【作者】 苏治军；

【导师】 李新海； 张世煌；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2009， 博士

【摘要】 玉米是重要的粮食、饲料和工业原料作物,在国民经济发展中占有重要地位。干旱是限制玉米生产的主要因素之一,对玉米造成的危害超过其他逆境胁迫。培育耐旱品种是提高玉米耐旱能力的有效途径,而发掘耐旱基因资源是培育耐旱品种的前提和基础。玉米种质资源中蕴藏有丰富的基因等位变异,鉴定出与玉米耐旱性相关且对玉米产量有较大贡献的优异等位变异有助于了解玉米耐旱的遗传机制,在此基础上开发耐旱相关分子标记,将极大地促进玉米遗传改良和种质创新。与连锁分析相比,应用关联分析方法可鉴定出与玉米表型性状密切相关的优异等位基因变异,因而成为研究玉米耐旱性的有效途径。本文以我国玉米生产上常用的196份自交系为材料,通过两年两个地点的田间试验鉴定和评估相关性状的耐旱性,结合196份自交系的群体结构分析和水分胁迫密切相关的三个基因(dbf1、nced、rab28)开展基于候选基因策略的关联分析,旨在发掘与耐旱性状相关的优异等位变异及耐旱单体型。获得的主要研究结果如下:1. 2007年冬季和2008年春季分别在海南三亚和新疆乌鲁木齐对196份玉米自交系进行田间耐旱鉴定,调查分析了株高、穗位高、雄穗长度、结实株数百分率、穗行数、行粒数、秃尖度、单穗粒重、百粒重、持绿度、叶片卷曲度和雌雄开花间隔天数(Anthesis-silking interval, ASI)等12个性状。通过典型相关分析,发现株高、ASI、单穗粒重和结实株数百分率四个性状可以作为玉米自交系耐旱性评价指标。采用因子分析方法,计算株高、ASI、单穗粒重和结实株数百分率四个性状的综合耐旱系数,对196份玉米自交系进行耐旱性评价,将试验材料分为耐旱、中度耐旱、中度干旱敏感和干旱敏感4种类型。两点试验耐旱级别完全一致的材料有58份,其中耐旱自交系7份,即H201,Mo113,英64,H21,早49,丹598,吉842;中度耐旱自交系有丹黄02,8902,中106,郑22,中黄68,K22等14份。这些材料为耐旱育种提供了基础。2.通过三个耐旱候选基因的序列多态性分析,发现(1)dbf1基因在175份玉米自交系中共检测到9个SNP变异(平均每60 bp一个),没有检测到Indel变异,在3个多态性位点间存在较高程度的连锁不平衡(r2>0.5);(2)nced基因在162份玉米自交系中共检测到20个SNP(平均每88 bp一个)和4个Indel(平均每441 bp一个),在8个位点间存在较高程度的连锁不平衡(r2>0.5);(3)rab28基因在131份玉米自交系中,共检测到52个SNP(平均每22 bp一个)和16个Indel(平均每72 bp一个),在8个位点间存在较高程度的连锁不平衡(r2>0.5)。3.通过关联分析,发现(1)在干旱胁迫下,dbf1基因中的多态性位点366与ASI和单穗粒重显著关联(P<0.05),位点452与单穗粒重和结实株数百分率存在显著关联(P<0.05)。位点366对ASI和单穗粒重的贡献率分别为6.94%和10.03%;位点452对单穗粒重和结实株数百分率的贡献率分别是1.01%和3.66%。根据2个位点将175份自交系分成4种单体型,其中单体型4含有能使ASI减小、结实株数百分率增加的等位基因位点,包含于早49、丹598、丹黄02等耐旱和中度耐旱自交系,推测单体型4可能是dbf1基因的耐旱单体型。(2)nced基因的6个多态性位点与ASI、单穗粒重、结实株数百分率和株高存在显著关联(P<0.05),其中位点739对单穗粒重的贡献率最大6.18%。选取与性状显著关联(P<0.05)且具有较大贡献的4个位点将162份自交系分为12种单体型,其中单体型3含有对单穗粒重较大贡献的位点739和位点386,包含于早49、丹黄02、中综4C1-3等耐旱和中度耐旱自交系,推断单体型3可能为nced基因的耐旱单体型。(3)在干旱胁迫下,rab28基因中多个序列多态性位点与株高、单穗粒重和结实株数百分率呈显著关联(P<0.05),其中与单穗粒重呈显著关联的位点为10个;与株高和结实株数百分率呈显著相关的位点均为6个。位点1113对单穗粒重的贡献率最大5.48%。选取具有较大贡献的4个位点将131份自交系分为10种单体型,其中单体型2含有对结实株数百分率较大贡献的位点1108和位点1092,包含于英64、M0113、K22、中黄68等耐旱和中度耐旱自交系,推断该单体型可能为rab28基因的耐旱单体型。4.对三个基因的耐旱单体型进行联合分析,发现玉米自交系早49和丹黄02含有dbf1和nced两个基因的耐旱候选单体型4和3。在上述自交系中可能蕴含丰富的耐旱优异等位基因变异,这些变异对干旱胁迫的多条调控途径可能有共同响应。本文研究结果为进一步开发耐旱功能标记及分子育种提供了基础。更多还原

【Abstract】 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.更多还原