Bulked Segregant Analysis to Detect QTL Related to Heat Tolerance in Rice(Oryza sativa L.) Using SSR Markers

The study was undertaken to assess the genetic effect of quantitative trait loci （QTLs） conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats （SSR）. The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis （SMA）. The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

Quantitative trait loci detection of E dwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese fl ounder( Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. o livaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis(BSA) and quantitative trait loci(QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333(♀: F0768 ×♂: F0915)(Nomenclature rule: F+year+family number) were used to detect simple sequence repeats(SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers(Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group(LG)-1 exhibited a signifi cant difference between DNA, pooled/bulked from the resistant and susceptible groups( P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of signifi cance in LG1, where 17 and 18 SSR markers were identifi ed, respectively. Each model found three resistance-related QTLs by composite interval mapping(CIM). These six QTLs, designated q E1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, q E-2 and q E-4, were located at the 66.7 c M region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese fl ounder in the future.

Evaluation and Bulked Segregant Analysis of Major Yield QTL qtl12.1 Introgressed into Indigenous Elite Line for Low Water Availability under Water Stress

Near isogenic lines carrying large-effect QTL （qtl2.1）, which has a consistent influence on grain yield under upland drought stress conditions in a wide range of environments, were evaluated under water stress in the fields. The line which gave higher yield under drought was crossed with a local elite line, PMK3, and forwarded to F2：3 generation. Significant variation was found among the F2：3 lines for agronomic traits under water stress in the fields. Low to high broad sense heritability （H） for investigated traits was also found. Water stress indicators such as leaf rolling and leaf drying were negatively correlated with plant height, biomass and grain yield under stress. Bulked segregant analysis （BSA） was performed with the markers in the vicinity of qUl2.1, and RM27933 was found to be segregated perfectly well in individual components of drought resistant and drought susceptible bulks which were bulked based on yield under water stress among F2：3 lines. Hence, this simple and breeder friendly marker, RM27933, may be useful as a potentially valuable candidate marker for the transfer of the QTL qtl12.1 in the regional breeding program. Bioinformatic analysis of the DNA sequence of the qtl12.1 region was also done to identify and analyze positional candidate genes associated with this QTL and to ascertain the putative molecular basis of qUl2.1.

Tagging of Brown Planthopper Resistance Genes in F_2s of IR50 × Ptb33 of Rice by Using Bulked Segregant Analysis

Brown planthopper （Nilaparvata lugens Stal） is one of the most damaging pests causing hopper burn in rice, and thereby reducing the productivity and also the quality of the product. The effective management strategy to control this pest is the identification and transfer of desirable genes to local rice cultivars. The most important approach for developing resistant cultivars is the identification of markers, which can help in marker-assisted selection of more durable resistant genotype. The susceptible parent IR50 and the resistant parent Ptb33, and their F2 populations were used in bulked segregant analysis for identification of resistant genes with random amplified polymorphic DNA marker （RAPD） primers. The primers OPC7 and OPAG14 showed both dominant and susceptible specific banding pattern so called co-dominant markers. Moreover, OPC7697 and OPAG14680 showed resistant specific bands and thus being in coupling phase, whereas OPC7846 and OPAG14650 showed susceptible specific genotypic bands in bulked segregant analysis. Therefore, the coupling phase markers, OPC7697 and OPAG14680, are considered to be more useful in marker-assisted selection of rice genotypes in crop improvement.

RNA-Seq and Bulked Segregant Analysis of Genes Related to High Growth in <i>Ginkgo biloba</i>Half-Sibling Families

The lifetime of G. biloba is very long, and its growth is relatively slow. However, little is known about growth-related genes in this species. We combined mRNA sequencing (RNA-Seq) with bulked segregant analysis (BSA) to fine map significant agronomic trait genes by developing polymorphism molecular markers at the transcriptome level. In this study, transcriptome sequencing of high growth (GD) and low growth (BD) samples of G. biloba half-sib families was performed. After assembling the clean reads, 601 differential expression genes were detected and 513 of them were assigned functional annotations. Single nucleotide polymorphism (SNP) analysis identified SNPs associated with 119 genes in the GD and BD groups;58 of these genes were annotated. Two Homeobox-leucine zipper protein genes were up-regulated in the GD group compared with the BD group;therefore, these are very likely related to high growth of G. biloba. This study provides molecular level data that could be used for seed selection of high growth G. biloba half-sib families for future breeding programs.

