sequence for marker PHM741.

[0137] SEQ ID NO:103 is the reference sequence for marker PHM6289.

[0138] SEQ ID NO:104 is the reference sequence for marker PHM13089.

[0139] SEQ ID NO:105 is the reference sequence for marker PHM2305.

[0140] SEQ ID NO:106 is the reference sequence for marker PHM9867.

[0141] SEQ ID NO:107 is the reference sequence for marker PHM7775.

[0142] SEQ ID NO:108 is the reference sequence for marker PHM16407.

[0143] SEQ ID NO:109 is the reference sequence for marker PHM14523.

[0144] SEQ ID NO:110 is the reference sequence for marker PHM2916.

[0145] SEQ ID NO: 11 is the reference sequence for marker PHM4583.

[0146] SEQ ID NO:112 is the PHM10028 forward external primer.

[0147] SEQ ID NO:113 is the PHM10028 forward internal primer.

[0148] SEQ ID NO:114 is the PHM10028 reverse internal primer.

[0149] SEQ ID NO:115 is the PHM10028 reverse external primer.

[0150] SEQ ID NO:116 is the PHM741 forward external primer.

[0151] SEQ ID NO:117 is the PHM741 forward internal primer.

[0152] SEQ ID NO:118 is the PHM741 reverse internal primer.

[0153] SEQ ID NO:119 is the PHM741 reverse external primer.

[0154] SEQ ID NO:120 is the PHM6289 forward external primer.

[0155] SEQ ID NO:121 is the PHM6289 forward internal primer.

[0156] SEQ ID NO:122 is the PHM6289 reverse internal primer.

[0157] SEQ ID NO:123 is the PHM6289 reverse external primer.

[0158] SEQ ID NO:124 is the PHM13089 forward external primer.

[0159] SEQ ID NO:125 is the PHM13089 forward internal primer.

[0160] SEQ ID NO:126 is the PHM13089 reverse internal primer.

[0161] SEQ ID NO:127 is the PHM13089 reverse external primer.

[0162] SEQ ID NO:128 is the PHM2305 forward external primer.

[0163] SEQ ID NO:129 is the PHM2305 forward internal primer.

[0164] SEQ ID NO:130 is the PHM2305 reverse internal primer.

[0165] SEQ ID NO:131 is the PHM2305 reverse external primer.

[0166] SEQ ID NO:132 is the PHM9867 forward external primer.

[0167] SEQ ID NO:133 is the PHM9867 forward internal primer.

[0168] SEQ ID NO:134 is the PHM9867 reverse internal primer.

[0169] SEQ ID NO:135 is the PHM9867 reverse external primer.

[0170] SEQ ID NO:136 is the PHM7775 forward external primer.

[0171] SEQ ID NO:137 is the PHM7775 forward internal primer.

[0172] SEQ ID NO:138 is the PHM7775 reverse internal primer.

[0173] SEQ ID NO:139 is the PHM7775 reverse external primer.

[0174] SEQ ID NO:140 is the PHM16407 forward external primer.

[0175] SEQ ID NO:141 is the PHM16407 forward internal primer.

[0176] SEQ ID NO:142 is the PHM16407 reverse internal primer.

[0177] SEQ ID NO:143 is the PHM16407 reverse external primer.

[0178] SEQ ID NO:144 is the PHM14523 forward external primer.

[0179] SEQ ID NO:145 is the PHM14523 forward internal primer.

[0180] SEQ ID NO:146 is the PHM14523 reverse internal primer.

[0181] SEQ ID NO:147 is the PHM14523 reverse external primer.

[0182] SEQ ID NO:148 is the PHM2916 forward external primer.

[0183] SEQ ID NO:149 is the PHM2916 forward internal primer.

[0184] SEQ ID NO:150 is the PHM2916 reverse internal primer.

[0185] SEQ ID NO:151 is the PHM2916 reverse external primer.

[0186] SEQ ID NO:152 is the PHM4583 forward external primer.

[0187] SEQ ID NO:153 is the PHM4583 forward internal primer.

[0188] SEQ ID NO:154 is the PHM4583 reverse internal primer.

[0189] SEQ ID NO:155 is the PHM4583 reverse external primer.

[0190] SEQ ID NO:156 is the reference sequence for marker PHM14165.

[0191] SEQ ID NO:157 is the reference sequence for marker PHM15960.

[0192] SEQ ID NO:158 is the reference sequence for marker PHM8438.

[0193] SEQ ID NO:159 is the reference sequence for marker PHM9867.

[0194] SEQ ID NO:160 is the reference sequence for marker PHM14523.

[0195] SEQ ID NO:161 is the PHM14165 forward external primer.

[0196] SEQ ID NO:162 is the PHM14165 forward internal primer.

[0197] SEQ ID NO:163 is the PHM14165 reverse internal primer.

[0198] SEQ ID NO:164 is the PHM14165 reverse external primer.

[0199] SEQ ID NO:165 is the PHM15960 forward external primer.

[0200] SEQ ID NO:166 is the PHM15960 forward internal primer.

[0201] SEQ ID NO:167 is the PHM15960 reverse internal primer.

[0202] SEQ ID NO:168 is the PHM15960 reverse external primer.

[0203] SEQ ID NO:169 is the PHM8438 forward external primer.

[0204] SEQ ID NO:170 is the PHM8438 forward internal primer.

[0205] SEQ ID NO:171 is the PHM8438 reverse internal primer.

[0206] SEQ ID NO:172 is the PHM8438 reverse external primer.

[0207] SEQ ID NO:173 is the PHM9867 forward external primer.

[0208] SEQ ID NO:174 is the PHM9867 forward internal primer.

[0209] SEQ ID NO:175 is the PHM9867 reverse internal primer.

[0210] SEQ ID NO:176 is the PHM9867 reverse external primer.

[0211] SEQ ID NO:177 is the PHM14523 forward external primer.

[0212] SEQ ID NO:178 is the PHM14523 forward internal primer.

[0213] SEQ ID NO:179 is the PHM14523 reverse internal primer.

[0214] SEQ ID NO:180 is the PHM14523 reverse external primer.

[0215] Grain moisture is an important trait for maize production. If the grain is too moist when the grower wants to harvest, then the grower may have to leave the crop in the field for a longer period of time, thereby exposing the crop to adverse weather and field conditions that could affect yield. Furthermore, once the grain is harvested, artificial drying may be needed to achieve a desired grain moisture level, requiring access to drying equipment, transportation to move the grain to the dryers, and power to run the dryers. Thus, it is desirable to identify and select maize plants with reduced grain moisture.

[0216] The present invention provides maize marker loci that demonstrate statistically significant co-segregation with grain moisture. Detection of these loci or additional linked loci can be used in marker assisted maize breeding programs to produce maize plants with reduced grain moisture.

[0217] The following definitions are provided as an aid to understand this invention.

[0218] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, terms in the singular and the singular forms “a”, “an” and “the”, for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “plant”, “the plant” or “a plant” also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules.

[0219] Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

[0220] The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.

[0221] “Allele frequency” refers to the frequency (proportion or percentage) at which an allele is present at a locus within an individual, within a line, or within a population of lines. For example, for an allele “A”, diploid individuals of genotype “AA”, “Aa”, or “aa” have allele frequencies of 1.0, 0.5, or 0.0, respectively. One can estimate the allele frequency within a line by averaging the allele frequencies of a sample of individuals from that line. Similarly, one can calculate the allele frequency within a population of lines by averaging the allele frequencies of lines that make up the population. For a population with a finite number of individuals or lines, an allele frequency can be expressed as a count of individuals or lines (or any other specified grouping) containing the allele.

[0222] An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

[0223] The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

[0224] The term “assemble” applies to BACs and their propensities for coming together to form contiguous stretches of DNA. A BAC “assembles” to a contig based on sequence alignment, if the BAC is sequenced, or via the alignment of its BAC fingerprint to the fingerprints of other BACs. Public assemblies can be found using the Maize Genome Browser, which is publicly available on the internet.

[0225] An allele is “associated with” a trait when it is part of or linked to a DNA sequence or allele that affects the expression of the trait. The presence of the allele is an indicator of how the trait will be expressed.

