中国三系杂交稻恢复系资源的遗传多样性

以来自全国6个稻区16个省（市、自治区）及外引的128份三系杂交稻恢复系为试验材料，采用均匀分布于水稻12条染色体的36对SSR引物及9个表型性状标记进行遗传多样性分析。根据SSR标记检测结果，基于遗传距离的Neibor-jointing聚类显示128份恢复系可分为6个类群，籼亚种（83.2%）和野败型（82.6%）大多数分布于类群1和类群2，粳亚种（81.5%）和BT型（78.6%）大部分聚在类群3，红莲型（75%）相对集中于类群1。系谱分析显示，含有IR24血缘的材料分布于4个聚类群，而明恢63及其衍生系则分布非常集中。36对SSR引物在128份恢复系中共检测出281个等位变异，平均每个位点8.0个，平均遗传多样性指数(He)为0.6190，其中籼亚种（0.5770）略高于粳亚种（0.5656），但未达显著水平。按恢保类型，BT型(0.5816)>野败型(0.5705)>红莲型(0.4989)，野败型与BT型无显著差异，但二者均显著高于红莲型。按不同地理来源，He呈现南方稻区大于北方稻区，以华中双单季稻稻区(0.6057)最高，与其他稻区差异极显著差异；其次为西南高原单双季稻稻区(0.5326)；华北单季稻稻区(0.3902)最小，与其他稻区也差异极显著。表型性状检测的标记表明，平均Shannon-Weiner多样性指数为1.3980，其中籼、粳亚种分别为1.3746和1.3789，未达显著差异；不同恢保类型表现为BT型(1.4026)>野败型(1.3567)>红莲型(1.1732)。表型性状与SSR标记结果表现出一致性，相关系数分别为0.9981*（按亚种）、0.9418（按恢保类型）和0.8835**（按稻区）。     

长雄野生稻(Oryza longistaminata)全基因组Fosmid文库构建

长雄野生稻(O.longistaminata)起源于非洲,具有和亚洲栽培稻相同的AA基因组,是栽培稻改良的重要遗传资源。本研究构建了长雄野生稻全基因组fosmid文库,共获得33.6万克隆,文库的平均插入片段大小约为40kb,其中94.7%的克隆插入片段大小在30~50kb之间。挑取其中约11万克隆并保存,可覆盖10倍栽培稻基因组,对任意基因或序列的筛选概率达到了99.99%。将剩余的22万个克隆进行了末端配对测序,显示其有助于长雄野生稻全基因组测序的拼接。所构建的fosmid文库为筛选和克隆长雄野生稻的有利基因提供了研究材料。     

基于广西普通野生稻染色体单片段代换系的Rf3和Rf4复等位基因的恢复效应分析

本研究以5份广西普通野生稻材料DP15、DP29、DP30、DP50和DP63为供体亲本,以栽培稻品种9311为受体亲本,通过杂交及多代回交获得携带Rf3或Rf4基因座位的染色体单片段代换系8个。进一步将分别携带Rf3和Rf4基因座位的单片段代换系两两杂交、自交,筛选出同时具有这2个基因座位的双片段聚合系2个。通过野败型细胞质不育系(WA-CMS)盟抗A对单片段代换系和双片段聚合系进行恢复力测定。研究结果显示:(1)8个单片段代换系对盟抗A的恢复能力有显著不同:来自DP15的Rf3基因与来自DP15、DP30、DP50和DP63的Rf4基因具有强恢复力,它们的F1植株花粉育性达到86.18%~91.13%;来自DP29的Rf4基因具有较强的恢复力,其F1植株花粉育性为81.21%;来自DP29的Rf3基因具有弱恢复力,其F1植株花粉育性为7.00%;而来自DP30的Rf3基因没有恢复能力,其F1植株花粉育性仅为0.33%。(2)Rf3或Rf4基因的聚合效应在不同供体间表现不同:来自DP29的较强恢复基因(Rf429)与弱恢复基因(Rf329)间聚合呈现显著的累加效应;而来自DP15的2个强恢复基因(Rf315和Rf415)聚合后没有进一步提高恢复能力。研究结果表明,利用广西普通野生稻挖掘育性恢复基因是有效的,获得了多个对野败型细胞质不育系(CMS-WA)盟抗A具有强恢复力的Rf3或Rf4位点复等位基因,可供育种研究利用。     