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 A (high cocoon shell ratio) and C.Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis(BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis, DNA from the parents (B20 A, C.Nichi), F 1 and F 2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F 1, F 2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

Fine-mapping and primary analysis of candidate genes associated with seed coat color in mung bean(Vigna radiata L.)

Seed coat color affects the appearance and commodity quality of mung beans(Vigna radiata L.).The substances that affect mung bean seed coat color are mainly flavonoids,which have important medicinal value.Mapping the seed coat color gene in mung beans would facilitate the development of new varieties and improve their value.In this study,an F2 mapping population consisting of 546 plants was constructed using Jilv9(black seed coat)and BIS9805(green seed coat).Using bulk segregated analysis(BSA)sequencing and kompetitive allele-specific PCR(KASP)markers,the candidate region related to seed coat color was finally narrowed to 0.66 Mb on chromosome(Chr.)4 and included eight candidate genes.Combined transcriptome and metabolome analyses showed that three of the eight candidate genes(LOC106758748,LOC106758747,and LOC106759075)were differentially expressed,which may have caused the differences in flavonoid metabolite content between Jilv9 and BIS9805.These findings can provide a research basis for cloning the genes related to seed coat color and accelerate molecular markerassisted selection breeding in mung beans.

Discovering Candidate Chromosomal Regions Linked to Kernel Size-Related Traits via QTL Mapping and Bulked Sample Analysis in Maize

Kernel size-related traits,including kernel length,kernel width,and kernel thickness,are critical components in determining yield and kernel quality in maize(Zea mays L.).Dissecting the phenotypic characteristics of these traits,and discovering the candidate chromosomal regions for these traits,are of potential importance for maize yield and quality improvement.In this study,a total of 139 F2:3 family lines derived from EHel and B73,a distinct line with extremely low ear height(EHel),was used for phenotyping and QTL mapping of three kernel sizerelated traits,including 10-kernel length(KL),10-kernel width(KWid),and 10-kernel thickness(KT).The results showed that only one QTL for KWid,i.e.,qKWid9 on Chr9,with a phenotypic variation explained(PVE)of 13.4%was detected between SNPs of AX-86298371 and AX-86298372,while no QTLs were detected for KL and KT across all 10 chromosomes.Four bulked groups of family lines,i.e.,Groups I to IV,were constructed with F2:3 family lines according to the phenotypic comparisons of KWid between EHel and B73.Among these four groups,Group I possessed a significantly lower KWid than EHel(P=0.0455),Group II was similar to EHel(P=0.34),while both Group III and Group IV were statistically higher than EHel(P<0.05).Besides,except Group IV exhibited a similar KWid to B73(P=0.11),KWid of Groups I to III were statistically lower than B73(P<0.00).By comparing the bulked genotypes of the four groups to EHel and B73,a stable chromosomal region on Chr9 between SNPs of AX-86298372 to AX-86263154,entirely covered by qKWid9,was identified to link KWid with the positive allele of increasing phenotypic effect to KWid from B73,similar to that of qKWid9.A large amount of enzyme activity and macromolecule binding-related genes were annotated within this chromosomal region,suggesting qKWid9 as a potential QTL for KWid in maize.

Integrated genomic and transcriptomic analysis reveals genes associated with plant height of foxtail millet

Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.

DeepBSA: A deep-learning algorithm improves bulked segregant analysis for dissecting complex traits

Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for BSA have not been systematically evaluated and are complex and fallible to operate.We developed a BSA method driven by deep learning,DeepBSA,for QTL mapping and functional gene cloning.DeepBSA is compatible with a variable number of bulked pools and performed well with various simulated and real datasets in both animals and plants.DeepBSA outperformed all other algorithms when comparing absolute bias and signal-to-noise ratio.Moreover,we applied DeepBSA to an F2 segregating maize population of 7160 individuals and uncovered five candidate QTLs,including three well-known plant-height genes.Finally,we developed a user-friendly graphical user interface for DeepBSA,by integrating five widely used BSA algorithms and our two newly developed algorithms,that is easy to operate and can quickly map QTLs and functional genes.The DeepBSA software is freely available to noncommercial users at http://zeasystemsbio.hzau.edu.cn/tools.html and https://github.com/lizhao007/DeepBSA.