[0226] A “BAC”, or bacterial artificial chromosome, is a cloning vector derived from the naturally occurring F factor of Escherichia coli, which itself is a DNA element that can exist as a circular plasmid or can be integrated into the bacterial chromosome. BACs can accept large inserts of DNA sequence. In maize, a number of BACs each containing a large insert of maize genomic DNA from maize inbred line B73, have been assembled into contigs (overlapping contiguous genetic fragments, or “contiguous DNA”), and this assembly is available publicly on the internet.

[0227] A BAC fingerprint is a means of analyzing similarity between several DNA samples based upon the presence or absence of specific restriction sites (restriction sites being nucleotide sequences recognized by enzymes that cut or “restrict” the DNA). Two or more BAC samples are digested with the same set of restriction enzymes and the sizes of the fragments formed are compared, usually using gel separation.

[0228] “Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene/genes, locus/loci, or specific phenotype to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the F1 generation; the term “BC1” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.

[0229] A centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.

[0230] As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.

[0231] A “chromosome” is a single piece of coiled DNA containing many genes that act and move as a unity during cell division and therefore can be said to be linked. It can also be referred to as a “linkage group”.

[0232] The phrase “closely linked”, in the present application, means that recombination between two linked loci occurs with a frequency of equal to or less than about 10% (i.e., are separated on a genetic map by not more than 10 cM). Put another way, the closely linked loci co-segregate at least 90% of the time. Marker loci are especially useful in the present invention when they demonstrate a significant probability of co-segregation (linkage) with a desired trait (e.g., reduced grain moisture). Closely linked loci such as a marker locus and a second locus can display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination a frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” each other. In some cases, two different markers can have the same genetic map coordinates. In that case, the two markers are in such close proximity to each other that recombination occurs between them with such low frequency that it is undetectable.

[0233] The term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence, i.e. the sequences are related by the Watson-Crick base-pairing rules.

[0234] The term “contiguous DNA” refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.

[0235] When referring to the relationship between two genetic elements, such as a genetic element contributing to grain moisture and a proximal marker, “coupling” phase linkage indicates the state where the “favorable” allele at the grain moisture locus is physically associated on the same chromosome strand as the “favorable” allele of the respective linked marker locus. In coupling phase, both favorable alleles are inherited together by progeny that inherit that chromosome strand.

[0236] The term “crossed” or “cross” refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).

[0237] A plant referred to herein as “diploid” has two sets (genomes) of chromosomes.

[0238] A plant referred to herein as a “doubled haploid” is developed by doubling the haploid set of chromosomes (i.e., half the normal number of chromosomes). A doubled haploid plant has two identical sets of chromosomes, and all loci are considered homozygous.

[0239] An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.

[0240] An “exotic maize strain” or an “exotic maize germplasm” is a strain derived from a maize plant not belonging to an available elite maize line or strain of germplasm. In the context of a cross between two maize plants or strains of germplasm, an exotic germplasm is not closely related by descent to the elite germplasm with which it is crossed. Most commonly, the exotic germplasm is not derived from any known elite line of maize, but rather is selected to introduce novel genetic elements (typically novel alleles) into a breeding program.

[0241] A “favorable allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, e.g., reduced grain moisture, and that allows the identification of plants with that agronomically desirable phenotype. A favorable allele of a marker is a marker allele that segregates with the favorable phenotype.

[0242] “Fragment” is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.

[0243] A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by how frequently their alleles appear together in a population (their recombination frequencies). Alleles can be detected using DNA or protein markers, or observable phenotypes. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. Genetic distances between loci can differ from one genetic map to another. However, information can be correlated from one map to another using common markers. One of ordinary skill in the art can use common marker positions to identify positions of markers and other loci of interest on each individual genetic map. The order of loci should not change between maps, although frequently there are small changes in marker orders due to e.g. markers detecting alternate duplicate loci in different populations, differences in statistical approaches used to order the markers, novel mutation or laboratory error.

[0244] A “genetic map location” is a location on a genetic map relative to surrounding genetic markers on the same linkage group where a specified marker can be found within a given species.

[0245] “Genetic mapping” is the process of defining the linkage relationships of loci through the use of genetic markers, populations segregating for the markers, and standard genetic principles of recombination frequency.

[0246] “Genetic markers” are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and the like. The term also refers to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also know for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).

[0247] “Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis.

[0248] “Genome” refers to the total DNA, or the entire set of genes, carried by a chromosome or chromosome set.

[0249] The term “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci. Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.

[0250] “Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection). The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells, that can be cultured into a whole plant.

[0251] “Grain moisture” is a property of grain that is measured in order to determine the optimal time to harvest. It can be measured using any of a number of methods, one of which is the use of a meter that measures the electrical properties of grain. While exact values for grain moisture can be obtained, grain moisture as used herein is a relative term. Thus, a plant with favorable alleles at either or both chromosome 9 QTL will have “reduced grain moisture” as compared to plants that do not have favorable alleles at these QTL.

[0252] A plant referred to as “haploid” has a single set (genome) of chromosomes.

[0253] A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to alleles at a particular locus, or to alleles at multiple loci along a chromosomal segment.

[0254] The term “heterogeneity” is used to indicate that individuals within the group differ in genotype at one or more specific loci.

[0255] The heterotic response of material, or “heterosis”, can be defined by performance which exceeds the average of the parents (or high parent) when crossed to other dissimilar or unrelated groups.

[0256] A “heterotic group” comprises a set of genotypes that perform well when crossed with genotypes from a different heterotic group (Hallauer et al. (1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley (ed.) Corn and corn improvement). Inbred lines are classified into heterotic groups, and are further subdivided into families within a heterotic group, based on several criteria such as pedigree, molecular marker-based associations, and performance in hybrid combinations (Smith et al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely used heterotic groups in the United States are referred to as “Iowa Stiff Stalk Synthetic” (also referred to herein as “stiff stalk”) and “Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, or non-Stiff Stalk).

[0257] Some heterotic groups possess the traits needed to be a female parent, and others, traits for a male parent. For example, in maize, yield results from public inbreds released from a population called BSSS (Iowa Stiff Stalk Synthetic population) has resulted in these inbreds and their derivatives becoming the female pool in the central Corn Belt. BSSS inbreds have been crossed with other inbreds, e.g. SD 105 and Maiz Amargo, and this general group of materials has become known as Stiff Stalk Synthetics (SSS) even though not all of the inbreds are derived from the original BSSS population (Mikel and Dudley (2006) Crop Sci: 46:1193-1205). By default, all other inbreds that combine well with the SSS inbreds have been assigned to the male pool, which for lack of a better name has been designated as NSS, i.e. Non-Stiff Stalk. This group includes several major heterotic groups such as Lancaster Surecrop, Iodent, and Leaming Corn.

[0258] An individual is “heterozygous” if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles).

[0259] The term “homogeneity” indicates that members of a group have the same genotype at one or more specific loci.

[0260] An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes).

[0261] The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.

[0262] “Hybridization” or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.

[0263] The term “hybridize” means to form base pairs between complementary regions of nucleic acid strands.

[0264] An “IBM genetic map” can refer to any of following maps: IBM, IBM2, IBM2 neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2 2005 neighbors, IBM2 2005 neighbors frame, IBM2 2008 neighbors, IBM2 2008 neighbors frame, or the latest version on the maizeGDB website. IBM genetic maps are based on a B73×Mo17 population in which the progeny from the initial cross were random-mated for multiple generations prior to constructing recombinant inbred lines for mapping. Newer versions reflect the addition of genetic and BAC mapped loci as well as enhanced map refinement due to the incorporation of information obtained from other genetic maps or physical maps, cleaned date, or the use of new algorithms.

[0265] The term “inbred” refers to a line that has been bred for genetic homogeneity.

[0266] The term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an inserted nucleotide or piece of DNA relative to a second line, or the second line may be referred to as having a deleted nucleotide or piece of DNA relative to the first line.

[0267] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0268] The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times.

[0269] A “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic). A “subline” refers to an inbred subset of descendents that are genetically distinct from other similarly inbred subsets descended from the same progenitor.

