贵州地方水稻种质资源籼-粳遗传分化的InDel分子标记研究

本研究利用36对InDel分子标记引物对贵州地方水稻种质的籼-粳遗传分化和亲缘关系进行分析,结果表明,82份贵州地方栽培稻中49份为粳稻,33份为籼稻,贵州地方栽培稻“禾”品种主要属于粳稻,而“谷”品种主要为籼稻。基于Nei氏遗传距离的亲缘关系分析表明在粳稻群体和籼稻群体中均存在与野生稻亲缘关系近的品种,其中的粳稻品种与野生稻的遗传关系比之籼稻品种近。而基于MCMC算法的遗传结构分析揭示了贵州地方籼稻品种中存在较为复杂的遗传结构。分子变异分析显示,粳稻和籼稻品种的遗传变异主要来自亚种内,遗传多样性分析表明其亚种内籼稻品种的遗传多样性略高于粳稻品种。研究结果揭示了贵州省黔东南地区栽培稻种质资源的籼-粳分化程度、遗传关系及其遗传多样性。     

基于线粒体基因片段核苷酸多态性的亚洲栽培稻起源进化研究

亚洲栽培稻的祖先是普通野生稻,已成为世界公认的观点,然而亚洲栽培稻的2个亚种:粳稻和籼稻是一次起源还是二次起源仍存在很大争议,其起源地是国内还是国外依然是国际学者间争论的焦点。本文通过对184份亚洲栽培稻和203份普通野生稻3段基因序列cox3、cox1、orf 224和2段基因间序列ssv-39/178、rps2-trnfM的多样性研究,验证了以下观点:1)粳稻起源于中国,籼稻起源于中国和国外;2)亚洲栽培稻的起源为二次起源,即普通野生稻存在偏籼和偏粳2种类型,亚洲栽培稻的2个亚种籼稻和粳稻在进化过程中分别由偏籼型的普通野生稻和偏粳型的普通野生稻进化而来。     

江永普通野生稻遗传多样性和籼粳基因频率监测分析

以栽培稻的8个籼-粳测验种为对照,采用39对SSR引物检测了江永野生稻居群在1982年、2008年、2017年的遗传多样性,采用38对In Del引物检测了江永野生稻居群在1982年、2008年、2017年的籼-粳基因频率。结果表明:在1982年取样保存在异位圃的40份样本的遗传多样性稍高于2008年、2017年原位保护区样本的遗传多样性;2008年取的样本数虽然比2017年多,但两次取的样本之间遗传多样性几乎没差异。不同年份取的样本之间的遗传分化系数Fst都很小,基因流Nm都较大,分化不明显。通过聚类分析和主坐标分析(PCo A),发现野生稻居群与4份栽培粳稻聚为一类,4份栽培籼稻单独聚成一类,显示江永野生稻与粳稻的血缘近于籼稻;籼-粳基因频率的分析表明,野生稻样本多属粳稻型,少数属偏粳稻型,原位保护区的偏粳稻类型单株数占取样单株总数的比例,2008年比1982年的增加了10.0%,2017年比2008年的增加了1.6%,显示江永野生稻原位保护区生境条件有利野生稻从粳稻型向偏粳稻型变异,随着野生稻产生环境适应性变异,籼型基因频率在提高。     

水稻耐寒基因CTB4a的核苷酸多样性及区域适应性

为探究CTB4a基因的自然变异是否与栽培稻苗期耐低温相关, 本研究以133份栽培稻(Oryza sativa L.)及35份普通野生稻(O. rufipogon Griff.)为实验材料, 对CTB4a编码区的核苷酸多样性及其单倍型与地理分布的关系进行分析。结果表明CTB4a基因编码区有14个核苷酸变异, 组成33个单倍型。Network分析发现, 这些单倍型的286 bp处的SNP变异(+2,035,097 bp, G > C; +96 aa, Ala→Pro)将其分成Group A和Group B两组。Group A (CTB4a^jap)共有56份样品, 其中有43份(76.79%)是种植于中高纬度地区的粳稻; Group B (CTB4a^ind)有77份样品, 有63份(83.12%)是种植于热带和亚热带国家的籼稻。Group A中样品苗期黄叶率的平均值比Group B样品低30%, 且两者耐寒性具有显著差异(P = 1.25e-09)。位于286 bp处的变异只在Group A中出现, 在Group B中均不存在, 说明随着水稻种植区域向北迁移的育种过程中, 栽培稻中出现了CTB4a^jap并被固定下来。该研究为理解水稻对低温适应的分子遗传机制和培育耐寒品种提供了理论基础。     

