应用分子标记辅助选择培育抗稻瘟病杂交稻亲本

多性状的分子标记辅助选择具有重要的育种应用价值。研究先应用与水稻抗稻瘟病基因Pi25紧密连锁的分子标记对394个重组自交系进行筛选,再应用育性恢复基因连锁标记检测中选株系,获得20份候选恢复系和27份候选保持系。经田间接种稻瘟病菌、考查测交F1育性,表明利用该研究策略可以同步对多个性状进行选择,提高杂交稻亲本选育的效率。     

利用分子标记辅助选择聚合Pi-1和fgr基因改良水稻恢复系

以含有广谱稻瘟病抗性基因Pi-1的籼稻品系BL122为稻瘟病基因的供体,籼稻恢复系香型中间材料XH74为香味基因的供体,三系优良恢复系R290为受体亲本,通过杂交、多次回交、基因聚合和多次自交,并同时利用与Pi-1基因紧密连锁的SSR标记RM144以及香味基因fgr的功能标记GRFM04在不同世代分离群体中进行分子标记辅助选择,最终筛选获得6个双基因纯合的改良株系。利用30个广东代表性稻瘟病菌株接种鉴定,6个改良株系的抗性频率为100%,而对照R290抗性频率仅为66.67%;自然病圃诱发鉴定6个改良株系,其叶瘟和穗颈瘟均表现高抗。检测表明6个改良株系叶片和米粒均具有较浓的香味。统计分析表明,除1个株系结实率稍低与对照差异显著外,其余改良株系与对照相比,在所有农艺性状上均差异不显著。通过分子标记辅助选择将稻瘟病基因Pi-1和香味基因fgr聚合到恢复系R290中,不仅显著地提高了恢复系的稻瘟病抗性,而且稻米有了较浓的香味,利用这批改良株系育成杂交稻新组合荣优633和五优633已参加省区试。     

分子标记辅助选择改良三系杂交稻恢复系R225稻瘟病抗性

稻瘟病是水稻的主要病害,培育抗稻瘟病品种是防治稻瘟病的有效途径。本研究通过分子标记辅助选择与杂交育种相结合的方式,将稻瘟病抗性基因Pi1、Pi2和Pi9导入到三系杂交稻恢复系R225。对BC3F3代材料进行苗期和成熟期稻瘟病抗性鉴定,携带1个或2个抗性基因的目标株系抗性达到中抗以上水平,稻瘟病抗性显著高于各自的轮回亲本。SSR标记分析表明,改良株系的遗传背景回复率达到86.1%~95.3%。通过标记辅助选择获得的改良材料为三系杂交稻恢复系的培育提供了稻瘟病抗性亲本。     

抗稻瘟病基因Pi9的STS连锁标记开发及在分子标记辅助育种中的应用

利用带有广谱抗稻瘟病基因Pi9的籼稻品系75-1-127作为抗病基因供体亲本,用于扬稻6号和R6547抗病性的回交育种。通过比较75-1-127、扬稻6号和日本晴的Pi9基因位点的DNA序列,开发出了与Pi9基因紧密连锁的共显性STS(序列标记位点)标记PB9-1,用于Pi9基因的分子标记辅助选择。结合田间农艺性状选择和分子标记辅助选择,培育出8个Pi9基因纯合的回交后代株系。其中,具有扬稻6号和R6547遗传背景的株系各4个。经湖北恩施和宜昌的病圃鉴定,携带有抗病基因株系的稻瘟病抗性水平较受体品种扬稻6号和R6547有不同程度的提高。具有R6547遗传背景的株系08C893配制的杂交组合在上述病区的抗性表现也明显优于对照品种扬两优6号。上述结果说明,共显性标记PB9-1在Pi9抗稻瘟病基因分子标记辅助育种中具有应用价值,并且Pi9基因作为稻瘟病抗源之一可以在湖北稻区进行有效利用。     

抗稻瘟病基因Pi9的STS连锁标记开发及在分子标记辅助育种中的应用

 利用带有广谱抗稻瘟病基因Pi9的籼稻品系75 1 127作为抗病基因供体亲本,用于扬稻6号和R6547抗病性的回交育种。通过比较75 1 127、扬稻6号和日本晴的Pi9基因位点的DNA序列,开发出了与Pi9基因紧密连锁的共显性STS（序列标记位点）标记PB9 1,用于Pi9基因的分子标记辅助选择。结合田间农艺性状选择和分子标记辅助选择,培育出8个Pi9基因纯合的回交后代株系。其中,具有扬稻6号和R6547遗传背景的株系各4个。经湖北恩施和宜昌的病圃鉴定,携带有抗病基因株系的稻瘟病抗性水平较受体品种扬稻6号和R6547有不同程度的提高。具有R6547遗传背景的株系08C893配制的杂交组合在上述病区的抗性表现也明显优于对照品种扬两优6号。上述结果说明,共显性标记PB9 1在Pi9抗稻瘟病基因分子标记辅助育种中具有应用价值,并且Pi9基因作为稻瘟病抗源之一可以在湖北稻区进行有效利用。     