[0270] As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus. The linkage relationship between a molecular marker and a locus affecting a phenotype is given as a “probability” or “adjusted probability”. Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map (a genetic map based on a population that has undergone one round of meiosis, such as e.g. an F₂; the IBM2 maps consist of multiple meioses). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “in proximity to” each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

[0271] The term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype. A marker locus can be “associated with” (linked to) a trait. The degree of linkage of a marker locus and a locus affecting a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype (e.g., an F statistic or LOD score).

[0272] Linkage disequilibrium is most commonly assessed using the measure r², which is calculated using the formula described by Hill, W. G. and Robertson, A, Theor. Appl. Genet. 38:226-231(1968). When r²=1, complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. The r² value will be dependent on the population used. Values for r² above ⅓ indicate sufficiently strong LD to be useful for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibrium when r² values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

[0273] As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).

[0274] A “locus” is a position on a chromosome, e.g. where a nucleotide, gene, sequence, or marker is located.

[0275] The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science 255:803-804 (1992)) is used in genetic interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage. LOD scores can also be used to show the strength of association between marker loci and quantitative traits in “quantitative trait loci” mapping. In this case, the LOD score's size is dependent on the closeness of the marker locus to the locus affecting the quantitative trait, as well as the size of the quantitative trait effect.

[0276] “Maize” refers to a plant of the Zea mays L. ssp. mays and is also known as “corn”.

[0277] The term “maize plant” includes whole maize plants, maize plant cells, maize plant protoplast, maize plant cell or maize tissue culture from which maize plants can be regenerated, maize plant calli, maize plant clumps and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips and the like.

[0278] A “marker” is a means of finding a position on a genetic or physical map, or else linkages among markers and trait loci (loci affecting traits). The position that the marker detects may be known via detection of polymorphic alleles and their genetic mapping, or else by hybridization, sequence match or amplification of a sequence that has been physically mapped. A marker can be a DNA marker (detects DNA polymorphisms), a protein (detects variation at an encoded polypeptide), or a simply inherited phenotype (such as the ‘waxy’ phenotype). A DNA marker can be developed from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA). Depending on the DNA marker technology, the marker will consist of complementary primers flanking the locus and/or complementary probes that hybridize to polymorphic alleles at the locus. A DNA marker, or a genetic marker, can also be used to describe the gene, DNA sequence or nucleotide on the chromosome itself (rather than the components used to detect the gene or DNA sequence) and is often used when that DNA marker is associated with a particular trait in human genetics (e.g. a marker for breast cancer). The term marker locus is the locus (gene, sequence or nucleotide) that the marker detects.

[0279] Markers that detect genetic polymorphisms between members of a population are well-established in the art. Markers can be defined by the type of polymorphism that they detect and also the marker technology used to detect the polymorphism. Marker types include but are not limited to, e.g., detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), detection of simple sequence repeats (SSRs), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, or detection of single nucleotide polymorphisms (SNPs). SNPs can be detected e.g. via DNA sequencing, PCR-based sequence specific amplification methods, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase assays, Flap endonudeases, 5′ endonucleases, primer extension, single strand conformation polymorphism (SSCP) or temperature gradient gel electrophoresis (TGGE). DNA sequencing, such as the pyrosequencing technology has the advantage of being able to detect a series of linked SNP alleles that constitute a haplotype. Haplotypes tend to be more informative (detect a higher level of polymorphism) than SNPs.

[0280] A “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.

[0281] “Marker assisted selection” (of MAS) is a process by which individual plants are selected based on marker genotypes.

[0282] “Marker assisted counter-selection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.

[0283] A “marker haplotype” refers to a combination of alleles at a marker locus.

[0284] A “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., one that affects the expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a genetically or physically linked locus.

[0285] A “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus (“all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus.

[0286] The term “molecular marker” may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non-collinear region described herein. This is because the insertion region is, by definition, a polymorphism vis a vis a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g. SNP technology is used in the examples provided herein.

[0287] An allele “negatively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.

[0288] “Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and “nucleic acid fragment” are used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed, and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

[0289] The term “phenotype”, “phenotypic trait”, or “trait” can refer to the observable expression of a gene or series of genes. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., weighing, counting, measuring (length, width, angles, etc.), microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait” or a “simply inherited trait”. In the absence of large levels of environmental variation, single gene traits can segregate in a population to give a “qualitative” or “discrete” distribution, i.e. the phenotype falls into discrete classes. In other cases, a phenotype is the result of several genes and can be considered a “multigenic trait” or a “complex trait”. Multigenic traits segregate in a population to give a “quantitative” or “continuous” distribution, i.e. the phenotype cannot be separated into discrete classes. Both single gene and multigenic traits can be affected by the environment in which they are being expressed, but multigenic traits tend to have a larger environmental component.

[0290] A “physical map” of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc.) on chromosome DNA. However, in contrast to genetic maps, the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination (that can vary in different populations).

[0291] A “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.

[0292] A maize plant “derived from an inbred in the Stiff Stalk Synthetic population” may be a hybrid.

[0293] A “polymorphism” is a variation in the DNA between two or more individuals within a population. A polymorphism preferably has a frequency of at least 1% in a population. A useful polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an “indel”.

[0294] An allele “positively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.

[0295] The “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a locus and a phenotype are associated. The probability score can be affected by the proximity of the first locus (usually a marker locus) and the locus affecting the phenotype, plus the magnitude of the phenotypic effect (the change in phenotype caused by an allele substitution). In some aspects, the probability score is considered “significant” or “nonsignificant”. In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of association. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.001.

[0296] A “production marker” or “production SNP marker” is a marker that has been developed for high-throughput purposes. Production SNP markers are developed to detect specific polymorphisms and are designed for use with a variety of chemistries and platforms. The marker names used here begin with a PHM prefix to denote ‘Pioneer Hi-Bred Marker’, followed by a number that is specific to the sequence from which it was designed, followed by a “.” or a “-” and then a suffix that is specific to the DNA polymorphism. A marker version can also follow (A, B, C etc.) that denotes the version of the marker designed to that specific polymorphism.

[0297] The term “progeny” refers to the offspring generated from a cross.

[0298] A “progeny plant” is a plant generated from a cross between two plants.

[0299] The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question. An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. A haplotype can be defined by the unique fingerprint of alleles at each marker within the specified window.

[0300] A “reference sequence” or a “consensus sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence for a PHM marker is obtained by sequencing a number of lines at the locus, aligning the nucleotide sequences in a sequence alignment program (e.g. Sequencher), and then obtaining the most common nucleotide sequence of the alignment. Polymorphisms found among the individual sequences are annotated within the consensus sequence. A reference sequence is not usually an exact copy of any individual DNA sequence, but represents an amalgam of available sequences and is useful for designing primers and probes to polymorphisms within the sequence.

[0301] In “repulsion” phase linkage, the “favorable” allele at the locus of interest is physically linked with an “unfavorable” allele at the proximal marker locus, and the two “favorable” alleles are not inherited together (i.e., the two loci are “out of phase” with each other).

[0302] A “topcross test” is a test performed by crossing each individual (e.g. a selection, inbred line, clone or progeny individual) with the same pollen parent or “tester”, usually a homozygous line.

[0303] The phrase “under stringent conditions” refers to conditions under which a probe or polynucleotide will hybridize to a specific nucleic acid sequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C., depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references.

[0304] An “unfavorable allele” of a marker is a marker allele that segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants that can be removed from a breeding program or planting.

[0305] The term “yield” refers to the productivity per unit area of a particular plant product of commercial value. For example, yield of maize is commonly measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is affected by both genetic and environmental factors. “Agronomics”, “agronomic traits”, and “agronomic performance” refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like. Yield is, therefore, the final culmination of all agronomic traits.

[0306] Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

[0307] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

[0308] It has been recognized for quite some time that specific genetic loci correlating with particular phenotypes, such as grain moisture, can be mapped in an organism's genome. The plant breeder can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co-segregation with a desired phenotype, manifested as linkage disequilibrium. By identifying a molecular marker or clusters of molecular markers that co-segregate with a trait of interest, the breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (a process called marker-assisted selection, or MAS).

[0309] A variety of methods well known in the art are available for detecting molecular markers or clusters of molecular markers that co-segregate with a trait of interest, such as grain moisture. The basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes. Thus, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes (or alleles) or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference.