普遍野生稻和亚洲栽培稻遗传多样性的研究

用 ４４个 ＲＦＬＰ标记对来自中国、印度、泰国等亚洲 １０个国家的普通野生稻（简称普野,下同）和来自多个国家的７５个栽培稻品种,从多态位点的比率、等位基因数、基因型数、平均杂合度及平均基因多样性等多个方面,比较了不同国家和不同地区的普通野生稻、栽培稻籼粳亚种及栽培稻与普野之间遗传多样性的差异。结果表明：中国普野的遗传多样性最大;其次是印度普野;南亚普野的平均基因多样性大于东南亚普野,而多态位点的比率、等位基因数及基因型数等却低于东南亚普野;栽培稻的遗传多样性明显小于普通野生稻。在所检测的４４个位点中,栽培稻的多态位点数仅为野生稻的３／４,等位基因数约为野生稻的６０％,基因型种类约为野生稻的１／２。栽培稻中籼稻的遗传多样性高于粳稻。在平均每个位点的实际杂合度上,以中国普野杂合度最高,普通野生稻是栽培稻的２倍。说明从野生稻演化成栽培稻的过程中,经过自然选择和人工选择,杂合度降低,等位基因减少,基因多样性下降。     

基于大样本数据的中国水稻扇型植硅体形态特征研究

水稻扇型植硅体在水稻起源研究中起着重要的作用,但由于野生稻和栽培稻扇型植硅体的区分存在不确定性,限制了扇型植硅体在早期稻作研究中的应用。本研究通过对植硅体大样本数据的统计分析得出:扇型植硅体长宽大小趋势是,普通野生稻大于栽培稻,粳稻大于籼稻;普通野生稻B/A显著大于栽培稻,粳稻B/A又显著小于籼稻。水稻扇型植硅体的长(VL)与宽(HL)呈现正相关的规律,扇型植硅体主要分布在VL<41μm和HL<39μm与VL>41μm和HL>39μm的区间内;在遗址水稻鉴定中,如发掘的扇型植硅体中多数B/A小于等于1.20就有很大可能是粳稻;如B/A大于1.2,可再根据扇型植硅体的大小区分野生稻和籼稻,多数植硅体在VL<41μm和HL<39μm的区间内可能是籼稻,在VL>41μm和HL>39μm的区间内则可能是普通野生稻。     

野生稻基因组随机扩增多态性DNA(RAPD)分析

用18个随机引物对2份栽培稻、12份包含有六个基因组型的野生稻DNA进行了扩增,共获得147个多态性DNA片断,把这些多态性DNA片断作为遗传位点用UPGMA法计算出各材料间的遗传相似性系数,并作了聚类分析．主要结果如下：1普通野生稻同栽培稻的亲缘关系很近,其中江永普通野生稻更接近于粳稻．2．CCDD组的Oryzalatifolia和EE组的O．australiensis遗传多态性相似。3．B、C、D、E组的遗传多态性相似,组成一个复合体,此复合体与A组的遗传多态性也相似,而F组则相距较远．4．O．mcyeriana和Rhynchofyzasabulata尚未确定组型,RAPD测定结果表明,前者与其它组型的种亲缘关系较远,后者则与AC复合体的种较近．     

基于叶绿体基因多样性的中国水稻起源进化研究

针对目前亚洲栽培稻起源地和进化途径学说众多、分歧巨大的现状,本研究选择原产中国的98份亚洲栽培稻和125份普通野生稻为材料,对叶绿体中atpA序列、rps16内含子序列、trnP-rpl33间隔区、trnG-trnfM序列、trnT-trnL间隔区序列的五段高突变序列进行测序,利用生物信息学方法进行比对分析,绘制Network网络图,构建系统发育树。结果表明,普通野生稻的Indel和SNP数目均比亚洲栽培稻多,序列多样性丰富;基于单倍型的Network网络图和系统发育树可将所有参试材料归为3个类群,类群I主要为粳稻与普通野生稻,类群II主要为籼稻,类群III主要为普通野生稻,而类群II和类群III亲缘关系较近,提示粳、籼两个亚种可能由偏粳、偏籼的普通野生稻分别进化而来,支持二次起源学说;所有与亚洲栽培稻亲缘关系较近的普通野生稻均来源于华南地区,支持华南地区为我国亚洲栽培稻起源中心的论点。     

云南栽培稻种SSR 遗传多样性比较

采用64个SSR标记对96份云南水稻(Oryz a sativa)地方品种和选育品种的遗传多样性进行比较分析。结果发现64个标记都具有多态性, 共检测到741个等位基因, 每个多态性位点检测到的等位基因数为2-29个, 平均11.57个; Nei基因多样性指数(He)范围在0.345(RM321)-0.932(RM1)之间, 平均为0.56。水稻品种的遗传多样性并非按地理位置均匀分布, 而是在相 似系数为0.17的水平上明显分为2个不同类群, 即籼稻类群和粳稻类群, 且籼粳亚种间的SSR多样性差异不明显, 籼稻平均等位基因数(Ap)和Nei基因多样性指数(Ap=10.6, He=0.46)与粳稻品种(Ap=10.7, He=0.48)十分接近, 可能与这些品种间存在一定频率的基因交流有关。糯稻和非糯稻在籼稻群和粳稻群中都有表现, 没有特别的分布规律。云南栽培稻选育品种与地方稻亲缘关系较近, 其遗传基础可能来源于云南水稻地方品种。本研究结果表明, SSR标记能较好地区分云南栽培稻品种, 且云南水稻地方品种遗传多样性丰富, 存在大量的优质性状可供育种实践选择。     

