【作者】 吴永升；

【导师】 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。氮肥是农作物生产上使用最多的大量营养元素肥料,氮肥利用效率低是世界农业研究领域的难题,提高玉米氮肥利用效率具有重要研究意义。培育耐低氮品种是提高玉米氮利用效率的有效途径,而发掘耐低氮基因资源是培育耐低氮品种的前提和基础。玉米Gln1-3和Gln1-4基因编码茎秆与穗部GS同工酶,与全株氮向籽粒蛋白质同化、氮利用效率及产量紧密相关。与连锁分析相比,关联分析方法可鉴定出与玉米表型性状密切相关的功能等位基因变异,因而成为研究玉米氮利用效率的有效途径。本文以我国玉米生产上常用的189份自交系为材料,通过两年一点田间试验鉴定和评估相关性状的耐低氮性,结合180份自交系群体结构分析和氮代谢2个关键基因(Gln1-3、Gln1-4)开展基于候选基因策略的关联性分析,旨在发掘与氮利用效率性状相关的功能等位变异及氮高效单体型。获得的主要研究结果如下:1.2007年和2008年冬季分别在海南三亚对189份玉米自交系进行田间耐低氮评价,调查分析大喇叭口期叶片叶绿素含量、开花期和开花后10天穗三叶叶绿素含量、株高、穗位高、结实株数百分率、ASI、穗长、穗粗、穗行数、穗粒数、秃尖度、单穗重、百粒重和小区产量等19个表型性状。通过典型相关分析和因子分析,发现大喇叭口期叶片叶绿素含量、开花期穗上叶叶绿素含量、株高、百粒重、穗粒数和小区产量6个性状可以作为玉米自交系耐低氮性评价指标。以大喇叭口期叶片叶绿素含量、开花期穗上叶叶绿素含量、株高、百粒重、穗粒数、小区产量耐低氮系数的加权平均值作为评价玉米自交系耐低氮能力的综合指数,对189份玉米自交系进行耐低氮性评价,将试验材料分为高度耐低氮、中度耐低氮、中度敏感和高度敏感4种类型。两点试验耐低氮级别典型一致的材料有59份,其中高度耐低氮自交系17份,即齐205、7595-2、早49、郑28、郑30、CA339、川273、川321、合344、获唐黄、沈137、品1P6Co、辽2345、吉818、x178、H201和E28;一致中度耐低氮的材料有19份,即1029、临系11、郑22、铁7922、502、CML292、昌7-2、77、H152、金黄96B、CN962、冀53、鲁原133、4379、辽白371、196、CA335、丹340和郑58;一致中度敏感的材料有12份,即龙抗11、31778、掖107、掖515、金黄55、PI143、中自01、200B、M0113、东91、374和吉81162;一致高度敏感的材料有11份,即CML206、齐209、65232宽、QB80、四F1、y75、7379-2、塔5、长3、吉8415和吉842。这些材料为玉米耐低氮育种奠定基础。2.采用PCR与接头步移(walking)方法分离得到Gln1-3、Gln1-4基因区域基因组DNA全长。Gln1-3基因全长4571 bp,起始密码子至终止密码子序列长3062 bp;Gln1-4基因全长3724 bp,起始密码子至终止密码子序列长2858 bp。对这两个基因的结构进行分析并确定保守功能域,为关联分析奠定了基础。3.在180份玉米自交系中,对Gln1-3、Gln1-4基因的5′非编码区、氨离子结合功能域与ATPase活性保守功能域等重要分析区间分析表明,(1)Gln1-3基因中,共检测到182个位点的等位变异,其中140个位点为SNP, 42个位点为Indel。等位基因频率大于5%的位点为80个,其中58个位点为SNP,平均每39 bp一个;22个位点为indel,平均每104 bp一个。(2)在Gln1-4基因中,共检测到186个位点的等位变异,其中148个位点为SNP,38个位点为Indel。等位基因频率大于5%的位点为58个,其中32个位点为SNP,平均每71 bp一个;26个位点为indel,平均大约每88 bp一个。连锁不平衡结构分析表明,这两个基因的5′端、氨离子结合域和ATP酶结合结构域都分布着较强的LD结构(r2>0.5)。4.通过关联分析发现,在低氮胁迫下,(1)Gln1-3基因中19个序列多态性位点与开花期穗上叶叶绿素含量、株高、穗粒数、百粒重和小区产量显著关联。其中,与小区产量关联显著的位点10个;与株高呈显著关联的位点8个。位点2069对小区产量的效应最大,其值为26.8%,10位点对小区产量的总贡献率达到32.7%。选取具有较大贡献的5个位点将180份自交系分为11种单体型,其中单体型7含有对小区产量贡献较大的位点-172的C位、889的C位、2069的C位和2072的0位,包含于郑30、CA339和CA156等多个高度耐低氮和中度耐低氮自交系,推断该单体型可能为Gln1-3基因的耐低氮单体型。(2)Gln1-4基因中14个序列多态性位点与大喇叭口期叶片叶绿素含量、开花期穗上叶叶绿素含量、株高、穗粒数和小区产量显著关联。其中,与穗粒重关联显著的位点有11个;与株高和大喇叭口期叶片叶绿素含量呈显著关联的位点分别有2个。位点-218对小区产量的效应最大,其值为22.1%,11位点对穗粒数的总贡献率为36.7%。选取具有较大贡献的5个位点将180份自交系分为10种单体型,其中单体型1中含有对穗粒数贡献较大的位点-574的A位、-218的A位和-199的1位;对应4个高度耐低氮和中度耐低氮自交系齐205、7595-2、吉1037、吉495,推断该单体型可能为Gln1-3基因的耐低氮单体型。(3)两个基因的33个位点分别与氮利用效率相关性状存在显著关联,其中一些单体型累积可解释表型变异达20%以上,具有功能分子标记开发与育种利用前景。更多还原

【Abstract】 Maize (Zea mays L.) is one of the most important crops as food, feed and industry use. Nitrogen is the important factor in the crop production. The low nitrogen use efficiency of cereals is difficult to handle in the agricultural research around the world. Therefore, it has become of major importance to improve nitrogen use efficiency of maize. Development of high nitrogen use efficiency varieties using maize diverse germplasm is an available way to resolve the yield loss caused by nitrogen stress. Discovering those inbred lines with high nitrogen use efficiency is the basis of maize breeding. Gln1-3 and Gln1-4 are members of GS (Glutamine synthetase)gene family that encoded the isozyme expressed in stalk and ear of maize. To generate new information for maize breeding programs, association mapping is an effective way to identify functional natural allelic variations related to high nitrogen use efficiency traits. Gln1-3 and Gln1-4 were chosen as the candidate genes in this study, which were certified to responsible for the kernel number and kernel weight of maize respectively. 