【作者】 丁泽红；

【导师】 余四斌；

【作者基本信息】 华中农业大学 ， 作物生物技术， 2011， 博士

【摘要】 水稻作为重要的粮食作物,是全世界一半以上人口主食的重要来源。氮是植物生长所必需的大量营养元素之一,是农业生产中限制作物产量的重要因素。氮肥的过量施用不仅造成了严重的环境污染,而且还提高了水稻的生产成本。水稻拥有遗传资源丰富多样的自然群体,建立在自然群体之上的关联分析方法,已成为目前水稻重要基因发掘与利用的热点和焦点。关联分析不仅是发掘新基因的一种有效手段,还是联系结构基因组学和表型组学的一座桥梁,可以直接鉴定出与表型相关的目标基因或等位变异。随着分子生物学和测序技术的不断发展,许多基因被克隆并得到功能验证。本研究主要是利用测序技术,分析水稻氮代谢基因在水稻微核心种质和普通野生稻中的核苷酸多样性,分析群体中各基因及基因之间的连锁不平衡(LD)程度,并利用关联分析研究氮代谢基因与正常和低氮条件下的产量性状之间的关系,旨在发掘氮代谢基因的重要等位变异,为选育水稻氮高效品种提供理论依据。此外,本研究还为水稻氮代谢基因的起源和进化研究奠定了一定的试验数据。主要结果如下：1.利用PCR扩增以及测序技术,获得7个氮代谢基因(包括OsAMT1;1、OsNRT1、OsNR1、OsGS1;1、OsNADH-GOGAT1、OsAS1和OsGDH1)在216份水稻品种(包括籼稻、粳稻和普通野生稻等)中的核苷酸序列信息。经比对拼接之后,各基因的测序序列长度范围为1608 bp (OsNADH-GOGAT1)-3506bp (OsGDH1),总的测序分析长度为16,899 bp。在7个氮代谢基因中总共检测到72个Inde1,它们主要分布在内含子和5’或3’非编码区序列；检测到330个SNP,在籼稻中有97个,粳稻中有71个,栽培稻中有103个,普通野生稻(O. rufipogon)中有240个。2.对7个氮代谢基因的核苷酸多样性进行了比较分析。整体上,普通野生稻的核苷酸多样性要显著地高于籼稻和粳稻。籼稻和粳稻分别保留了普通野生稻大约33.5%和23.4%的核苷酸多样性。3.将样本分为普通野生稻、地方品种和优良品种,进行核苷酸多样性分析后发现,氮代谢基因将近一半(45.7%)的遗传多样性都在水稻驯化(从普通野生稻到地方品种)的过程中丢失了,而在水稻品种改良(从地方品种到优良品种)的过程中基本没有什么变化。4.在190份栽培稻中,对6个氮代谢基因(除了OsAMT1;1)的LD分析结果表明：OsGDH1的LD水平最高,其r2均值为0.96;OsAS1(r2=0.62);OsNRT1(r2= 0.45)和OsNADH-GOGAT1(r2=0.44)表现出中等的LD水平；OsNR1(r2=0.35)和OsGS1;1(r2=0.28)则表现出相对较低的LD水平。而且,在5个氮代谢基因(OsNR1、OsNRT1、OsGS1;1、OsNADH-GOGAT1和OsGDH1)之间也检测到较高的LD水平。5.在190份栽培稻中,7个氮代谢基因共检测到24个主要单倍型(样本数>5),单倍型数目的变化范围为1到5个,平均每个基因有3.4个单倍型。每种单倍型(除了H1)都有明显的偏籼或偏粳的属性。单倍型分析结果表明：尽管这些氮代谢基因分别位于不同的染色体上,但在每一个亚路径中,由于较强的LD水平,籼型的单倍型总是与籼型的在一起,粳型的单倍型总是与粳型的在一起。这可能与籼、粳亚种有不同的地理起源相关。6.利用一般线性模型(考虑群体结构)对各氮代谢基因等位变异或单倍型与性状之间进行关联分析。整体上,OsNRT1与单株产量和结实率相关,OsNR1与千粒重相关,OsGS1:1与结实率和单株产量相关,OsNADH-GOGAT1与单株有效穗数和千粒重相关,OsAS1与单株有效穗数、每穗实粒数和单株产量相关,OsGDH1与千粒重相关。针对各氮代谢基因,分别找到了其影响表型的最优等位基因和单倍型。利用近等基因系对其中部分等位基因的遗传效应进行了验证。这些信息可应用于水稻育种实践中。7.与普通野生稻相比,OsAMT1;1在栽培稻(包括籼稻和粳稻)中的核苷酸多样性极低(仅为普通野生稻的2.3%),且Tajima’s D和Fu和Li’s D*都达到了负的显著或极显著水平,暗示OsAMT1;1受到了正选择作用。绝大部分(182/190)栽培稻含有相同的OsAMT1;1等位基因；与两个其它氮代谢基因(Loc_Os01g48960和Loc_Os02g50240)相比,OsAMT1;1在地方品种和优良品种中的核苷酸多样性显著下降。8.在94份栽培稻和19份普通野生稻中,对OsAMT1;1附近7个DNA片段进行了测序。结果表明,与普通野生稻相比,栽培稻的核苷酸多样性在OsAMT1;1前后大约100 kb的区域内急剧减少；EHH(Extended Haplotype Homozygosity)分析表明,在这100 kb的区域内,LD在栽培稻中维持着较高的水平而在普通野生稻中衰减得非常迅速。因此,认为OsAMT1;1受到了强的选择作用。更多还原