一个AA基因组特异的串联重复序列的克隆及其在中国普通野生稻和栽培稻中的分化特征

从籼稻（ＯｒｙｚａｓａｔｉｖａＬ．ｓｐｐ．ｉｎｄｉｃａ）“窄叶青”中克隆到了１个重复序列（ｐＯｓ１３９）。经分子杂交证明,ｐＯｓ１３９为一稻属内ＡＡ基因组特异的串联重复序列。序列分析表明,ｐＯｓ１３９以３５５ｂｐ为一重复单位。以ｐＯｓ１３９为探针对２９份中国普通野生稻和４３份中国栽培稻的基因组ＤＮＡ进行的分子杂交表明,籼、粳亚种之间具有明显的差异,籼稻杂交带数明显多于粳稻,普通野生稻与籼稻相似,具有较多的杂交带数。拷贝数测定结果表明,ｐＯｓ１３９在普通野生稻和籼稻中丰度均较高,在粳稻中丰度较低。结合ｐＯｓ１３９的Ｓｏｕｔｈｅｒｎ杂交结果和以前的ＲＡＰＤ结果,认为籼稻和粳稻共同起源于普通野生稻。     

云南栽培稻种SSR遗传多样性比较

采用64个SSR标记对96份云南水稻（Oryza sativa）地方品种和选育品种的遗传多样性进行比较分析。结果发现64个标记都具有多态性,共检测到741个等位基因,每个多态性位点检测到的等位基因数为2—29个,平均11．57个：Nei基因多样性指数（He）范围在0．345（RM321）-0．932（RM1）之间,平均为0．56。水稻品种的遗传多样性并非按地理位置均匀分布,而是在相似系数为0．17的水平上明显分为2个不同类群,即籼稻类群和粳稻类群,且籼粳亚种间的SSR多样性差异不明显,籼稻平均等位基因数（Ap）和Nei基因多样性指数（Ap=10．6,He=0．46）与粳稻品种（Ap=10．7,He=0．48）十分接近,可能与这些品种间存在一定频率的基因交流有关。糯稻和非糯稻在籼稻群和粳稻群中都有表现,没有特别的分布规律。云南栽培稻选育品种与地方稻亲缘关系较近,其遗传基础可能来源于云南水稻地方品种。本研究结果表明,SSR标记能较好地区分云南栽培稻品种,且云南水稻地方品种遗传多样性丰富,存在大量的优质性状可供育种实践选择。     

Microsatellite diversity within Oryza sativa with emphasis on indica-japonica divergence

The molecular evolution of cultivated rice Oryza sativa L. has long been a subject of rice evolutionists. To investigate genetic diversity within and differentiation between the indica and japonica subspecies, 22 accessions of indica and 35 of japonica rice were examined by five microsatellite loci from each chromosome totalling 60 loci. Mean gene diversity value in the indica rice (H=0.678) was 1.18 times larger than in the japonica rice (H=0.574). Taking the sampling effect into consideration, average allele number in the indica rice was 1.40 times higher than that in the japonica rice (14.6 vs 10.4 per variety). Chromosome-based comparisons revealed that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. An overall estimate of F(ST) was 0.084-0.158, indicating that the differentiation is moderate and 8.4-15.8% of the total genetic variation resided between the indica and japonica groups. Our chromosome-based comparisons further suggested that the extent of the indica-japonica differentiation varied substantially, ranging from 7.62% in chromosome 3 to 28.72% in chromosome 1. Cluster analyses found that most varieties formed merely two clusters for the indica and japonica varieties, in which two japonica varieties and five indica varieties were included in the counterpart clusters, respectively. The 12 chromosome-based trees further showed that 57 rice varieties cannot be clearly clustered together into either the indica or japonica groups, but displayed relatively different clustering patterns. The results suggest that the process of indica japonica differentiation may have proceeded through an extensive contribution by the alleles of the majority in the rice genome.     