引进水稻资源对野败型不育胞质恢复力的鉴定研究

旨在明确来自亚洲、欧洲、非洲、北美洲、南美洲共441份水稻种质的恢保关系和强恢种质分布地区,为高效选育恢复系、保持系提供优异亲本。用野败型细胞质雄性不育系‘明218A’为母本与外引种质测交配制441份测验种。以自然结实率为指标,鉴别恢保类型。鉴定出恢复型种质53份,保持型种质43份,半恢半保型种质333份。分析53份恢复种质的分布地区,结果表明来源于亚洲国家（地区）种质最多,达到了33份,占恢复种质的比例为62.3%;筛选出美国强恢种质6份,占来源美国种质31.6%;来源于欧洲种质仅2份,占来源于欧洲种质的比例为5%,欧洲恢复种质相对较少。对53份强恢种质测交F1与测交父本7个重要性状均值进行相关分析,其中株高(r=0.97**)、千粒重(r=0.934**)和穗长(r=0.747**)3个性状达极显著相关,而其余的4个性状的相关不显著。相关程度的大小顺序为：株高＞千粒重＞穗长＞有效穗＞穗总粒数＞穗实粒数＞自然结实率。野败型不育系强恢的种质5大洲都有分布,主要集中在亚洲的东亚、南亚、东南亚地区以及美国。恢复系的株高、千粒重以及穗长对杂交种F1的影响较大。     

亚洲杂草稻红色果皮分子鉴定及其Rc-bHLH序列多态性分析

多数杂草稻的果皮是红色,该特征可用于稻田中杂草稻的早期的初步鉴定,也可用于水稻果皮Rc/rc基因的起源进化研究。本研究利用红色果皮Rc基因第六外显子的In Del标记RID14对来自亚洲不同稻作区的199份杂草稻(O.sativa f.spontanea)、24份栽培稻(O.sativa)和9份野生稻(O.rufipogon)进行基因型鉴定并与表型比对;同时对9份代表性杂草稻Rc-b HLH位点的DNA序列进行测序,并将其与下载的29份栽培稻(籼,粳)和14份野生稻的对应序列进行比对分析。研究结果表明:(1)亚洲杂草稻的果皮颜色的表型多样,多数(84.4%)为红色或褐色(由浅至深)、少数(15.6%)为白色,其基因型与表型鉴定结果高度一致;杂草稻红色果皮Rc基因第六外显子均为无14 bp缺失的类型,仅有一份中国东北杂草稻除外(为14 bp缺失型),揭示用RID14标记可以早期快速的鉴定稻田中红色果皮杂草稻;(2)测试的9份杂草稻其Rc-b HLH位点的DNA序列均较为保守,其中出现4个突变位点;(3)在Rc-b HLH部分区域(1~115 bp),红色果皮的籼型栽培稻、杂草稻和野生稻的DNA序列相同,但红色果皮粳型栽培稻与白色果皮籼型栽培稻和杂草稻在106 bp处存在一个A/G替换;(4)通过对比野生稻和栽培稻,杂草稻在Rc-b HLH位点核苷酸多态性多态性最高,但是野生稻在该位点Tajima'D检验值呈显著,说明Rc-b HLH位点在野生稻中有选择性;通过聚类分析发现分为2个类群,类群1由白色果皮的杂草稻、籼型栽培稻和红/白果皮的粳型栽培稻组成;类群2由红色果皮的栽培稻(籼型)、杂草稻和野生稻组成。该结果暗示:在粳稻区,白色果皮的亚洲杂草稻可能由栽培稻退化返祖而形成的;在籼稻区,红色果皮的亚洲杂草稻可能经由栽培稻及野生稻杂交(如籼籼交或栽野交)演化而来。     

水稻孕穗期茎注射野生稻DNA变异株系的RAPD分析

利用野生稻有利基因是培育超高产栽培稻(Oryza sativa L, 2n=24AA染色体组型)的主 要技术路线之一。 本研究通过穗茎注射法将小粒野生稻O. minuta(2n=4x=48, BBCC染 色体组型)总DNA导入杂交稻当家亲本V20B, 获得了变异株的3个高世代株系; 用RAPD方 法 对上述3个株系、 供、 受体及对照品种明恢63共6个材料进行了多态性研究     