分子标记辅助选择Pi9基因改良R288的稻瘟病抗性

根据已克隆的广谱持久抗瘟基因Pi9的DNA序列设计功能标记Clon2-1,通过分子标记辅助选择开展回交育种实践,定向改良水稻恢复系R288的稻瘟病抗性。获得如下结果:Clon2-1为共显性标记,在Pi9基因供体亲本75-1-127和受体亲本R288之间多态性明显且稳定;Clon2-1标记基因型对稻瘟病抗性表型的选择效率达100%。通过分子标记辅助选择和连续回交自交,获得了含Pi9基因的BC6F3群体,在此基础上筛选鉴定出1个高抗稻瘟病水稻新品系‘R288-Pi9’,用其与培矮64S配组获得的杂交组合同样表现出高水平稻瘟病抗性。     

利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性

【目的】CRISPR/Cas9基因编辑技术是作物遗传改良的有效工具。本研究通过对水稻Pita、Pi21和ERF922稻瘟病相关基因进行定点编辑,以期获得能够稳定遗传的抗稻瘟病水稻材料。【方法】利用CRISPR/Cas9基因编辑技术,以Pita、Pi21和ERF922为靶基因,构建共编辑载体pC1300-2×35S::Cas9-g~(Pita)-g~(Pi21)-g~(ERF922),用农杆菌转化长粒粳稻恢复系L1014,筛选获得稳定遗传的纯合突变体用于稻瘟病抗性鉴定。【结果】在T_0代转基因株系中,Pita、Pi21和ERF922突变频率分别为75%、85%和65%,突变基因型多为双等位突变。筛选到的不含T-DNA成分的T_1代能够稳定遗传给T_2代,并从中获得Pi21单突变纯合株系及Pita、Pi21和ERF922的三突变纯合株系。稻瘟病抗性鉴定结果表明,与野生型相比,突变株系的抗性显著提高。同时,接种后纯合突变体株系内水杨酸、茉莉酸和乙烯等信号转导途径相关基因的表达量均上调。据此,我们推测纯合突变株系对稻瘟病的抗性增强可能与其对稻瘟病菌的响应被激活有关。【结论】利用CRISPR/Cas9技术获得了能够稳定遗传和具有较高稻瘟病抗性的纯合突变株系,为水稻稻瘟病抗性改良提供了良好的材料。     

利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性

[目的]CRISPR/Cas9 基因编辑技术是作物遗传改良的有效工具。本研究通过对水稻Pita、Pi21和ERF922稻瘟病相关基因进行定点编辑,以期获得能够稳定遗传的抗稻瘟病水稻材料。[方法]利用 CRISPR/Cas9基因编辑技术,以 Pita、Pi21 和 ERF922 为靶基因,构建共编辑载体 pC1300-2×35S::Cas9-g^Pita-g^Pi21-g^ERF922 ,用农杆菌转化长粒粳稻恢复系L1014,筛选获得稳定遗传的纯合突变体用于稻瘟病抗性鉴定。[结果]在T0代转基因株系中,Pita、Pi21和ERF922 突变频率分别为 75%、85%和 65%,突变基因型多为双等位突变。筛选到的不含 T-DNA成分的T1代能够稳定遗传给T2代,并从中获得 Pi21单突变纯合株系及Pita、Pi21和ERF922的三突变纯合株系。稻瘟病抗性鉴定结果表明,与野生型相比,突变株系的抗性显著提高。同时,接种后纯合突变体株系内水杨酸、茉莉酸和乙烯等信号转导途径相关基因的表达量均上调。据此,我们推测纯合突变株系对稻瘟病的抗性增强可能与其对稻瘟病菌的响应被激活有关。[结论]利用 CRISPR/Cas9 技术获得了能够稳定遗传和具有较高稻瘟病抗性的纯合突变株系,为水稻稻瘟病抗性改良提供了良好的材料。     

