植物细胞质雄性不育基因的鉴定及育性调控机理

细胞质雄性不育（cytoplasmic male sterility,CMS）是作物杂交种生产的主要授粉控制系统,研究细胞质雄性不育分子基础及调控机理对利用杂种优势提高作物产量具有重要的指导意义。本文从植物的线粒体基因与细胞质雄性不育的关系着手,列举了植物CMS基因的鉴定情况,重点介绍了研究较多的矮牵牛、玉米、水稻和油菜细胞质雄性不育基因的鉴定进展及其对不育性状的调控。同时根据恢复基因与不育基因的相互作用情况阐述了育性恢复的可能机理,并对植物CMS分子机理的研究前景进行了展望。     

DEP1与NRT1.1B基因的遗传互作对水稻氮素利用的影响

提高氮素利用效率一直是水稻遗传改良攻关的重点方向。直立穗等位基因dep1及籼稻等位基因nrt1.1b均有利于提高水稻氮素利用效率,因此,阐明DEP1与NRT1.1B基因的互作关系对水稻氮素利用的影响对培育氮高效水稻品种具有重要指导意义。以携带不同DEP1与NRT1.1B基因型组合的重组自交系为供试材料,在低、中及高氮条件下（分别记为LN、MN和HN）,分析了DEP1与NRT1.1B基因间不同的遗传互作方式对水稻氮素利用、产量及其构成因素的影响。结果表明,不同氮素条件下,基因型组合dep1/nrt1.1b具有最大的氮素收获指数;LN条件下,nrt1.1b基因有利于提高氮素利用效率,在MN和HN条件下dep1基因有利于提高氮素利用效率;携带dep1基因株系有效穗数和穗粒数显著提升,其产量均值显著高于其他株系,而NRT1.1B基因对产量无显著影响。     

滇型水稻细胞质雄性不育恢复基因Rf-D1（t）的克隆与遗传特性分析

CMS/Rf测交F1结实率与花粉可育率检测表明,Ansanbyeo和南34对三种不同细胞质来源的粳稻不育系(CMS-DT1,CMS-BT和CMS-HL)都有较强的恢复力。根据已公布的水稻基因组序列,在水稻恢复基因定位区段内设计引物,确定出一对与两个滇型CMS的恢复系Ansanbyeo和南34恢复基因紧密连锁的分子标记引物M43804和M43558,首次克隆了滇型恢复基因Rf-D1(t),编码16个PPR蛋白。序列比对表明,该基因与BT型恢复基因Rf-1相似度达99%,两个恢复基因之间在ORF区仅存在一个碱基差异,即Rf-D1(t)第1149bp位的A变成Rf-1的C,并产生一个氨基酸的改编即K(赖氨酸)变成N(天冬酰胺)。该差异处于一个PPR结构域中,暗示该氨基酸是恢复基因的关键位点之一,它的改变可能影响对水稻CMS育性恢复力的变化。     

大麦中与胞质雄性不育性的育性恢复位点（Rfml）紧密连锁的基于AFLP的STS标记

胞质雄性不育（CMS）系统是杂交种生产中的需要性状。CMS系统已被用于大规模地生产玉米、油菜、高梁、水稻杂交种。CMS在大麦上尚未进行商业化应用，但是其F1杂交种的杂种优势效应已得到认可。Favret和Ryan最早报道大麦雄性不育胞质，他们在一个突变群体中找到这种雄性不育胞质。Schooler报道，大麦的雄性不育胞质来源于芒麦草（Hordeum jubatum），但是，尚未找到满意的恢复这种胞质育性的恢复基因。     

滇型水稻细胞质雄性不育恢复基因Rf-D1(t)克隆与遗传特性分析

CMS/Rf测交F1结实率与花粉可育率检测表明,Ansanbyeo和南34对三种不同细胞质来源的粳稻不育系(CMS-DT1,CMS-BT和CMS-HL)都有较强的恢复力.根据已公布的水稻基因组序列,在水稻恢复基因定位区段内设计引物,确定出一对与两个滇型CMS的恢复系Ansanbyeo和南34恢复基因紧密连锁的分子标记引物M43804和M43558,首次克隆了滇型恢复基因Rf-D1(t),编码16个PPR蛋白.序列比对表明,该基因与BT型恢复基因Rf-1相似度达99%,两个恢复基因之间在ORF区仅存在一个碱基差异,即Rf-D1(t)第1 149 bp位的A变成Rf-1的C,并产生一个氨基酸的改编即K(赖氨酸)变成N(天冬酰胺).该差异处于一个PPR结构域中,暗示该氨基酸是恢复基因的关键位点之一,它的改变可能影响对水稻CMS育性恢复力的变化.     