油菜Nsa CMS候选恢复基因的来源及表达

根据同源序列法克隆的Nsa CMS候选恢复基因PPR618的序列,采用PCR方法在Nsa CMS不育系、恢复系、原始体细胞杂交亲本以及其他甘蓝型油菜品种中共克隆出22个同源序列。序列分析表明,野芥亲本野油18、甘蓝型油菜亲本中双4号各含有2个同源序列,而Nsa CMS 4个恢复系则分别含有3、1、1、1个同源序列。恢复系中候选恢复基因的序列与野芥亲本野油18的同源序列的一致性在93%以上,其中恢1、恢3和恢4中至少有一个同源序列与野油18的第一个同源序列完全相同,恢2中的同源序列与野油18的第2个同源序列完全相同;但与甘蓝型油菜的同源序列一致性均低于80%,说明候选恢复基因来源于新疆野芥,而不是甘蓝型油菜。除了原来的PPR618外,获得了3个来源于恢复系的新候选恢复基因。候选恢复基因在序列上与萝卜和矮牵牛的恢复基因一致性较高。半定量RT-PCR分析表明,候选恢复基因在恢复系中多个组织都有表达,但在根、茎中表达量特别少。随着营养器官到生殖器官的发育逐渐升高,候选恢复基因在恢复系中表达量最高的是雄性败育关键时期, 即1.5~2.5 mm的花蕾中,但不育系中的同源基因在茎中的表达量则相对高于其他组织。     

野生稻与栽培稻之间第6染色体的遗传比较

用具有Ｗｘ１的“Ｔａｉｃｈｕｎｇ６５”（Ｔ６５ｗｘ１栽培稻粳型品种）为轮回亲本和多年生近缘野生稻种（Ｏｒｙｘａｒｕｆｉｐｏｇｏｎ）回交，考察了它们在第６染色体上的遗传差异，野生稻携带的配子排除（ｇａｍｅｔｅ ｅｌｉｍｉｎａｔｏｒ）基因Ｓ６证明了第６染色体上的Ｗｘ基因具有松散的连锁关系。Ｓ６相对位点基因Ｓ６＾ａ会导致配子败育，只有杂合体（Ｓ６Ｓ６＾ａ）以及非Ｓ６＾ａ的配子能产生后代。基因排列顺序为Ｗ     

Bph15在非洲栽培稻、药用野生稻和宽叶野生稻中的BAC-FISH比较物理定位

用覆盖抗褐飞虱基因Bph15的两个BAC克隆,即20M14 (27 kb)和64O9 (36 kb)作为探针,对非洲栽培稻、药用野生稻和宽叶野生稻体细胞染色体进行荧光原位杂交(FISH)。两个BAC克隆均被定位于非洲栽培稻和药用野生稻第4染色体的短臂上,杂交信号的百分距离分别为37.03±4.11和81.22±3.62,相应的信号检出率为41.18%和38.22%。在宽叶野生稻中,有两对同源染色体同时检出信号,分别定位于染色体的短臂和着丝粒区,信号距着丝粒平均百分距离分别为87.78±5.23和0,信号检出率为52.58%。由此推知,这两个BAC克隆在非洲栽培稻和药用野生稻的第4染色体分布同线且同区,并且在宽叶野生稻的DD基因组也存在Bph15基因的同源序列。在未封阻的情况下,BAC克隆在非洲栽培稻的多条染色体上有杂交信号,表明它和栽培稻C_0t-1 DNA在一定程度上具有同源性。上述结果初步显示Bph15在3个稻种染色体中的相对位置。文章讨论了Bph15在3个种间的关系,为有效分离和利用Bph15基因提供了有益的依据,对不同基因组及二倍体和四倍体中功能基因可能进化机制的分析提供了线索。     

云南地方老品种水稻中恢复基因位点遗传差异分析

目前生产上使用的水稻恢复系之间的遗传基础过于狭窄,应积极开发水稻新恢复系的选育工作。云南地理气候条件特殊,地方老品种资源丰富,是筛选新恢复系的理想材料。以61份云南地方老品种水稻资源为父本,通过测交筛选到6份可以恢复野败型不育系的品种。再利用5对特异引物,对这6份恢复材料和7份恢复系的恢复基因位点遗传差异进行分析。结果表明：5对特异引物对6份恢复材料的扩增结果各不一样,而与对照恢复系仅有2.8 kb的一条扩增带型一致。说明云南地方老品种水稻中蕴藏的恢复基因与目前生产上应用的恢复基因的亲缘关系较远。从云南地方老品种水稻中发掘利用新型恢复基因和资源,是突破水稻恢复基因遗传基础狭窄、筛选新质源恢复系的一种有效策略。     