利用CRISPR/Cas9系统定向改良水稻稻瘟病抗性

【目的】CRISPR/Cas9基因编辑技术是作物遗传改良的有效工具。本研究通过对水稻Pita、Pi21和ERF922稻瘟病相关基因进行定点编辑，以期获得能够稳定遗传的抗稻瘟病水稻材料。【方法】利用CRISPR/Cas9基因编辑技术，以Pita、Pi21和ERF922为靶基因，构建共编辑载体pC1300-2×35S::Cas9-gPita-gPi21-gERF922 ，用农杆菌转化长粒粳稻恢复系L1014，筛选获得稳定遗传的纯合突变体用于稻瘟病抗性鉴定。【结果】在T0代转基因株系中，Pita、Pi21和ERF922突变频率分别为75%、85%和65%，突变基因型多为双等位突变。筛选到的不含T-DNA成分的T1代能够稳定遗传给T2代，并从中获得Pi21单突变纯合株系及Pita、Pi21和ERF922的三突变纯合株系。稻瘟病抗性鉴定结果表明，与野生型相比，突变株系的抗性显著提高。同时，接种后纯合突变体株系内水杨酸、茉莉酸和乙烯等信号转导途径相关基因的表达量均上调。据此，我们推测纯合突变株系对稻瘟病的抗性增强可能与其对稻瘟病菌的响应被激活有关。【结论】利用CRISPR/Cas9技术获得了能够稳定遗传和具有较高稻瘟病抗性的纯合突变株系，为水稻稻瘟病抗性改良提供了良好的材料。     

太湖流域粳稻地方品种黑壳子粳抗稻瘟病基因的分子定位

以广谱、高抗稻瘟病的太湖流域粳稻地方品种黑壳子粳与感病品种苏御糯杂交,产生F1、F2、F2∶3及F5∶6重组自交系群体,用日本稻瘟病鉴别菌系北1接种鉴定。黑壳子粳对北1的抗性是由1对显性主效基因控制的,定名为Pi hk1(t) 。根据不同杂交世代群体对北1的抗、感反应,结合SSR分子标记,将黑壳子粳中的Pi hk1(t) 基因定位在水稻第11染色体长臂末端,与RM7654和RM27381两个标记的遗传距离分别为0.9 cM和1.6 cM。     

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight(BB) diseases,the two most prevalent diseases jeopardizing rice production.Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food.An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions.Only one pair of parents,DH146 × TM487,showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes,Xa21,xa13 and xa5,thus it was used in the marker-assisted selection(MAS).F2 individuals of DH146 × TM487 were genotyped using flanking markers of RM3330 and sequence tagged site(STS) marker SA7 for Pi25.The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21,xa13 and xa5 identified by the markers pTA248,RM264 and RM153,respectively in subsequent generations.Finally,after selection for agronomic traits and restoration ability among 12 pyramided lines,we acquired an elite restorer line,R8012 including all four target genes(Pi25+Xa21+xa13+xa5).Hybrid Zhong 9A/R8012 derived from the selected line showed stronger resistance to blast and BB,and higher grain yield than the commercial checks uniformally in experimental plots,2007 state-wide yield trial and 2008 nation-wide yield trial.This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes.The resultant restoring line and its hybrid would play an important role in securing rice production in China.     

Avoidance of Linkage Drag Between Blast Resistance Gene and the QTL Conditioning Spikelet Fertility Based on Genotype Selection Against Heading Date in Rice

Previous study showed that a linkage drag between a blast resistance gene Pi25(t) and QTLs conditioning spikelet fertility (qSF-6) and number of filled grains per panicle (qNFGP-6) was detected on the short arm of chromosome 6. A larger population was used for further verification, and the results confirmed the linkage drag between the blast resistance gene and QTL conditioning spikelet fertility, other than QTL conditioning number of filled grains per panicle. Breakdown or avoidance of the linkage drag could be achieved by selection against the genotype background of a heading-date gene (qHD-7) that resided in the region between RM2 and RM214 on chromosome 7. For further validation, two lines with almost identical genotypes on all chromosomal regions except the Pi25(t) region on chromosome 6 were chosen to develop a new population. The results showed that qSF-6 could be further subdivided into qSF-6-1 and qSF-6-2. When the genotype of the region between RM2 and RM214 was from rice variety Zhong 156, the linkage drag between Pi25(t) and qSF-6-2 was detected and the allele of qSF-6-2 from rice variety Gumei 2 reduced the spikelet fertility. When the genotype of the region between RM2 and RM214 was from Gumei 2, no linkage drag was detected. This indicates that the linkage drag between the blast resistance gene and the QTL conditioning spikelet fertility could be broken down or avoided under a certain background genotype selection against heading-date and provides a marker aided solution for high level of blast resistance and yield breeding in rice and other crops as well.     