Distribution and Organization of AA Genome- specific and Tandemly Repetitive Sequences in Chinese Common Wild Rice and Cultivated Rice

Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species diversity. The authors report the isolation and characterization of repetitive DNA sequences (pOs139) from Oryza sativa cuhivars "Zhaiyeqing". By Southern blot analysis, the authors discovered that pOs139 sequences were organized not only tandemly, but also highly specifc for the AA genome of Oryza genus. Sequence analysis revealed that the clone pOs139 contains a 355 bp repetitive unit. The genomic DNA of 29 Chinese common wild accessions, and 43 cultivated rice accessions, were analyzed by Southern blot with pOs139 as a probe. The results illustrated that there was significant difference in hybridization patterns between japonica and indica subspecies. Hybridization bands of indica subspecies were much more than those of japonica, and the Chinese common wild rice was similar to indica in hybridization patterns. The copy number estimated by dot blot hybridization analysis indicated that a considerable degree of variation existed among different accessions of O. sativa and the Chinese common wild rice. It is interesting to note that japonica subspecies contains relatively low copy numbers of pOs139-related repetitive DNA sequences, while the indica and Chinese common wild rice contain relatively high copy numbers.     

Nonindependent domestication of the two rice subspecies, Oryza sativa ssp. indica and ssp. japonica, demonstrated by multilocus microsatellites

The origins of the Asian cultivated rice Oryza sativa from its wild ancestor O. rufipogon have been debated for decades. The question mainly concerns whether it originated monophyletically or polyphyletically. To shed light on the origins and demographic history of rice domestication, we genotyped a total of 92 individual plants from the two O. sativa subspecies and O. rufipogon for 60 microsatellites. An approximate Bayesian method was applied to estimate demographic parameters for O. rufipogon vs. O. sativa ssp. indica and O. rufipogon vs. O. sativa ssp. japonica. We showed that the japonica subspecies suffered a more severe bottleneck than the indica subspecies and thus a greater loss of genetic variation during its domestication. Across microsatellite loci there is a significant positive correlation in the reduction of genetic diversity between the two subspecies. The results suggest that completely independent domestication of indica and japonica subspecies may not explain our data and that there is at least partial sharing of their ancestral populations and/or recent gene flow between them.     

稻属谷氨酰胺合成酶家族的系统分析

比较籼粳栽培稻和野生稻中谷氨酰胺合成酶(GS)基因和蛋白质的结果表明,水稻GS蛋白编码区序列高度保守,而非编码序列变异较大。GS2基因的进化比GS1基因保守。短药野生稻中GS基因进化主要是内含子的变异,但此种内含子的变异在籼粳栽培稻中幅度要小得多。     

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence(399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb,respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon(including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.     

香稻资源遗传多样性的比较

利用60个水稻SSR标记, 对来自国内外的370份香稻材料的遗传多样性进行了比较分析。结果共检测到361个等位基因, 每个位点的等位基因变幅为2~10个, 平均Nei基因多样性指数(He)为0.663, 变幅为0.104(RM308)~0.885(RM2634)。籼粳亚种间的遗传多样性具有明显差异, 籼稻的等位基因数和Nei基因多样性指数均高于粳稻。地方品种的遗传多样性高于选育品种, 选育品种等位基因数仅为地方品种的86.5%。分子方差分析表明, 香稻材料中总变异的43.08%是由于亚种间的遗传差异引起的。不同稻区的遗传分化程度总体介于1.69%~14.40%之间。其中, 华南与西南、华中与西南地方品种间遗传差异的分化程度达显著水平。聚类分析将参试材料明显分为籼粳两大类, 同时地域相同(稻区)、相邻省份的香稻材料基本归为同一类群。     

Genetic differentiation of wild relatives of rice as assessed by RFLP analysis

To study genetic diversity and relationships of wild relatives of rice, 58 accessions of Oryza rufipogon, Oryza nivara, Oryza sativa f. spontanea and the cultivated Oryza sativa, representing a wide range of their distribution, were analyzed using the restriction fragment length polymorphism (RFLP) technique. All 30-used RFLP probes detected polymorphisms among the Oryza accessions, with an average of 3.8 polymorphic fragments per probe. Considerable genetic diversity was scored among the Oryza accessions with a similarity coefficient ranging from 0.28 to 0.93; but the cluster analysis of the accessions did not show an apparent grouping based on the species classification, instead they were scattered randomly in different groups. Noticeably, the Oryza accessions from the same geographic region, or near-by geographic regions, tended to be clustered in the same groups. The indica rice varieties showed relatively high genetic diversity and were scattered in different groups of their wild relatives, but the japonica varieties showed a relatively low variation and formed an independent group. It is concluded from the molecular analytical result that: (1) the four Oryza taxa have a remarkably close relationship and their independent species status need to be carefully reviewed; (2) geographic isolation has played a significant role in the differentiation of the Oryza accessions; therefore, a wide geographic range needs to be covered in collecting wild rice germplasm for ex situ conservation; and (3) the conventional conclusion of indica rice being directly domesticated from its ancestral wild species, and japonica rice being derived from indica, gains support from our data.