[0310] Two such methods used to detect trait loci of interest are: 1) Population-based association analysis and 2) Traditional linkage analysis. In a population-based association analysis, lines are obtained from pre-existing populations with multiple founders, e.g. elite breeding lines. Population-based association analyses rely on the decay of linkage disequilibrium (LD) and the idea that in an unstructured population, only correlations between genes controlling a trait of interest and markers closely linked to those genes will remain after so many generations of random mating. In reality, most pre-existing populations have population substructure. Thus, the use of a structured association approach helps to control population structure by allocating individuals to populations using data obtained from markers randomly distributed across the genome, thereby minimizing disequilibrium due to population structure within the individual populations (also called subpopulations). The phenotypic values are compared to the genotypes (alleles) at each marker locus for each line in the subpopulation. A significant marker-trait association indicates the close proximity between the marker locus and one or more genetic loci that are involved in the expression of that trait.

[0311] The same principles underlie traditional linkage analysis; however, LD is generated by creating a population from a small number of founders. The founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of cosegregation with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is an interval mapping approach (Lander and Botstein, Genetics 121:185-199 (1989), in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position. The genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a threshold value, there is significant evidence for the location of a gene controlling the trait of interest at that position on the genetic map (which will fall between two particular marker loci).

[0312] The present invention provides maize marker loci that demonstrate statistically significant co-segregation with grain moisture, as determined by traditional linkage analysis and by whole genome association analysis. Detection of these loci or additional linked loci can be used in marker assisted maize breeding programs to produce plants with reduced grain moisture.

[0313] Activities in marker assisted maize breeding programs may include but are not limited to: selecting among new breeding populations to identify which population has the highest frequency of favorable nucleic acid sequences based on historical genotype and agronomic trait associations, selecting favorable nucleic acid sequences among progeny in breeding populations, selecting among parental lines based on prediction of progeny performance, and advancing lines in germplasm improvement activities based on presence of favorable nucleic acid sequences.

[0314] Two QTL on chromosome 9 were identified from a linkage mapping analysis (Example 1). The first QTL, identified herein as QTL9-1, is located at 72-79 cM on a single meiosis map (e.g. the PHB map), which approximately corresponds to 200.4-230.6 cM on an IBM2 map, the most recent version of which is the IBM2 2008 map. QTL9-1 was found to be associated with both grain moisture and yield. The second QTL, identified herein as QTL9-2, is located at 93-98 cM on a single meiosis map (e.g. the PHB map), which approximately corresponds to 317-323 cM on an IBM2 map, the most recent version of which is the IBM2 2008 map. QTL9-2 was found to be associated with grain moisture. These QTL were further validated by a whole genome association analysis.

[0315] Chromosomal intervals that correlate with grain moisture are provided. A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene(s) controlling the trait of interest. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for grain moisture. Tables 1 and 2 show markers within the QTL9-1 and QTL9-2 regions, respectively, that are shown herein to associate with grain moisture and that are linked to a gene(s) controlling grain moisture. Reference sequences for each of the PHM markers listed in Tables 1 and 2 are represented by SEQ ID NOs:1-20 and the primers are represented by SEQ ID NOs:21-100.

TABLE 1
QTL9-1 Markers
			B73
			BAC(s)
	PHB	IBM2	on which	Position in
	Map	Map	marker is	most recent	internal
Marker	Position	Position	located	B73 assembly	contig
PHM18206	71.5	N/A	c0152O14	24,337,082-	9004
				24,385,520
PHM6567	75.2	220.7	c0412K22	28,512,451-	9004
				28,460,814
PHM4691	75.3	220.1	b0428G16	28,901,175-	9004
				28,901,074
PHM229	74.0	220.1	c0202D12	30,129,229-	9005
				30,129,128
PHM9929	75.8	226.3	c0504I01;	43,460,951-	9007
				43,460,850;
PHM3159	77.0	226.3*	b0605B16	44,264,873-	9007
			b0131J16	44,264,772
				44,384,523-
				44,350,417
PHM15758	76.1	226.3	c0245002	47,163,201-	9008
				47,223,179
PHM5208	75.9	226.3*	N/A	N/A	9011
PHM6667	76.2	226.3	c0242C10	60,969,688-	9011
				60,966,572
PHM17246	77.0	226.3	b0631G11	62,629,552-	9011
				62,629,451
PHM14370	76.9	226.3	c0150K08	N/A	9011
PHM6726	77.3	230.6	c0194F10	84,272,694-	9013
				84,399,377
PHM4910	78.8	230.6	c0131H04;	89,601,648-	9013
			c0009L19	89,601,547;
				89,745,216-
				89,854,317
*Map position is estimated based on relative physical map position.

TABLE 2
QTL9-2 Markers
			B73
			BAC(s)
	PHB	IBM2	on which	Position in
	Map	Map	marker is	most recent	Internal
Marker	Position	Position	located	B73 assembly	contig
PHM4598	94.5	317.0*	b0321m22	123,407,712-	9018
				123,456.512
PHM3507	94.6	317.0	b0321m22	123,407,712-	9018
				123.456,512
PHM18292	94.4	317.0	b0377g24	124,131,940-	9018
				124,131,839
PHM3004	94.7	317.0	c0062B12;	124,178,372-	9018
			b0377G24	124,254,793:
				124,131,940-
				124,131,839
PHM14122	97.0	320.6	c0286K12	126,975,669-	9018
				127,011,266
PHM10270	97.5	322.6	b0594A18	127,451,022-	9018
				127,545,826
PHM14092	97.8	322.6	c0286G14	128.070,700-	9018
				128,070,599
*Map position is estimated based on relative physical map position.

[0316] Each interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL. Close proximity of multiple QTL in the same interval may obfuscate the correlation of a particular marker with a particular QTL, as one marker may demonstrate linkage to more than one QTL. Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identify the same QTL or two different QTL. Regardless, knowledge of how many QTL are in a particular interval is not necessary to make or practice the invention.

[0317] The intervals described below encompass markers that co-segregate with grain moisture. The clustering of markers that co-segregate with grain moisture within a localized region occurs in relatively small domains on the chromosomes, indicating the presence of one or more QTL in those chromosome regions. The interval was drawn to encompass markers that co-segregate with grain moisture. The intervals are defined by the markers on their termini, where the interval encompasses markers that map within the interval as well as the markers that define the termini. An interval described by the terminal markers that define the endpoints of the interval will include the terminal markers and any marker localizing within that chromosomal domain, whether those markers are currently known or unknown.

[0318] The QTL9-1 interval may encompass any of the markers identified herein as being associated with the grain moisture trait including: PHM18206, PHM9929, PHM6726, PHM4910, PHM6667, PHM229, PHM6567, PHM5208, PHM15758, PHM14370, PHM3159, PHM17246, and PHM4691. The QTL9-1 interval, for example, may be defined by markers PHM18206 and PHM4910, which are separated by the greatest distance on the physical map, wherein the interval encompasses that markers that map within the interval as well as markers PHM18206 and PHM4910. Any marker located within these intervals can find use as a marker for grain moisture and can be used in the context of the methods presented herein to identify and/or select maize plants that have reduced grain moisture.

[0319] The QTL9-2 interval may encompass any of the markers identified herein as being associated with the grain moisture trait including: PHM18292, PHM4598, PHM3507, PHM3004, PHM14122, PHM10270, and PHM14092. The QTL9-2 interval, for example, may be defined by markers PHM4598 and PHM14092, which are separated by the greatest distance on the physical map, wherein the interval encompasses that markers that map within the interval as well as markers PHM4598 and PHM14092. Any marker located within these intervals can find use as a marker for grain moisture and can be used in the context of the methods presented herein to identify and/or select maize plants that have reduced grain moisture.

[0320] Chromosomal intervals can also be defined by markers that are linked to (show linkage disequilibrium with) a QTL marker, and r² is a common measure of linkage disequilibrium (LD) in the context of association studies. If the r² value of LD between a chromosome 9 marker locus lying within either of the QTL9-1 or QTL9-2 intervals, for example, and another chromosome 9 marker locus in close proximity is greater than ⅓ (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)), the loci are in linkage disequilibrium with one another.

[0321] A common measure of linkage is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.

[0322] Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.

[0323] The closer a marker is to a gene controlling a trait of interest, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci (e.g., a marker locus and a target locus) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.