水稻优良恢复系明恢63两个恢复基因恢复力的单独评价

野败型细胞质雄性不育系统是选配杂交稻组合广泛应用的主要不育细胞质资源，野败型细胞质雄性不育的育性恢复能力由两个恢复基因控制。以前的研究表明，明恢63具有2个恢复基因Rf3和Rf(μ)，分别位于第1和第10染色体上。为了分别准确估计这两个恢复基因的遗传效应，根据分子标记基因型，从珍汕97／明恢63衍生的241个F9重组自交系群体中选择两个自交系R124和R1183，它们分别含有单个恢复基因Rf3和Rf(M)，将R124和R1183与珍汕97A杂交，分别得到F1A和F1B，再自交得到F2A和F2B。在武汉和海南分别考察F1的育性，F1A的自然结实率海南和武汉分别为53．4％和60．2％，F1B的自然结实率海南和武汉分别为70．5％和75．7％。而珍汕97A／明恢63的杂种汕优63结实率为81．4％。F2A和F2B群体育性分离均符合1个主基因1：3的孟德尔期望分离比，表明，R124和R1183分别只含有一个恢复基因Rf3和Rf(M)。Rf(M)的效应较大，恢复力强，它单独几乎可以使育性恢复正常。利用标记辅助选择方法，转移两个恢复基因可以快速选育优良恢复系。     

Development and use of a two-gene marker-aided selection system for fertility restorer genes in rice

We have established marker-aided selection strategies for the two major Rf genes (Rf3 and Rf4) governing fertility restoration of␣cytoplasmic-genetic male sterility (CMS) in rice. Polymorphisms between restorer and non-restorer␣lines were observed using RG140/PvuII for Rf3 located on chromosome 1 and S10019/BstUI for Rf4 located on chromosome 10. DNA polymorphisms associated with these two loci in restorer lines of wild abortive (WA), Dissi, and Gambiaca cytoplasm are conserved, suggesting that similar biological processes control pollen fertility in this diverse cytoplasm. Because of their close linkage to Rf genes and distinct banding patterns, STS markers RG140/PvuII and S10019/BstUI are well suited for marker-aided selection, enhanced backcross procedures, and pyramiding of Rf genes in agronomically superior non-restorer lines. The combined use of markers associated with these two loci improved the efficiency of screening for putative restorer lines from a set of elite lines. Positional analyses of Rf4 and the inheritance pattern of the polymorphism in S10019/BstUI suggest that Rf4, governing fertility restoration in WA-CMS in rice, is likely to be the same gene governing fertility restoration in BT- and HL-CMS that has a gametophytic effect, which explains why 100% pollen fertility in hybrids is impossible to attain.     

Frequency and fertility restoration efficiency of Rf3 and Rf4 genes in Indian rice

Identification of new parental lines is crucial for developing ecology‐specific hybrids with ideal agronomic performance. We screened a total of 570 different ecology‐specific Indian rice varieties for the presence of fertility restorer genes, Rf3 and Rf4 using tightly linked markers DRRM Rf3‐10 and RM6100, respectively. Among these varieties, 13% carried Rf3Rf3/Rf4Rf4, 31% carried rf3rf3/rf4rf4, 6% carried Rf3Rf3/rf4rf4 and remaining 50% carried Rf4Rf4/rf3rf3 allelic combinations. A mini set of 40 varieties with variable allelic combinations of fertility restorer genes were testcrossed with WA and Kalinga‐based CMS lines. All the 80 F₁s were evaluated for spikelet fertility and fertility restoration ability. Rf3Rf3/rf4rf4 genotypes mostly behaved as partial maintainers or partial restorers. In contrast, rf3rf3/Rf4Rf4 genotypes were partial or effective restorers. However, double dominant genotypes showed better fertility restoration than the genotypes containing Rf3 or Rf4 individually. Some of the genotypes showed unexpected restoration pattern implying occurrence of other fertility restorer(s) apart from Rf3 and Rf4. The perfect restorers and maintainers identified in this study can be directly used in hybrid rice breeding.     

一个位于水稻Rf-1基因的InDel标记遗传分离对海拔和细胞质的响应

水稻细胞质雄性不育育性恢复基因仪存在于适于热带亚热带气候条件的野生稻和籼稻中,不存在适于气候冷凉广泛生长在温带气候条件的粳稻种质资源中.为研究恢复基因与温度适应性的关系,选用来自云南2650 m高海拔的地方粳稻老品种小麻谷(基因型为 rf/rf)与具有籼稻细胞质背景的杂交粳稻恢复系南34(基因型为Rf/Rf)进行正反交...     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.     