鄂西南地区稻瘟病菌AVR-Pia动态变化研究

为了解鄂西南地区稻瘟病菌AVR-Pia的分布和动态变化,于2017—2020年在鄂西南9个地点同时种植特定100个水稻品种,采集感病稻杆并分离稻瘟病菌;利用水稻单基因系IRBLa-A进行致病性鉴定,设计无毒基因AVR-Pia特异性引物进行PCR扩增和序列分析。结果表明2017—2020年分离保存的661株稻瘟病菌菌株中,有49株含有无毒基因AVR-Pia（占7.4%）,不同年份及不同地点AVR-Pia出现频率差异明显,2017—2020年分别为7.5%、16.0%、3.1%、5.0%,AVR-Pia出现频率最高的是野三关(15.8%),其次是柏杨和来凤（都是14.6%）,咸丰、建始和太平未出现。含Pia的单基因系IRBLa-A对其中91个菌株(13.8%)具有抗性。49个菌株能扩增出463 bp的特异性条带,序列分析发现其CDS区域未发现突变,起始密码子上游109 bp位点发生碱基突变G/T,该位点变异是否与表达相关有待进一步研究。说明鄂西南地区无毒基因AVR-Pia存在较少,抗性基因Pia不适合在鄂西南地区作为主效抗病基因来选择育种。     

不同水稻种质中渗透胁迫抗性基因DREB2A的遗传多样性分析

本研究旨在分析转录因子DREB2A基因在不同水稻种质中的遗传多样性,以期为水稻耐渗透胁迫遗传改良提供分子工具。利用单倍型分析、系统进化树、遗传距离和密码子偏好性分析,对85份不同类型水稻种质中DREB2A基因的功能性核苷酸序列变异及遗传多样性进行了研究。共鉴定出55个核苷酸变异位点,其中12个位于编码区,43个位于非编码区;鉴定出12个DREB2A等位基因型,其中来自非洲栽培稻(Oryza glaberrima)的3个等位基因型表现大片段变异;根据DREB2A基因的序列变异鉴定出39个单倍型,其中33个为新单倍型;将所有单倍型分为三组（Group I、II和III）,其中非洲栽培稻(Oryza glaberrima)中的10个单倍型单独分为一组(Group III);系统进化树、遗传距离和密码子偏好性分析均表明Group III与其他两组具有较大差异。对85份不同类型水稻种质中DREB2A基因的序列分析表明,非洲栽培稻(Oryza glaberrima)中的DREB2A等位基因在序列变异、系统进化关系和密码子偏好性方面均明显不同于其他种质材料中的等位基因。     

植物细胞质雄性不育恢复基因的克隆研究进展

细胞质雄性不育的恢复涉及线粒体基因组中的不育基因与核编码的育性恢复基因的相互作用,而恢复基因与CMS相关基因相互作用机理的最终阐明,有赖于Rf基因的分离与克隆.本文综述了目前已克隆的植物Rf基因,这些基因主要包括玉米的Rf 2基因、矮牵牛的Rf基因、萝卜的Rfo和Rfk 1基因以及水稻的Rf-1基因等.     

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。     

甘蓝型油菜BnGS3和BnGhd7的同源克隆及其与油菜产量相关性状的关系

甘蓝型油菜为异源四倍体, 基因组结构复杂, 而水稻基因组与油菜基因组具有一定的共线性。本研究利用水稻产量相关基因GS3和Ghd7序列信息, 通过同源克隆方法, 获得了甘蓝型油菜的同源基因BnGS3和BnGhd7。BnGS3有6个外显子, ORF全长666 bp, 编码222个氨基酸。BnGS3蛋白具有水稻GS3四个保守结构域中的VWF结构, 属于A型。BnGhd7含有1个外显子, ORF全长1014 bp, 编码337个氨基酸。BnGhd7蛋白具有N端的B-Box和C端的CCT两个重要的结构域。BnGS3和BnGhd7分别位于A2和A10连锁群, 比较测序得到BnGS3的多态性标记brgs-16及BnGhd7的多态性标记brghd-3和ghd7-7, 其中brghd-3与千粒重(P < 0.05)、ghd7-7与株高(P < 0.01)正相关, ghd7-7与开花期负相关(P < 0.05)。上述结果表明, 利用水稻功能基因序列信息克隆甘蓝型油菜的同源基因是可行的, 为油菜功能基因研究提供了一种有效途径。     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