Mapping of wheat stripe rust resistance gene Yr041133 by BSR-Seq analysis

Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing(BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 c M genetic interval on the chromosome arm 7 BL that corresponded to a 0.8 Mb physical interval(608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.

Strategy for the mapping of interactive genes using bulked segregant analysis method and Mapmaker/Exp software

A qualitative trait is usually controlled by a single gene, but it may be sometimes controlled by two or even more genes. This phenomenon is called gene interaction. Rapidly searching for linked mo- lecular markers via bulked segregant analysis (BSA) and then constructing regional linkage map with Mapmaker/Exp has become a common approach to mapping single major genes. However, methods and computer programs developed for mapping single major genes cannot be simply applied to interactive genes because the genetic patterns of gene interac- tions are quite different from that of single-gene in- heritance. Up to now, experimental methods for quickly screening molecular markers linked to inter- active genes and statistical methods and corre- sponding computer softwares for simultaneously analyzing the linkage relationships of multiple mo- lecular markers to an interactive gene have not been available. To solve this problem, in this paper, we propose a strategy for mapping interactive genes using BSA and Mapmaker/Exp. We demonstrate that all interactive genes can be mapped by the 'BSA + Mapmaker/Exp' strategy using F2 generation (in a few cases, F3 generation is also needed). As BSA and Mapmaker/Exp have been broadly used in gene mapping studies and are well known by many re- searchers, the strategies proposed in this paper will be useful for practical researches.

利用东乡普通野生稻染色体片段置换系定位水稻苗期耐盐性QTL

本实验室前期以东乡普通野生稻和日本晴为亲本创制了强耐盐染色体片段置换系CSSL91,本研究将其与日本晴和强耐盐种质Pokkati比较,结果显示CSSL91耐盐性与Pokkali相当。以CSSL91与日本晴构建的F2:3群体为试验材料,日本晴和CSSL91为对照,以耐盐等级和幼苗存活率为指标。结果表明2个指标均成正态分布,QTL连锁定位分析共检测到5个耐盐相关QTL,分别分布于第4、9、10号染色体上,LOD值介于2.95~3.97,表型贡献率为9.83%~18.48%;其中耐盐等级QTL-q ST4的表型贡献率最高,其定位在第4号染色体DX-C4-1~DX-S4-16标记间。分离群体分组分析法(BSA,bulked segregation analysis)分析检测到第4号染色体0~5.0 Mb区间有一个超过阈值的QTL,该区间与QTL-q ST4重合,QTL连锁分析方法和BSA方法均在第4号染色体的0~5.0 Mb区间定位到耐盐等级QTL,说明QTL-qST4是可靠的耐盐位点;耐盐等级QTL-qST4-1和幼苗存活率QTLqSSR4均定位在第4号染色体DX-C4-12和DX-C4-13标记间,LOD值分别为3.36和3.92,表型贡献率分别为13.97%和9.49%;在第9号、10号染色体还定位到两个耐盐等级QTL-qST9和QTL-qST10;其中QTL-qST4-1、QTL-qSSR4和QTL-qST10是本研究新定位的耐盐性QTL。本研究结果将为水稻耐盐性相关基因克隆和分子标记辅助改良水稻品种的耐盐性奠定基础。

玉米籽粒发育突变体emp35的表型分析与基因定位

为解析玉米籽粒形成的遗传基础,探究Emp35基因在玉米籽粒发育中的作用,对籽粒缺陷突变体empty pericarp35(emp35)进行表型鉴定、胚乳细胞显微观察、胚乳贮藏物质含量测定及图位克隆。结果显示:突变体籽粒发育缓慢,明显小于同期发育的正常籽粒,成熟籽粒干瘪呈空皮状;胚乳细胞显微观察发现emp35的胚和胚乳发育严重滞后,胚乳细胞中线粒体结构异常;淀粉和蛋白质积累减少;F_(2)代分离果穗上正常籽粒与发育缺陷籽粒呈3∶1分离,表明籽粒缺陷表型由单个隐性核基因突变所致。采用集团分离分析法(bulked segregant analysis,BSA)将Emp35定位于第8染色体127.90~163.36 Mb区间,在该区间内开发了4个InDel标记,连锁作图将Emp35精细定位于139571117~146176858区间。