189 maize inbred lines used in breeding programs of China were chosen for SNP (Single nucleotide polymorphism) genotyping and nitrogen use efficiency phenotyping. The objectives of this study were to identify functional alleles and haplotypes of high nitrogen use efficiency. The major results obtained were as follows:1. A total of 189 maize inbred lines were grown in the field , to evaluate for nitrogen use efficiency, on two levels of nitrogen fertilization (low nitrogen and normal nitrogen) in Hainan province over two consecutive years (2007 and 2008). The 19 phenotypic traits including chlorophyll content in pre-tassel stage, chlorophyll content of three leaves of the ear in pollination stage, chlorophyll content of three leaves of the ear in ten days after pollination stage, percentage of plant with seed, ASI (Anthesis-silking interval), plant height, ear height, kernel weight per ear, ear length, ear width, ear row number, kernel number, bared tip, ear weight, a hundred kernel weight and grain yield of the plot were investigated in these trials. The descriptive statistics from 19 traits investigated showed that 6 traits, chlorophyll content in pre-tassel stage, chlorophyll content of the top leave of the ear in pollination stage, plant height, a hundred kernel weight, kernel number and grain yield of the plot, were identified to evaluate inbred lines for nitrogen use efficiency. Based on factor analysis, integrated NTSI (Nitrogen tolerance synthesis index) of each inbred line was calculated to classify the maize inbreds tested into 4 different Nitrogen-tolerant types: highly nitrogen tolerance, medium nitrogen tolerance, medium nitrogen susceptible and highly nitrogen susceptible. 59 maize inbred lines had the same responses for nitrogen tolerance, including 17 highly nitrogen tolerant inbreds, 19 medium tolerant, 12 medium susceptible and 11 hghly susceptible.2. 4571 bp of genomic region of Gln1-3 was sequenced using PCR combined PCR walking strategy. The 3062 bp coding region of the gene was comprised of ten exons that were separated by nine introns. At the same time, 3824 bp of genomic region of Gln1-4 was sequenced using the same method. The full length of the coding region was 2858 bp, which was comprised of ten exons separated by nine introns. Gene structure of these two genes were analysed and two conserved domains were found through blast, this is the research base for association analysis.3. Software ClustalX was used to analyse the sequence polymorphism of these two candidate genes in 180 maize inbred lines. 1) A total of 140 SNPs and 42 Indels (Insertion and deletion) were identified in Gln1-3 gene. The number of sites’frequency more than 5% were 80, including 58 SNPs and 22 Indels, of which there was one SNP variation every 39 bp and one Indel every 104 bp, respectively. 