[0324] Although particular marker alleles can co-segregate with grain moisture, it is important to note that the marker locus is not necessarily responsible for the expression of the grain moisture phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that is responsible for the grain moisture phenotype (for example, is part of the gene open reading frame). The association between a specific marker allele and reduced grain moisture is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral maize line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the parent having reduced grain moisture that is used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.

[0325] Methods presented herein include detecting the presence of one or more marker alleles associated with reduced grain moisture in a maize plant and then identifying and/or selecting maize plants that have favorable alleles at those marker loci. Markers listed in Tables 1 and 2 have been identified herein as being associated with grain moisture and hence can be used to predict grain moisture in a maize plant. Any marker within 50 cM, 40 cM, 30 cM, 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM of any of the markers in Tables 1 and 2 could also be used to predict grain moisture in a maize plant.

[0326] Molecular markers can be used in a variety of plant breeding applications (e.g. see Staub et al. (1996) Hortscience 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS). A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a ‘perfect marker’.

[0327] When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. This “linkage drag” may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite maize line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0328] The availability of integrated linkage maps of the maize genome containing increasing densities of public maize markers has facilitated maize genetic mapping and MAS. See, e.g. the IBM2 Neighbors maps, which are available online on the MaizeGDB website.

[0329] The key components to the implementation of MAS are: (i) Defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

[0330] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6) Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press. pp 75-135).

[0331] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment. An SSR service for maize is available to the public on a contractual basis by DNA Landmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

[0332] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

[0333] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ‘ultra-high-throughput’ fashion, as they do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode™ (Qiagen), Invader®. (Third Wave Technologies) and Invader Plus®, SnapShot®. (Applied Biosystems), Taqman®. (Applied Biosystems) and Beadarrays®. (Illumina).

[0334] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. For example, a single SNP may be allele ‘T’ for a specific line or variety with reduced grain moisture, but the allele ‘T’ might also occur in the maize breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. See, for example, WO2003054229. Using automated high throughput marker detection platforms known to those of ordinary skill in the art makes this process highly efficient and effective.

[0335] Many of the PHM markers presented herein can readily be used as FLP markers to select for the gene loci on chromosome 9, owing to the presence of insertions/deletion polymorphisms. Primers for the PHM markers can also be used to convert these markers to SNP or other structurally similar or functionally equivalent markers (SSRs, CAPs, indels, etc.), in the same regions. One very productive approach for SNP conversion is described by Rafalski (2002a) Current opinion in plant biology 5 (2): 94-100 and also Rafalski (2002b) Plant Science 162: 329-333. Using PCR, the primers are used to amplify DNA segments from individuals (preferably inbred) that represent the diversity in the population of interest. The PCR products are sequenced directly in one or both directions. The resulting sequences are aligned and polymorphisms are identified. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). Specifically with respect to the fine map information described herein, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers listed in this disclosure. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

[0336] In addition to SSR's, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid based markers.

[0337] Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

[0338] Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the maize species, or even across other species that have been genetically or physically aligned with maize, such as rice, wheat, barley or sorghum.

[0339] In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a phenotype, such as grain moisture. Such markers are presumed to map near a gene or genes that give the plant its grain moisture phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with reduced grain moisture can be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region (i.e. a genotype associated with reduced grain moisture) is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as having reduced grain moisture.

[0340] PHM markers were identified from both linkage mapping and association analysis as being associated with grain moisture in Stiff Stalk Synthetic populations. Reference sequences for each of the PHM markers are represented by SEQ ID NOs:1-20 and the primers for the PHM marker loci are represented by SEQ ID NOs:21-100. For PHM marker analysis, nested PCR reactions can be performed, using the external and internal primers for each PHM marker. In the first PCR reaction, 0.90 μl of 10×PCR buffer, 0.18 μl of 10 mM dNTP mix, 0.27 μl of 50 mM MgCl₂, 1.50 μl of 2.5 μM external forward primer, 1.50 μl of 2.5 μM external reverse primer, 0.04 μl of Platinum Taq, 1.61 μl of ddH2O, and 3 μl of 1.5 ng/μl DNA are used. The thermocycling conditions constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, repeated for 24 cycles; 72° C. for 10 minutes; and a hold at 4° C. The DNA produced from the first round of PCR is then diluted 1:9 with TE for use in the second round of PCR. The reaction mix for the second round of PCR is the same except the internal sets of primers are used, and the DNA is the diluted DNA from the first round of PCR. The thermocycling conditions for the second round of PCR constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, repeated for 28 cycles; 72° C. for 10 minutes; and a hold at 4° C. The PCR products are then sequenced directly.

[0341] PCR products can be sequenced directly in one or both directions, and the resulting sequences can be aligned and polymorphisms can be identified. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats).

[0342] SNPs identified within the PHM marker sequences that are associated with grain moisture are presented herein including: PHM18206-5, PHM9929-8, PHM6726-10, PHM4910-5, PHM6667-11, PHM229-15, PHM6567-10, PHM5208-11, PHM15758-6, PHM14370-9, PHM3159-14, PHM17246-16, PHM4691-9, PHM10270-13, PHM4598-22, PHM14092-77, PHM18292-9, PHM3507-13, PHM3004-13, and PHM14122-22. The marker names begin with a PHM prefix to denote ‘Pioneer Hi-Bred Marker’, followed by a number that is specific to the sequence from which it was designed, followed by a “.” or a “-” and then a numeric suffix that is specific to the DNA polymorphism.

[0343] Tables 8 and 9 list SNP markers and the corresponding favorable alleles that could be used in marker assisted selection to identify and select the favorable QTL alleles (the alleles associated with reduced grain moisture) at QTL9-1 and QTL9-2, respectively, in a maize plant. The maize plant may be an inbred in the Stiff Stalk Synthetic population or may be derived from an inbred in the Stiff Stalk Synthetic population.

[0344] The SNP markers could be used alone or in combination (i.e. a SNP haplotype) to select for favorable QTL alleles associated with reduced grain moisture. For example, a SNP haplotype at QTL9-1 can comprise: a “T” at PHM9929-8, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, and a “T” at PHM17246-16, or any combination thereof; and a SNP haplotype at QTL9-2 can comprise: a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, and a “G” at PHM14122-22, or any combination thereof.

[0345] The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around the chromosome 9 markers identified herein, wherein one or more polymorphic sites is in linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a QTL allele of interest. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2 cM, or 1 cM of the grain moisture QTL.

[0346] The skilled artisan would understand that allelic frequency (and hence, haplotype frequency) can differ from one germplasm pool to another. Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.

[0347] Maize plants identified and/or selected by any of the methods described above are also of interest. This includes any plant from the species Zea mays that has within its genome a haplotype at QTL9-1 consisting of: a “T” at PHM9929-8, a “T” at PHM6667-11, a “C” at PHM229-15, a “T” at PHM6567-10, a “T” at PHM5208-11, a “C” at PHM15758-6, a “G” at PHM14370-9, a “C” at PHM3159-14, and that exhibits reduced grain moisture when compared to a maize plant that does not have the haplotype in its genome. This also includes any maize plant that has within its genome a haplotype at QTL9-2 consisting of: a “C” at PHM10270-13, a “C” at PHM4598-22, a “C” at PHM14092-77, an “A” at PHM18292-9, a “G” or an “A” at PHM3507-13, a “G” at PHM3004-13, and a “G” at PHM14122-22; and that exhibits reduced grain moisture and no decrease in yield when compared to a maize plant that does not have the haplotype in its genome. The maize plant may be an inbred in the Stiff Stalk Synthetic population or may be a progeny plant derived from a Stiff Stalk Synthetic population.

[0348] Preferred haplotypes and QTL identified by the present disclosure may be advanced as candidate genes for inclusion in expression constructs, i.e., transgenes. Nucleic acids underlying haplotypes or QTL of interest may be expressed in plant cells by operably linking them to a promoter functional in plants. Nucleic acids underlying haplotypes or QTL of interest may have their expression modified by double-stranded RNA-mediated gene suppression, also known as RNA interference (“RNAi”), which includes suppression mediated by small interfering RNAs (“siRNA”), trans-acting small interfering RNAs (“ta-siRNA”), or microRNAs (“miRNA”).