控制玉米株高基因PHR1的基因克隆

株高属于玉米理想株型育种的一个重要指标,不但影响玉米机械化收获,更与玉米的倒伏性和生物产量密切相关。本研究以低剂量快中子(4.19 Gy)辐照诱变玉米自交系KWS39获得的矮秆低穗位突变体为研究对象,该突变体命名为plant height reducing mutant-1(phr-1),开展了表型性状的田间调查分析,并利用phr-1×B73获得的F2分离群体,借助极端性状混池测序分析法(BSA-seq)及目标区段重组交换鉴定的方法,基于B73参考基因组对目标区段内的基因进行挖掘和功能注释,定位候选基因。研究结果表明,在1号染色体Bin1.06区间可能存在变异位点,进而利用大的分离群体结合目标区段多态性标记开发,将目标区段精细定位分子标记到Umc1122和Umc1583a两个标记之间约600 kb区间,该区段内存在一个控制株高的已知基因Brachytic2(BR2),BR2编码一个调控玉米茎秆中生长素极性运输的糖蛋白。候选基因测序结果表明,phr-1是BR2基因在第4个外显子处插入了165 bp的序列,导致第547位氨基酸变为终止子,蛋白翻译提前终止。phr-1的基因突变位点和变异方式与已报道的br2-1单个碱基发生变异位点完全不同,通过等位杂交实验证明了phr-1突变体就是br2-1的一个新等位突变体,候选基因就是BR2基因。本研究为玉米BR2基因在玉米株高遗传改良中提供了新的种质资源。

四倍体小麦成株抗白粉病基因定位

四倍体小麦是普通小麦的祖先种,也是重要的粮食作物。发掘四倍体小麦抗病种质并鉴定其携带的抗病基因,旨在为小麦新品种选育提供新抗源。TDI-1是栽培二粒小麦,在多年的田间种植过程中表现出对白粉病免疫的表型。为了确定TDI-1携带的抗病基因,为小麦白粉病抗性遗传提供理论依据,首先将其与表现为感白粉病表型的硬粒小麦TDU-1进行杂交并构建了遗传群体,然后对亲本及其杂交F_(1)、F_(2)、F_(2:3)群体进行了白粉菌小种E09接种试验和抗病性分析,最后利用混合分离群体分析法(BSA)对抗白粉病基因进行了定位。结果表明,TDI-1在苗期对E09表现为感病,在成株期对E09表现为抗病;TDI-1和TDU-1的F_(1)植株在成株期对E09表现为抗病;F_(2)群体中,表现为抗病的单株数和感病的单株数的比例符合3∶1(χ^(2)_(3∶1)=0.11,P=0.74);F_(2∶3)家系中,纯合抗病、抗病性分离、纯合感病的家系数目之比符合1∶2∶1(χ^(2)_(1∶2∶1)=0.47,P=0.79),表明TDI-1成株期白粉病抗性由1个显性基因控制,暂将其命名为PmTDI-1。对TDI-1和TDU-1及其杂交后代F_(2)群体进行分子标记筛选,发现4个位于2A染色体短臂上的分子标记Xwmc407、NRM-2AS29、NRM-2AS45和NRM-2AS84与PmTDI-1紧密连锁,其中,NRM-2AS45和NRM-2AS84位于两侧,遗传距离分别为1.8,4.6 cM。由此,将成株期抗白粉病基因PmTDI-1初步定位于2A染色体短臂上。研究结果显示,从四倍体小麦TDI-1中鉴定到1个新的显性成株抗白粉病基因PmTDI-1。