2) A total of 148 SNPs and 38 Indels were identified in Gln1-3 gene. The number of sites’frequency more than 5% were 58, including 32 SNPs and 26 Indels, of which there was one SNP variation every 71 bp and one Indel every 88 bp, respectively. Relative higher LD (Linkage disequilibrium, r2>0.5) was identified in the region of 5’ UTR, ammonium ion binding domain and ATPase domain of these two genes, association analysis should be focused on these regions.4. The results of candidate gene association analysis between genotypes and phenotypes of nitrogen use efficiency were: 1) A total of 19 polymorphism sites of Gln1-3 gene were identified significantly to be associated with 5 traits (LOD>2.5), chlorophyll content of the top leaves of the ear in pollination stage, plant height, hundred kernel weight , kernel number and grain yield. Of them, 10 polymorphism sites were identified significantly to be associated with grain yield, 8 polymorphism sites were identified significantly to be associated with plant high. Nucleotide variation C/T on site 2069 was most significantly associated with grain yield with contribution of 26.8%.In total, the contribution of the 10 polymorphism sites associated with grain yield was 32.7%. Based on five sites significantly associated with grain yield, 11 haplotypes were identified among 180 maize inbred lines. Haplotype 7 contained three positive contribution sites associated significantly with grain yield was speculated to be candidate nitrogen tolerance haplotype, which was included in nitrogen tolerant inbreds, such as Zheng30, CA339, CA156, etc. 2) A total of 14 polymorphism sites of Gln1-4 gene were identified significantly to be associated with 5 traits (LOD>2.5), chlorophyll content in pre-tassel stage, chlorophyll content of the top leaves of the ear in pollination stage, plant height, kernel number and grain yield. Of them, 11 polymorphism sites were identified significantly to be associated with kernel number and chlorophyll content in pre-tassel stage. 2 SNP sites were identified significantly to be associated with plant high respectively. SNP variation T/A on site -218 was most significantly associated with kernel number with contribution of 22.1%. In total, the contribution of the 11 polymorphism sites associated with kernel number was 36.7%. Based on five sites significantly associated with kernel number and chlorophyll content in pre-tassel stage, 10 haplotypes were identified among 180 maize inbred lines. Haplotype 1 contained three positive contribution sites associated significantly with kernel number was speculated to be candidate nitrogen tolerance haplotype, which was included in nitrogen tolerant inbreds, such as Qi205, 7595-2, Ji1037, Ji495, etc. 3) A total of 33 polymorphism sites of Gln1-3 and Gln1-4 were identified significantly to be associated with related trait of nitrogen use efficiency, the total contribution of some of the polymorphism sites were more than 20%, these results will be used to develop functional molecular markers in molecular assisted breeding in the future.更多还原