[0349] Methods are known in the art for assembling and introducing constructs into a cell in such a manner that the nucleic acid molecule for a trait is transcribed into a functional mRNA molecule that is translated and expressed as a protein product.

[0350] To protect and to enhance yield production and trait technologies, seed treatment options can provide additional crop plan flexibility and cost effective control against insects, weeds and diseases, thereby further enhancing the invention described herein. Seed material can be treated, typically surface treated, with a composition comprising combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematicides, avicides and/or molluscicides. These compounds are typically formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. The coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Examples of the various types of compounds that may be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C. D. S. Tomlin Ed., Published by the British Crop Production Council, which is hereby incorporated by reference.

[0351] Some seed treatments that may be used on crop seed include, but are not limited to, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, bacillus spp. (including one or more of cereus, firmus, megaterium, pumilis, sphaericus, subtilis and/or thuringiensis), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/or yuanmingense), captan, carboxin, chitosan, clothianidin, copper, cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil, fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, PCNB, penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin, prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and/or zinc. PCNB seed coat refers to EPA registration number 00293500419, containing quintozen and terrazole. TCMTB refers to 2-(thiocyanomethylthio)benzothiazole.

[0352] Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head smut susceptibility may benefit from the use of a seed treatment that provides protection against head smut, a variety with good yield potential but cyst nematode susceptibility may benefit from the use of a seed treatment that provides protection against cyst nematode, and so on. Likewise, a variety encompassing a transgenic trait conferring insect resistance may benefit from the second mode of action conferred by the seed treatment, a variety encompassing a transgenic trait conferring herbicide resistance may benefit from a seed treatment with a safener that enhances the plants resistance to that herbicide, etc. Further, the good root establishment and early emergence that results from the proper use of a seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment.

[0353] The following examples are offered to illustrate, but not to limit, the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.

[0354] Breeding populations are generated by crossing high value inbreds from within heterotic groupings for the purposes of generating the next generation of newly improved elite inbreds. The populations are tested across multiple locations for agronomic traits of interest to the breeder. When the same populations are marker profiled, QTLs can be mapped using such methods as regression or maximum likelihood.

[0355] Over several years (2003-2008), phenotypic and marker data were collected on breeding populations leading to an observation of a strong genetic signal in the region 60 to 100 cM (distances are based on a single meiosis map) on chromosome 9. The signal correlated with moisture and other agronomic traits in Stiff Stalk Synthetic (SSS) maize populations, in particular, in mid-US maturities. The strong signal warranted further investigation via populations specifically developed for more detailed, or ‘fine’, mapping of the QTL in this region.

[0356] A biparental backcross-derived Stiff Stalk Synthetic (SSS) population was created from a cross between inbred PHCA5 and inbred PH705, and the resulting F₁ progeny were crossed again to PH705. Another round of backcrossing to PH705 was subsequently performed, creating BC₂ lines which were then selfed over several generations, with individual ear selections being made at each generation. At the BC₂F₃ stage, 1700 lines were genotyped with nine SNP markers that were designed to detect recombinations occurring within the 57.5 cM to 97.5 cM region on chromosome 9. These 9 SNP markers were PHM10028-12, PHM18206-5, PHM9929-8, PHM6726-10, PHM4910-5, PHM741-38, PHM6289-369, PHM13089-15 and PHM10270-13 and are described in Table 3. The markers were designed for use with Invader Plus® reagents which are proprietary to Third Wave Technologies (Madison, Wis., USA). BC₂F₃ lines containing recombinations within the targeted region were further selected for selfing. Some additional lines that were non-recombinants within the region were also selected as controls. The resulting BC₂F₄ lines were crossed with inbred PHCND. PHCND is classified as a member of the non-Stiff Stalk (NSS) heterotic group; thus, the stiff-stalk BC₂F₄ lines crossed with PHCND generated hybrids, or test-crosses, for which grain moisture could be assessed. During the crossing procedure, the SNP markers were used to select individual BC₂F₄ plants with homozygous fixed recombinants, and following crossing with PHCND, only ears from the homozygous recombinant plants were harvested resulting in 336 BC₂F₄ lines. This seed formed the basis of the field experiments in 2009.

[0357] The BC₂F₄ lines were selfed to generate 220 BC₂F₅ lines that were further crossed to the non-Stiff Stalk inbred PHVAM to generate hybrid test-crosses materials for evaluation in 2010 experiments.

[0358] In 2009, the 336 test-cross entries (generated from BC₂F₄ PHCA5×PH705 recombinants crossed with PHCND) were evaluated for grain moisture and yield. The entries were planted in four yield test locations consisting of two-row test plots per entry that were combine harvested. Two locations were in Princeton, Ill.; one in Marion, Iowa; and one in Windfall, Ind. As agronomic controls within each experiment, 20% of the entries were replicated at each location (experimental design is described in Williams et al. 2011. Biometrical Journal 1:19-27). Six replicates of parental test-cross entries were also added as controls at each of the 4 locations; the parental test-cross entries consisted of the parental inbreds PH705 and PHCA5 being crossed to PHCND to generate hybrid materials. Yield was measured in bushels acre⁻¹ and grain moisture was measured at harvest using a calibrated electronic moisture meter.

[0359] In 2010, the hybrid test-cross materials generated by crossing the PHCA5×PH705 BC₂F₅ population to PHVAM were evaluated for yield and grain moisture in four yield test locations: two in Windfall, Ind. and two in Princeton, Ill. A series of 144 overlapping BC₂F₅ test-cross entries were selected to be grown at each location and approximately 20% of the selected entries were replicated in each experiment as agronomic controls. Eleven replicates of parental test-cross entries were also added as controls which consisted of the parental inbreds PH705 and PHCA5 being crossed to PHVAM to generate hybrid materials. Yield was measured in bushels acre⁻¹ and grain moisture was measured at harvest using a calibrated electronic moisture meter.

[0360] A spatial mixed linear model (Gilmour (1997) Journal of Agricultural, Biological and Environmental Statistics 2:269-293; Cullis (2006) Journal of Agricultural, Biological and Environmental Statistics 11:381) was fit to the field data as a base model for a likelihood ratio test (LRT). The base model fit a fixed effect for locations, random effects for ear group, entries within ear group, interactions of location with these two random effects, random effects for row and column at each location, and a first order separable autoregressive process (AR1×AR1) at each location (Gogel (1997) Spatial Analysis of multi-environment variety trials; Cullis et al. (1998) Biometrics 54:1-18). Markers listed in Tables 3 and 4 were utilized for mapping. Each marker was added one at a time to the base model, and a LRT statistic was calculated and plotted against the cM position of the marker being tested (FIG. 1). QTL were considered significant at an LRT threshold of 13.81; equivalent to a LOD of 3.

TABLE 3
Markers Used for Selection of Recombinants and Genotyping
in 2009 and 2010 Experiments and for Mapping
				SNP
				Position in			PHB
	Reference	Primer	SNP	Reference	PHCA5	PH705	Map
Marker	Sequence	Sequences	identifier	Sequence	allele	allele	Position*
PHM10028	SEQ ID	SEQ ID	PHM10028-12	367	C	T	57.5
	NO: 101	NOs: 112-115
PHM18206	SEQ ID	SEQ ID	PHM18206-5	334	T	C	71.5
	NO: 1	NOs: 21-24
PHM9929	SEQ ID	SEQ ID	PHM9929-8	163	T	A	75.8
	NO: 5	NOs: 37-40
PHM6726	SEQ ID	SEQ ID	PHM6726-10	397	A	T	77.3
	NO: 12	NOs: 65-68
PHM4910	SEQ ID	SEQ ID	PHM4910-5	212	G	A	78.8
	NO: 13	NOs: 69-72
PHM741	SEQ ID	SEQ ID	PHM741-38	277	T	C	79.4
	NO: 102	NOs: 116-119
PHM6289	SEQ ID	SEQ ID	PHM6289-369	190	T	C	80.8
	NO: 103	NOs: 120-123
PHM13089	SEQ ID	SEQ ID	PHM13089-15	263	G	A	83.2
	NO: 104	NOs: 124-127
PHM10270	SEQ ID	SEQ ID	PHM10270-13	260	T	C	97.5
	NO: 19	NOs: 93-96
*The PHB map represents a single meiosis map.