SSR-BSA技术对乌鳢性别差异标记的初步筛选

采用SSR结合BSA技术对1个家系96个乌鳢个体(雌雄各48个)进行性别差异标记的筛选。首先构建了雌雄基因池(各24个个体),然后用140对微卫星引物对其进行扫描,发现3对引物(HLJWL17,HLJWL59,HLJWL70)在雌雄基因池间扩增出差异条带,且都出现在雌性基因池中。用构建基因池的48个个体对3对引物进行第一轮单个体验证,HLJWL17和HLJWL70在基因池中扩增出的差异条带仅在极个别个体中出现,而HLJWL59的差异条带在绝大多数的雌性个体中被成功扩增出来,雄性个体皆无。用其余的48个个体对HLJWL59进行第二轮单个体验证,得到同样结果。对HLJWL59在8个雌性个体中扩增出来的差异基因片段进行克隆并测序。将8个个体的测序结果用Vector 8.0进行多序列比对,证实各个个体得到的条带是同一序列,该序列长度为243 bp,以TGC为重复单位,GC含量为51%。通过BLASTn比对,发现在GenBank数据库中无同源性序列存在。经统计,此差异标记在雌性乌鳢中出现的概率为62.5%,即该标记对该家系乌鳢性别的正确判别率为81.25%。

利用BSA法检测水稻条纹叶枯病高效应抗性位点

通过前期对江苏水稻地方品种抗条纹叶枯病资源的筛选获得的一份高抗条纹叶枯病水稻品种旱稻,配制其与条纹叶枯病感病品种武育粳3号杂交组合,采用田间自然接种鉴定方法,以病情指数为表型值,对141个F2单株的F2:3株系进行了条纹叶枯病抗性鉴定。同时利用群体分离分析法(Bulked segregant analysis,BSA法)对抗/感DNA池分析发现第11染色体SSR标记RM209、RM21与条纹叶枯病抗性有明显的连锁关系。利用基于完备复合区间作图方法的QTL检测软件(QTL IciMapping V3.2)在第11染色体上检测到一个条纹叶枯病抗性相关QTL,位于SSR标记RM209和RM21之间,LOD值为13.25,可解释表型变异38.39%,加性效应为负值,表明该数量性状基因座对水稻条纹叶枯病的抗性来自抗病亲本旱稻。

基于BSA-SSR技术初步筛选与叶用芥菜莱菔硫烷含量相关的分子标记

为获得与叶用芥菜莱菔硫烷含量相关的分子标记,本试验采用高效液相色谱法(HPLC)测定了80个叶用芥菜F2单株的莱菔硫烷含量,构建F2混合分离群体(BSA)基因池,利用50对引物进行PCR扩增。筛选得到2个显性标记SF12和SF28,SF12在低莱菔硫烷含量亲本圆叶春菜、低莱菔硫烷含量池(L)和低莱菔硫烷含量池单株中扩增出大小约250 bp的条带,而SF28在高莱菔硫烷含量亲本太乐四季青、高莱菔硫烷含量池(H)和高莱菔硫烷含量池单株中扩增出大小约250 bp的条带;并且通过F2部分单株验证了SF12、SF28与叶用芥菜莱菔硫烷含量具有相关性。

利用BSA-Seq方法鉴定谷丰B抗稻瘟病基因

【目的】挖掘和鉴定谷丰B稻瘟病抗性基因,了解谷丰B稻瘟病抗性遗传模式。【方法】以谷丰B和日本晴杂交获得F1和F2代遗传群体,接种稻瘟菌不同生理小种并分析抗病遗传模式;在F2群体中挑选极端抗/感单株构建DNA混合池,利用群体分离分析法(BSA)定位关联区域。【结果】谷丰B对KJ201、RB22、CHNOS、RB6、2Y838-1、501-3和IR16-1等菌株均表现高抗性,表明谷丰B基因组可能携带了广谱高抗稻瘟病基因。谷丰B和日本晴杂交,F1群体表现抗501-3和IR16-1,F2群体的抗病/感病分离比不符合3∶1,推测谷丰B基因组存在多个位点影响稻瘟病抗性。对F2群体的极端抗病、感病混合池及亲本DNA进行全基因组测序,鉴定了1756964个单核苷酸多态性(SNPs)标记。分析子代△SNP-index,定位到2个与抗病性显著关联区间,分别为Chr.6:10082-11397 kb和Chr.11:120-266 kb。其中,6号染色体的关联区间与Pi2/9抗病位点等位,区间内含有4006个SNPs和623个插入缺失(InDels)标记;11号染色体的关联区间含有752个SNPs和195个InDels标记。【结论】谷丰B对强致病力501-3菌株抗性可能是由第6号和11号染色体上的基因共同控制。研究结果为谷丰B抗性基因的精细定位及基因克隆奠定了基础,并为水稻抗稻瘟病分子标记辅助选择提供标记资源。