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。     

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。     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

野败型育性恢复基因在AA基因组野生稻中的分布与遗传

分析了野败型恢复基因在AA基因组野生稻的分布。结果表明：（1）在31份野生稻中，有16份含有恢复基因，分布频率达51.6%。（2）6个AA基因组野生稻种中有4个种存在恢复基因，但主要集中于O. rufipogon和O. nivara。（3）在所鉴定的16份野生稻恢复系中，对野败型花粉育性恢复力大于80%和50%～80%的各6份，小于50%的4份；强恢复     

Chromosomal location of fertility restoring genes for ‘wild abortive’ cytoplasmic male sterility using primary trisomics in rice

Summary Identification and location of fertility restoring genes facilitates their deployment in a hybrid breeding program involving cytoplasmic male sterility (CMS) system. The study aimed to locate fertility restorer genes of CMSWA system on specific chromosomes of rice using primary trisomics of IR36 (restorer), CMS (IR58025A) and maintainer (IR58025B) lines. Primary trisomic series (Triplo 1 to 12) was crossed as maternal parent with the maintainer line IR58025B. The selected trisomic and disomic F₁ plants were testcrossed as male parents with the CMS line IR58025A. Plants in testcross families derived from disomic F₁ plants (Group I crosses) were all diploid; however, in the testcross families derived from trisomic F₁ plants (Group II crosses), some trisomic plants were observed. Diploid plants in all testcross families were analyzed for pollen fertility using 1% IKI stain. All testeross families from Group I crosses segregated in the ratio of 2 fertile: 1 partially fertile+partially sterile: 1 sterile plants indicating that fertility restoration was controlled by two independent dominant genes: one of the genes was stronger than the other. Testcross families from Group II crosses segregated in 2 fertile: 1 partially fertile+ partially sterile: 1 sterile plants in crosses involving Triplo 1, 4, 5, 6, 8, 9, 11 and 12, but families involving triplo 7 and triplo 10 showed significantly higher X² values, indicating that the two fertility restorer genes were located on chromosome 7 and 10. Stronger restorer gene (Rf-WA-1) was located on chromosome 7 and weaker restorer gene (Rf-WA-2) was located on chromosome 10. These findings should facilitate tagging of these genes with molecular markers with the ultimate aim to practice marker-aided selection for fertility restoration ability.     

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.     

Tournefortii male sterility system in Brassica napus. Identification, expression and genetic characterization of male fertility restorers

A germplasm collection of 152 diverse rapeseed accessions from Canada, China, France, India, Poland and South Korea was assayed for identifying new fertility restorers and sterility maintainers for a Tournefortii (tour) cytoplasmic male sterility (CMS) system in rape‐seed. Only 16 (10.5%) genotypes showed complete fertility restoration following hybridization with tour CMS line NE 409A. Notable among these were GSL 8851, GSL 8953, Mokpo # 9, Mali, Buk‐wuk‐13, Kuju‐27 and Mokpo # 84. As many as 78 (51.3%) genotypes were perfect maintainers of sterility, the remaining 58 (38.2%) genotypes were classified as partial maintainers. To study the inheritance of fertility restoration, 20 CMS (tour) rapeseed lines were crossed with the four best fertility restorers, namely GSL 8851, GSL 8953, Kuju‐27 and Mokpo # 9, to obtain F₂ and test cross populations. Segregation data indicated that fertility restoration for tour CMS was governed by two genes, of which, one is stronger than the other (χ²_12:3:1). Differences in gene interactions were also observed (χ²_9:3:4) which could be explained on the basis of influence of female parent genotypes/or modified expression of the restorer gene(s) in different genetic backgrounds. Tests of allelism indicated that the restorer genes present in the four restorers evaluated were allelic.     

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。