TABLE 4
Additional Markers Used for Mapping
				SNP
				Position in			PHB
	Reference	Primer	SNP	Reference	PHCA5	PH705	Map
Marker	Sequence	Sequences	identifier	Sequence	allele	allele	Position*
PHM2305	SEQ ID	SEQ ID	PHM2305-17	452	T	C	67.3
	NO: 105	NOs: 128-131
PHM6667	SEQ ID	SEQ ID	PHNA6667-11	237	T	C	76.2
	NO: 9	NOs: 53-56
PHM9867	SEQ ID	SEQ ID	PHM9867-22	355	T	G	80.6
	NO: 106	NOs: 132-135
PHM7775	SEQ ID	SEQ ID	PHM7775-65	387	A	G	81.5
	NO: 107	NOs: 136-139
PHM16407	SEQ ID	SEQ ID	PHM16407-19	186	A	G	82.6
	NO: 108	NOs: 140-143
PHM14523	SEQ ID	SEQ ID	PHM14523-8	342	C	G	85.0
	NO: 109	NOs: 144-147
PHM2916	SEQ ID	SEQ ID	PHM2916-18	245	G	T	92.5
	NO: 110	NOs: 148-151
PHM4583	SEQ ID	SEQ ID	PHM4583-7	314	C	T	93.0
	NO: 111	NOs: 152-155
PHM4598	SEQ ID	SEQ ID	PHM4598-22	246	G	C	94.5
	NO: 14	NOs: 73-76
PHM14092	SEQ ID	SEQ ID	PHM14092-77	253	G	C	97.8
	NO: 20	NOs: 97-100
*The PHB map represents a single meiosis map

[0361] Prominent QTL (QTL on chromosome 9 location 1, or QTL 9-1) were identified for both grain moisture and yield between 71.7 and 79.0 cM on chromosome 9. The location range for QTL9-1 is based on 99% confidence intervals (CI) calculated for each year of data by a method which approximates the QTL distribution based on the LRT (Li, H. (2011) Journal of Genetics 90(2):355-360) (Table 5). Nearly identical QTL positions were identified in 2009 and in 2010, with the most likely position for both the grain moisture and yield QTLs being located at 75.8 cM in 2009, and at 77.3 and 76.2 cM for moisture and yield, respectively, in 2010 (Table 5). A second smaller QTL (QTL 9-2) for moisture was also apparent within the region 93.4-97.8 cM on chromosome 9, with the most likely position being located at 97.5 cM in agreement with both years of data (Table 5). A significant QTL was not found at this chromosomal location for yield.

TABLE 5
QTL positions and confidence intervals
		Major Peak (and 99% CI) in cM
Trait	Year	QTL 9-1	QTL 9-2
Moisture	2009	75.8 (71.7-76.7)	97.5 (93.4-97.8)
Yield	2009	75.8 (71.8-76.2)	—
Moisture	2010	77.3 (76.3-78.7)	97.5 (94.2-97.8)
Yield	2010	76.2 (74.6-79.0)	—

[0362] The spatial mixed linear base model with the addition of the two most likely QTL positions and an interaction between the positions was separately fit for the two years of data to assess the effect of these QTL positions for grain moisture and yield. Best linear unbiased predictions (BLUPs) were predicted from this model for all combinations of QTL 9-1 and QTL 9-2. At QTL 9-1, the PHCA5 alleles consistently resulted in an average decrease in kernel moisture across both years of testing when controlling for QTL 9-2 in the 93-98 cM region (Tables 6 and 7). There was also an associated decrease in yield within the same region (Tables 6 and 7). The effects at QTL 9-2, when controlling for QTL 9-1, showed that for this QTL the PH705 alleles decreased moisture levels but had no statistically significant effect on yield (Tables 6 and 7).

[0363] The greatest reduction in moisture can therefore be achieved through selection of the PHCA5 (designated as “B”) alleles at QTL 9-1 and PH705 (designated as “A”) alleles at QTL 9-2 (Tables 6 and 7). The QTL 9-1 region from 72-79 cM on a single meiosis map approximately corresponds with the region 200.4-230.6 cM on the IBM2 2008 map, and the QTL 9-2 region from 93-98 cM on a single meiosis map approximately corresponds with 317-323 cM on the IBM2 2008 map.

TABLE 6
Phenotypic BLUPs for moisture and yield based on allelic
combinations at the two QTL detected in 2009 and 2010.
AA = PH705 homozygous alleles and BB = PHCA5
homozygous alleles. QTL positions are as shown in Table 5.
			Moisture	Yield
	QTL	QTL	Predicted	Standard	Predicted	Standard
Year	9-1	9-2	Value	Error	Value	Error
2009	BB	BB	25.35	0.19	208.33	1.93
	BB	AA	24.97	0.19	207.81	1.94
	AA	BB	26.05	0.19	214.16	1.93
	AA	AA	25.80	0.19	213.48	1.96
2010	BB	BB	21.25	0.16	201.64	2.08
	BB	AA	21.08	0.16	201.64	2.08
	AA	BB	21.99	0.16	207.91	2.09
	AA	AA	21.82	0.16	207.91	2.09

TABLE 7
Average difference in phenotype BLUPs due to PH705 alleles at each
QTL, while controlling for alleles at the alternate QTL. QTL
positions are as shown in Table 5.
		QTL 9-1	QTL 9-2
		Average		Average
		difference		difference
		(PH705-	Standard	(PH705-	Standard
Region	Trait	PHCA5)	error	PHCA5)	error
2009	Moisture	0.77	0.05	−0.32	0.06
	Yield	5.75	0.52	−0.60*	0.43
2010	Moisture	0.74	0.04	−0.17	0.04
	Yield	6.27	0.58	0*	7.66E−04
*no significant effect

[0364] To identify favorable haplotypes associated with reduced grain moisture, PHCA5 and PH705 were compared to a group of 491 Stiff Stalk Synthetic inbreds, including Pioneer elite inbreds and key founder and public lines, based on SNP alleles in the regions of the moisture QTL. A set of 9 SNPs were identified that could be used for selection of the favorable QTL allele for moisture in PHCA5 between 73 and 78 cM and that showed consistent allelic inheritance across pedigrees (Table 8). A set of 7 SNPs were identified that could be used for selection of the favorable QTL allele for moisture at PH705 between 93 and 98 cM and that showed consistent allelic inheritance across pedigrees (Table 9).

TABLE 8
Markers that define a haplotype that differentiates PHCA5 from other inbreds in the region
between 73 and 78 cM (based on single meiosis map, e.g. the PHB map) on chromosome 9.
				SNP
				Position in	PHB	IBM2
	Reference	Primer	SNP	Reference	Map	Map	PHCA5
Marker	Sequence	Sequences	Identifier	Sequence	Position	Position	Allele
PHM229	SEQ ID	SEQ ID	PHM229-15	168	74.0	220.1	C
	NO: 4	NOs: 33-36
PHM6567	SEQ ID	SEQ ID	PHM6567-10	246	75.2	220.7	T
	NO: 2	NQs: 25-28
PHM9929	SEQ ID	SEQ ID	PHM9929-8	163	75.8	226.3	T
	NO: 5	NOs: 37-40
PHM5208	SEQ ID	SEQ ID	PHM5208-11	413	75.9	226.3	T
	NO: 8	NOs: 49-52
PHM15758	SEQ ID	SEQ ID	PHM15758-6	277	76.1	226.3	C
	NO: 7	NOs: 45-48
PHM6667	SEQ ID	SEQ ID	PHM6667-11	237	76.2	226.3	T
	NO: 9	NOs: 53-56
PHM14370	SEQ ID	SEQ ID	PHM14370-9	461	76.9	226.3	G
	NO: 11	NOs: 61-64
PHM3159	SEQ ID	SEQ ID	PHM3159-14	217	77.0	226.3	C
	NO: 6	NOs: 41-44
PHM17246	SEQ ID	SEQ ID	PHM17246-16	240	77.0	226.3	T
	NO: 10	NOs: 57-60