通过生育期基因型选择避免稻瘟病抗性与结实率的遗传累赘

利用重组自交系研究表明,在水稻第6染色体短臂上稻瘟病抗性基因Pi25(t)与控制结实率和每穗实粒数的QTL之间存在遗传累赘。为了验证这种关系,采用了更大的遗传群体进行分析,结果表明稻瘟病抗性与结实率存在遗传累赘,但未检测到稻瘟病抗性与每穗实粒数存在遗传累赘。通过对第7染色体长臂RM2-RM214区间抽穗期基因（qHD 7）型背景进行选择,可以打破或避免稻瘟病抗性与结实率的遗传累赘。为了进一步验证这种关系,选择Pi25(t)区间基因型不同、RM2-RM214区间基因型相同、其他染色体区间基本一致的两个株系发展新群体进行分析,除第6染色体的结实率QTL可以分解成2个效应较小的QTL（qSF 6 1和qSF 6 2）外,当第7染色体RM2-RM214区间基因型为中156背景时,Pi25(t)与结实率QTL（qSF 6 2）存在遗传累赘,且qSF 6 2来自父本谷梅2号的等位基因起减效作用;当第7染色体RM2-RM214区间基因型为谷梅2号背景时,第6染色体上没有检测到结实率QTL。上述结果说明在特定育种材料中对抽穗期基因进行选择可以成功打破或避免稻瘟病抗性与结实率的遗传累赘,为水稻以及其他作物的高产抗病育种提供了一种新途径。     

Improvement of Upland Rice Variety by Pyramiding Drought Tolerance QTL with Two Major Blast Resistance Genes for Sustainable Rice Production

Varalu is an early maturing rice variety widely grown in the rainfed ecosystem preferred for its grain type and cooking quality. However, the yield of Varalu is substantially low since it is being affected by reproductive drought stress along with the blast disease. The genetic improvement of Varalu was done by introgressing a major yield QTL, qDTY_12.1, along with two major blast resistance genes i.e. Pi54 and Pi1 through marker-assisted backcross breeding. Both traits were transferred till BC₂ generation and intercrossing was followed to pyramid the two traits. Stringent foreground selection was carried out using linked markers as well as peak markers (RM28099, RM28130, RM511 and RM28163) for the targeted QTL (qDTY_12.1), RM206 for Pi54 and RM224 for Pi1. Extensive background selection was done using genome-wide SSR markers. Six best lines (MSM-36, MSM-49, MSM-53, MSM-57, MSM-60 and MSM-63) having qDTY_12.1 and two blast resistance genes in homozygous condition with recurrent parent genome of 95.0%-96.5% having minimal linkage drag of about 0.1 to 0.7 Mb were identified. These lines showed yield advantage under drought stress as well as irrigated conditions. MSM-36 showed better performance in the national coordinated trials conducted across India, which indicated that improved lines of Varalu expected to replace Varalu and may have an important role in sustaining rice production. The present study demonstrated the successful marker-assisted pyramiding strategy for introgression of genes/QTLs conferring biotic stress resistance and yield under abiotic stress in rice.     

粳稻野败型细胞质雄性不育恢复系SWR78的恢复基因定位

 利用野败(WA)型粳稻广亲和不育系苏秋A和广亲和广谱型恢复系SWR78配组,根据F2与BC1F1群体的育性分离情况,初步推测WA型苏秋A的育性恢复至少由3对基因控制。选取F2群体中无染色花粉植株,采用隐性基因组分析法进行恢复基因定位,将其中1个主效基因Rf4定位于第10染色体长臂上,与标记RM5629、RM5373、STS10 17和STS10 18分别相距0.17、0.03、0.03和0.07 cM。Rf4位于标记RM5373与STS10 17之间,两标记间的物理距离为78 kb。     

优质抗病水稻新品种粤标5号的选育及特征特性

粤标5号是应用分子标记辅助选择与系谱选育相结合的方法,将稻瘟病广谱抗病基因Pi2导入粤丰占中,经多代选择育成的优质抗病常规水稻新品种。该品种高抗稻瘟病,对广东的优势菌群中B、中C的抗性频率分别为93.8%~100%、92.9%~100%,丰产性较好,米质达国标优质1级,抗倒性强。2015年5月通过广东省农作物新品种审定。     

利用野生稻选育水稻恢复系研究

为了利用野生稻优良基因选育强优势的水稻恢复系,从野生稻资源中筛选出抗性强、具有较好农艺性状且恢复能力强的野生稻,与传统优良恢复系远缘杂交,后代适当复交,采用“大群体、多世代”的系谱法处理杂交后代。经过17 a（1980—1997）的选育,获得了测25、测253、测258、测1012等4个优良恢复系。至2006年,用这些恢复系配组育成的14个杂交稻组合通过了省级以上品种审定,累计推广面积达693.3万hm^2。