| Maize is one of the important crops as food, feed and industry use. Yield loss resulted from drought stress annually exceeds the sum of other abiotic stress. Development of drought-tolerant varieties using maize diverse germplasm is an available way to resolve the yield loss caused by water stress. Maize genome is full of abundant allelic variations, and discovering these alleles from genes related to drought tolerance is the basis of maize breeding, as well as understanding the complex genetic and molecular mechanism underlying drought tolerance. Studies on maize drought tolerance through linkage mapping have resulted in major progress. To generate new information for maize breeding programs, association analysis is an effective method to identify functional allelic variations related to drought-tolerant traits. The objectives of this study are to use the natural variation inherent in maize inbred line population and drought tolerant candidate genes for identification of functional alleles and haplotypes.Candidate genes related to drought tolerance dbf1, nced and rab28 were chosen from the publications, which were validated to have effects on the drought-tolerant traits. One hundred and ninety six maize inbred lines used in breeding programs of China were chosen for SNP (Single nucleotide polymorphism) genotyping and drought-tolerant phenotyping. Furthmore, based on the population structure analysis, association analysis were conducted to identify the functional SNP variations and functional haplotypes related to drought tolerance within each gene by logistic model.The major results obtained were as follows:1. A total of 196 maize inbred lines were evaluated for drought tolerance with alpha-lattice field design in 2007, Hainan province and 2008, Xinjiang province, respectively. The 12 phenotypic traits including plant stay green, tassel length, plant height, ear height, leaf rolling, kenel weight per ear, hundred kernels weight, ear row numbers, bared tip, percentage of plant with seeds and kernel number per row were investigated in these trials. The descriptive statistics from 12 traits investigated showed that four traits of plant height, ASI (Anthesis-silking interval), kernel weight per ear and percentage of plant with seed, were identified to evaluate inbred lines for drought tolerance. Based on factor analysis, integrated DTI (Drought Tolerance Index) of each inbred line was calculated to classify the maize inbreds tested into 4 different drought-tolerant types: drought tolerance, medium drought tolerance, medium drought susceptible and drought susceptible. 58 maize inbred lines had the same responses for drought tolerance, including 7 drought tolerant inbreds, 14 medium tolerant, 24 medium susceptible and 13 susceptible.2. Sequence alignment using software ClustalX showed the sequence polymorphism of three candidate genes. 1) Nine SNPs in dbf1 (DRE binding factor) gene were identified among 175 maize inbred lines, and no Indel (insertion and deletion) was found. Averagely, there was a variation every 60 bp. Higher LD (Linkage disequilibrium, r2>0.5) was identified between three polymorphic site pairs. 2) Twenty SNPs and four Indels in nced (9-cis-epoxycarotenoid dioxygenases) gene were identified among 162 maize inbred lines. Averagely, there was a SNP variation every 88 bp and an Indel every 441 bp, respectively. Relative higher LD (r2>0.5) was identifed between eight polymorphisic site pairs. 3) Fifty two SNPs and sixteen Indels in rab28 (the 28th response gene to ABA) gene were identified among 131 maize inbred lines. Averagely, there was a SNP variation every 22 bp and an Indel every 72 bp, respectively. Relative higher LD (r2>0.5) was identifed between eight polymorphisic site pairs.3. Candidate-gene association analysis between genotypes and phenotypes of drought tolerance were: 1) Two SNP sites in dbf1 gene were identified significantly to be associated with three traits (P<0.05), ASI, kernel weight per ear and percentage of plant with seeds. Of them, nucleotide variation T/C on site 366 was significantly associated with traits ASI and kernel weight per ear with contribution of 6.94% and 10.03%, respectively. Nucleotide variation A/G on site 452 was significantly associated with traits percentage of plant with seeds and kernel weight per ear with contribution of 1.01% and 3.66%, respectively. Based on these two sites, four haplotypes were identified among 175 maize inbred lines. Haplotype 4 decreasing ASI and increasing percentage of plant with seeds was thought to be candidate drought tolerant haplotype, which was included in some drought tolerant and medium tolerant inbred lines such as Zao49, Dan598, Danhuang02, etc. 2) Six SNP sites of nced gene were identified significantly to be associated with four traits (P<0.05), ASI, kernel weight per ear, percentage of plant with seeds and plant height. Of them, nucleotide variation T/G on site 739 was most significantly associated with kernel weight per ear with contribution of 6.18%. Based on four sites significantly associated with ASI, kernel weight per ear, percentage of plant with seeds, 12 haplotypes were identified among 162 maize inbred lines. Haplotype 3 increasing keneral weight per plant was thought to be candidate drought tolerance haplotype, which was included in drought tolerant inbreds, such as Zao49, Danhuang02, Zhongzong4C1-3, etc. 3) Under drought stress condition, some polymorphic sites were identified in gene rab28 significantly associated with plant height, kernel weight per ear and percentage of plant with seeds (P<0.05). Of them, 10 sites were found associated with kernel weight per ear, and 6 sites were found associated with plant height and percentage of plants with seeds, respectively. The site 1113 was signigicantly associated with kernel weight per ear with contribution of 5.48%. Furthermore, 4 critical polymorphic sites with large contribution to the phenotype were chosen to classify 131 inbred lines into 10 haplotypes. Haplotype 2 with two sites 1108 and 1092 both increasing percentage of plant with seeds was deduced to be drought tolerant haplotypes in rab28, and was included in drought tolerant and medium tolerant inbreds, such as Ying64, M0113, K22, Zhonghuang68, etc.4. Combined haplotype analysis of three candidate genes showed that maize inbred lines Zao49 and Danhuang02 both contained candidate drought tolerant haplotypes 4 and 3 in dbf1and nced genes, respectively. The results obtained in this paper could be used in development of functional markers and molecular breeding for dought tolerance breeding program in maize. |
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