TABLE 9
Markers that define a haplotype that differentiates PH705 from other inbreds in the region
between 93 and 98 cM (based on a single meiosis map, e,g. the PHB map) on chromosome 9.
				SNP
				Position in	PHB	IBM2
	Reference	Primer	SNP	Reference	Map	Map
Marker	Sequence	Sequences	Identifier	Sequence	Position	Position	PH705
PHM18292	SEQ ID	SEQ ID	PHM18292-9	285	94.4	317	A
	NO: 16	NOs: 81-84
PHM4598	SEQ ID	SEQ ID	PHM4598-22	246	94.5	N/A	C
	NO: 14	NOs: 73-76
PHM3507	SEQ ID	SEQ ID	PHM3507-13	454	94.6	317	G
	NO: 15	NOs: 77-80
PHM3004	SEQ ID	SEQ ID	PHM3004-13	320	94.7	317	G
	NO: 17	NOs: 85-88
PHM14122	SEQ ID	SEQ ID	PHM14122-22	166	97.0	320.6	G
	NO: 18	NOs: 89-92
PHM10270	SEQ ID	SEQ ID	PHM10270-13	260	97.5	322.6	C
	NO: 19	NOs: 93-96
PHM14092	SEQ ID	SEQ ID	PHM14092-77	253	97.8	322.6	C
	NO: 20	NOs: 97-100

[0365] These SNP alleles, alone or in combination, can be used to select for reduced moisture in maize Stiff Stalk Synthetic populations. The reference sequences for the PHM markers shown in Tables 8 and 9 are represented by SEQ ID NOs:1-20, and the primers for the PHM markers shown in Tables 8 and 9 are represented by SEQ ID NOs:21-100.

[0366] An association mapping strategy was undertaken to identify markers associated with MST (grain moisture) in maize. The association analysis utilized SNP genotype data that was directly obtained from maize inbreds and breeding value scores for grain moisture that were obtained through the analysis of testcrosses of the inbreds with multiple testers.

[0367] A structure-based association analysis was conducted using standard association mapping methods, where the population structure is controlled using marker data. The model-based cluster analysis software, Structure, developed by Pritchard et al., (Genetics 155:945-959 (2000)) was used with haplotype data to estimate admixture coefficients and assign the inbreds to subpopulations. The structure analysis resulted in five subpopulations. Kuiper's statistic for testing whether two distributions are the same was then used to test a given marker for association between haplotype and phenotype in a given subpopulation (Press et al., Numerical Recipes in C, second edition, Cambridge University Press, NY (2002)).

[0368] Two peaks of significant marker-trait associations (FIG. 2) on chromosome 9, identified in a stiff stalk subpopulation, overlapped with the QTL 9-1 and QTL 9-2 intervals described in EXAMPLE 1. Markers in the region of chromosome 9 from 74 to 79 cM (based on a single meiosis map) having the most significant associations with grain moisture were PHM229-15, PHM6567-10, and PHM4691-9. For the second region (QTL 9-2), markers PHM3507-13 and PHM3004-13 had the most significant associations with the grain moisture phenotype.

[0369] Table 10 provides a listing of the top chromosome 9 markers significantly associated with the mechanical stalk strength phenotype based on association analysis. A reduction in moisture was associated with a “1” at PHM4691-9, a “T” at PHM6567-10, and a “C” at PHM229-15 in the QTL 9-1 region and an “A” at PHM3507-13 and a “G” at PHM3004-13 in the QTL 9-2 region. The SEQ ID NO: for each marker reference sequence is shown in Table 10. Primers for these marker sequences are represented by SEQ ID NOs:33-36 (PHM229), 25-28 (PHM6567), 29-32 (PHM4691), 77-80 (PHM3507), and 85-88 (PHM3004).

TABLE 10
Markers associated with grain moisture in a stiff stalk population
(identified using association analysis)
	Reference			Chr. 9
Marker	Sequence	SNP Identifier	P-value	Position
PHM229	SEQ ID	PHM229-15	6.579E−25	74.0
	NO: 4
PHM6567	SEQ ID	PHM6567-10	2.661E−27	75.2
	NO: 2
PHM4691	SEQ ID	PHM4691-9	1.19E−24	75.3
	NO: 3
PHM3507	SEQ ID	PHM3507-13	1.358E−22	94.6
	NO: 15
PHM3004	SEQ ID	PHM3004-13	1.623E−30	94.7
	NO: 17

[0370] The introgression of the reduced grain moisture QTL alleles can be achieved via repeated backcrossing to a recurrent parent accompanied by marker assisted selection to retain the alleles of the donor parent. This backcross procedure can be implemented at any stage in line development and can occur in conjunction with breeding for superior agronomic characteristics of one or more traits of interest, including transgenic and non-transgenic traits.

[0371] BC₂-F₃ (backcross) populations were created by backcrossing the QTL9-1 region from PHWRW (donor parent; has PHCA5 alleles) into PH134R, PHR1J, and PHCPR (RP=recurrent parents; do not have PHCA5 alleles). The markers used to genotype the region containing QTL9-1 were PHM14165-17, PHM15960-14, PHM5208-11 (see Table 8), PHM8438-6, PHM9867-22, and PHM14523-8 (See Table 11 for marker information). The BC2 lines were selfed twice and markers were used to select lines homozygous for the QTL9-1 PHWRW haplotype and lines homozygous for the PH134R, PHR1J, and PHCPR haplotypes. These lines were topcrossed to the tester PH1C1S1 to create hybrid seed for evaluation.

[0372] The effect of the QTL9-1 haplotype was evaluated by comparing grain moisture and yield of the topcrossed BC₂-F₃ lines with the QTL to the lines without the QTL in a hybrid yield experiment. The hybrid entries were planted in five locations, and BLUPS data from a spatial linear mixed model of moisture and yield was analyzed. The hybrid data for grain moisture and yield is shown in Table 12. The donor parent or recurrent parent genotype of the entries was determined using PHM5208-11. The p-value shown is the p-value of the difference between the haplotypes with respect to either yield (YLD) or grain moisture (MST).

TABLE 11
Markers used to Genotype the Region Containing QTL9-1
				SNP	Chr. 9
				Position	Position
				in	(single
	Reference	Primer	SNP	Reference	meiosis
Marker	Sequence	Sequences	Identifier	Sequence	map)
PHM14165	SEQ ID	SEQ ID	PHM14165-	432	72.5
	NO: 156	NOs:	17
		161-164
PHM15960	SEQ ID	SEQ ID	PHM15960-	246	73.02
	NO: 157	NOs:	14
		165-168
PHM8438	SEQ ID	SEQ ID	PHM8438-6	427	78.56
	NO: 158	NOs:
		169-172
PHM9867	SEQ ID	SEQ ID	PHM9867-22	355	80.29
	NO: 159	NOs:
		173-176
PHM14523	SEQ ID	SEQ ID	PHM14523-8	342	85
	NO: 160	NOs:
		177-180

TABLE 12
Yield and Moisture Phenotypic analysis of Topcross experiments.
						T-Test of
			SE		Differ-	Difference
Variable	N	Mean	Mean	StDev	ence	(P-vaue)
YLD-PH134R-RP	23	187.95	0.73	3.50
YLD-PH134R-DP	30	186.81	0.50	2.73	1.14	0.205
YLD-PHR1J-RP	30	188.89	0.65	3.55
YLD-PHR1J-DP	20	188.45	0.64	2.87	0.44	0.633
YLD-PHCPR-RP	26	173.82	0.75	3.84
YLD-PHCPR-DP	50	172.21	0.46	3.24	1.61	0.074
MST-PH134R-RP	23	25.14	0.20	0.94
MST-PH134R-DP	30	23.88	0.15	0.83	1.26	0.000
MST-PHR1J-RP	30	26.19	0.16	0.89
MST-PHR1J-DP	20	25.42	0.17	0.74	0.77	0.000
MST-PHCPR-RP	26	21.43	0.13	0.66
MST-PHCPR-DP	50	20.61	0.09	0.63	0.82	0.000

[0373] Lines having the PHWRW haplotype have grain moisture averages of 0.77-1.26 less than the grain moisture averages of lines not having the PHWRW haplotype but also show decreases in yield. The differences between the haplotypes with respect to grain moisture were statistically significant; however, the differences between the haplotypes with respect to grain yield were not significant. Thus, by selecting for the favorable alleles at QTL9-1 using marker assisted selection, grain moisture was significantly less in the lines that did not have the favorable alleles.