【Abstract】 Rice, one of the world’s most important crops, is the staple food of over half of the world’s population. Nitrogen, one of the macronutrients necessary for plant growth, is an important factor limiting the crops production. Excessive application of nitrogen fertilizer not only causes serious environmental pollution, but also increases the rice production cost. Rice has rich and diverse genetic resources in the natural population. Association mapping, which based on linkage disequilibrium (LD) and used natural population as the basic research material, has become a more and more popular method to discover and utilize important genes in rice. Association mapping is not only an effective approach to discover new genes, but also a bridge to link the structural genomics and phenomics. It could directly identify the alleles that associated with the target traits. With the rapid development of molecular biology and DNA sequencing technology, lots of genes have been cloned and functional characterized. The objectives of this study are (1) to investigate the nucleotide diversity of nitrogen metabolism genes in a rice mini-core collection and a panel of O. rufipogon accessions using sequencing technology, (2) to clarify the LD levels within and between different genes, and (3) to demonstrate the relationship of polymorphism sites and yield-related traits under normal and low nitrogen conditions. The alleles explored in this study would provide useful guides in rice breeding of nitrogen efficiency. In addition, this study is also aim to reveal origin and evolution of OsAMT1;1 in rice. The main results are as follows:1. We obtained the sequences of seven nitrogen metabolism genes (OsAMT1;1, OsNRT1, OsNR1, OsGS1;1, OsNADH-GOGAT1, OsAS1 and OsGDHl) in a panel of 216 rice accessions (including indica, japonica and its wild relatives) using the sequencing technology of PCR products. The alignment of the genes ranged from 1608 bp (OsNADH-GOGAT1) to 3506 bp(OsGDH1), with a total length of 16,899 bp. A total of 72 insertion/deletions (Indels) were identified exclusively in introns and 5’ or 3’ noncoding regions. Totally 330 single nucleotide polymorphisms (SNPs) were found across the 216 rice accessions, with the SNP number being 97 in indica,71 in japonica, 103 in O. sativa and 240 in O. rufipogon.2. The nucleotide diversity of the seven nitrogen metabolism genes was compared at the taxon level, including indica, japonica, O. sativa and O. rufipogon. Overall, the nucleotide diversity level of O. rufipogon was significantly higher than that in indica and japonica, and indica and japonica approximately maintained 33.5% and 23.4% as much diversity as O. rufipogon, respectively.3. The nucleotide diversity was also inverstigated in O. rufipogon, landraces and elite cultivars, and the results showed that about half (45.7%) of the nucleotide diversity of the nitrogen metabolism genes was lost during the transition from O. rufipogon to landraces while it was nearly unchanged during the transition from landraces to elite cultivars.4. LD plots were evaluated for six nitrogen metabolism genes (without OsAMT1;1) in 190 O. sativa, and different levels of LD were revealed. OsGDHl exhibited the highest LD level and the average value of r2 was 0.96. OsAS1 (average r2=0.62), OsNRTl (average r2=0.45) and OsNADH-GOGAT1 (average r2=0.44) showed medium LD levels, while OsNRl (average r2=0.35) and OsGSl;1 (average r2=0.28) showed the lowest LD levels. In addition, high LD levels were also observed among five genes of OsNR1, OsNRTl, OsGS1;1, OsNADH-GOGATl and OsGDH1.5. Totally twenty-four main haplotypes (n> 5) were observed for seven nitrogen metabolism genes in 190 O. sativa, the number of haplotypes ranged from one to five with an average of 3.4 per gene. In addition, the haplotypes (except haplotype H1) were indica-like or japonica-like. The results of haplotype analysis showed that in each sub-pathway, indica-like and japonica-like haplotypes were always linked with themselves-like haplotypes for strong LD levels, although these nitrogen metabolism genes were located on different chromosomes. This may be because indica and japonica have the different geographic origins.6. Based on general linear model considering population structure, association mapping was conducted between polymorphisms (or haplotypes) and the yield-related traits in seven environments. Overall, OsNRT1 associated with YPP and SS, OsNRl associated with TGW, OsGS1;1 associated with SS and YPP, OsNADH-GOGATl associated with PNPP and TGW, OsASl associated with PNPP, GNPP and YPP, and OsGDH1 associated with TGW. In addition, the favorable allele (or haplotype) of each nitrogen metabolism gene was identified, and the effects of some alleles (or haplotypes) were validated by using near isogenetic lines. The information explored in this study could be applied in the rice breeding programs.7. The nucleotide polymorphism (π) of OsAMT1; 1 in O. sativa (including indica and japonica) was as low as 2.3% of that in O. rufipogon, and the values of Tajima’s D and Fu and Li’s D* were both negative significant in O. sativa, suggesting OsAMT1;1 was under positive selection. Most (182/190) accessions of O. sativa have the same OsAMT1;1 allele. Compared with two other genes (Loc_Os01g48960 and Loc_Os02g50240) that were used as the control, the nucleotide diversity of OsAMT1;1 was severely reduced in landraces and elite cultivars.8. The nucleotide diversity of additional seven DNA fragments surrounding OsAMT1;1 was inverstigated in 94 O. sativa and 19 O. rufipogon, and the results showed that the nucleotide diversity of O. sativa was severely reduced in the OsAMT1;1 genomic region spanning approximately 100 kb long compared with O. rufipogon. EHH (Extended Haplotype Homozygosity) analysis showed that LD remained high levels across the 100 kb genomic region around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon. All the results suggest that OsAMT1;1 was under strong selection.更多还原