Molecular detection of WA‐CMS restorers from tropical japonica‐derived lines,their evaluation for fertility restoration and adaptation

Hybrid rice based on wild‐abortive cytoplasmic male sterility (WA‐CMS) is important in boosting rice production, which requires diverse parents to harness heterosis. For this, exploiting the diversity of japonica through tropical japonica (TRJ) lines is an excellent route. In this study, 310 TRJ‐based new plant type (NPT) lines were developed and evaluated for Rf3 and Rf4 genes. Gene‐based (DRRM‐Rf3‐5 and DRRM‐Rf3‐10) and functional marker (RMS‐SF21‐5) targeted Rf3 locus, while gene‐linked (RM6100) and functional marker (RMS‐PPR9‐1) targeted the Rf4 locus. The frequency of the restorer allele of Rf3 gene was lower when compared to that of Rf4. Combined phenotypic and molecular screening using gene‐based and functional markers identified 42 lines that carried Rf3 and/or Rf4 genes. All the selected lines produced fertile F₁s when crossed to a WA‐CMS line, “Pusa 6A”, but with varying levels of spikelet fertility. This is the first report of a marker‐cum‐phenotype‐based restorer selection using TRJ‐derived lines. Multilocation evaluation of these lines at three locations indicated better adaptation for grain yield in some of the lines.     

东乡野生稻细胞质雄性不育育性恢复基因的遗传分析及应用

细胞质雄性不育及其恢复系统在杂交水稻生产中必不可少,而从野生稻中鉴定、发掘细胞质雄性不育恢复源对促进杂交水稻的发展产生深远的影响。为了发掘东乡野生稻的育性恢复基因,应用协青早A/(协青早B//协青早B/东乡野生稻BC1F10)和中9A/(协青早B//协青早B/东乡野生稻BC1F10)两套测交群体,开展东乡野生稻细胞质雄性不育育性恢复经典遗传学研究。结果表明:东乡野生稻对矮败型(CMS-DA)和印尼水田谷败型(CMS-IA)的育性恢复表现为质量-数量性状,东乡野生稻对矮败型(CMS-DA)和印尼水田谷败型(CMS-IA)的育性恢复的控制存在主效恢复基因,同时还受到微效基因的影响。此外,还就东野恢复基因发掘及其在杂交水稻育种的生产应用进行了探讨。     

东乡野生稻细胞质雄性不育育性恢复的遗传研究

应用协青早A／（协青早B／／东乡野生稻／协青早B）BC1F5、中9A／（协青早B／／东乡野生稻／协青早B）BC1F5、协青早A/（协青早B／／协青早B／东乡野生稻）Be1F5和中9A／（协青早B／／协青早B／东乡野生稻）BC1F5四套测交群体,分析测交群体的育性表现及研究东乡野稻细胞质雄性不育育性恢复的遗传。结果表明：东乡野生稻对矮败（CMS—DA）和印尼水田谷败育（CMS—IA）的育性恢复表现为质量一数量性状,结合群体遗传结构、育性分布和东乡野生稻的偏粳性及广亲和性,可知东乡野生稻对CMS—DA和CMS—IA的恢复性由1对或2对恢复基因控制。     

野生稻与亚洲栽培稻的遗传多样性

为评价野生稻与亚洲栽培稻的遗传多样性及其变异关系,56对SSR引物被用于研究广泛地理分布的55份普通野生稻(其中32份O. rufipogon和23份O. nivara)和25份亚洲栽培稻(14份indica和11份japonica)样本。298个多态性位点被检出,占总扩增等位点的98.68%。野生稻多态性位点的百分比(平均达91%)及Nei’s遗传多样性值(h)明显高于亚洲栽培稻,表明普通野生稻比亚洲栽培稻具更丰富的遗传多样性。UPGMA聚类分析显示野生稻的两个类群(O. rufipogon和O. nivara)关系密切,但在遗传上存在明显的分化,支持其作为两个独立物种的分类观点。许多普通野生稻中籼粳分化尽管不很明显,然而亚洲栽培稻的籼粳亚种分化是明显的。亚洲栽培稻与多年生普通野生稻(O. rufipogon)关系更为密切,符合异源起源的遗传分化模式。     

Development and primary genetic analysis of a fertility temperature-sensitive polima cytoplasmic male sterility restorer in Brassica napus

Over the past decade, the polima cytoplasmic male sterility ( pol CMS) three-line and two-line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL-204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature-sensitive restorer (FTSR) in this paper. F₂, BC₁ as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL-204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene ( Rf ) and temperature-sensitive genes ( ts )] and the cytoplasm. The Rf gene in FL-204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL-204. However, the ts gene(s) could be non-functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL-204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL-204 for the production of hybrid seed in B. napus oilseed rape is proposed.