基于SLAF-Seq技术的辣椒抗炭疽病QTL定位分析及SSR分子标记开发

QTL localization analysis and SSR molecular marker develop⁃ ment of pepper resistance to anthracnose by SLAF-Seq technology

  • 摘要: 【目的】 利用SLAF-Seq技术对辣椒抗炭疽病QTL进行定位分析,开发与炭疽病抗性QTL紧密连锁的分子标记,为辣椒抗炭疽病分子标记辅助选择育种提供理论参考。【方法】 以抗病材料PPTC26-3-1-1-1-1-1和感病材料1287为亲本构建F2代群体,并采用SLAF-Seq技术进行分子标记开发,构建高密图遗传连锁图谱。结合表型数据,利用复合区间作图法(CIM)对辣椒果实转色期的炭疽病抗性进行QTL分析。利用Microsatellite(MISA)在定位区间识别SSR位点。【结果】 从亲本和子代样品共获得423351627条Reads,其中,来自PPTC26-3-1-1-1-1-1和1287分别为8130516和6621396条,F2代群体120个子代样品的平均Reads数量为34049987个。构建包含4671个SNP分子标记、分布于12个连锁群、总图距为1567.53 cM、平均图距为0.34 cM的辣椒高密度遗传图谱,检测到1个炭疽病抗性相关的QTL(CaR10.1),位于10号连锁群上187930592~189766111 bp物理区间内。共有23个基因定位在关联区域内,根据基因功能注释,其中1个基因编码假定晚疫病抗性蛋白同源物R1B-23异构体X2,1个基因编码PPR蛋白,可能与辣椒果实转色期炭疽病抗性相关。在关联到的QTL区间内开发出SSR分子标记T482,在F2代群体中的验证结果显示,T482引物在F2代群体单株中扩增结果与表型鉴定结果一致。F2代群体抗病和中抗单株中有53株具有抗病亲本的条带,有25株同时具有抗病亲本和感病亲本的条带;F2代群体感病单株中,有42株具有感病亲本的条带。【结论】 构建高密度遗传连锁图谱,将辣椒果实转色期炭疽病抗性定位于10号连锁群,在关联区间开发出SSR分子标记T482,可初步鉴别辣椒果实转色期炭疽病抗性,同时结合针刺接种法鉴定,可提高鉴定的准确率。

     

    Abstract: 【Objective】 The QTL localization analysis for resistance to anthracnose of pepper was analyzed by SLAFSeq technology,and the molecular markers closely linked with the QTL for resistance to anthracnose were developed to provide theoretical reference for molecular marker assisted selection breeding for resistance to anthracnose of pepper.【Method】The F2 generation population was constructed with the resistant material PPTC26-3-1-1-1-1-1 and susceptible material 1287 as the parents,and the molecular marker was developed by SLAF-Seq technology,and the high-density ge‐ netic linkage map was constructed,combined with the phenotypic data. QTL analysis was performed for anthracnose re‐ sistance of pepper fruit during the color transition stage by composite interval mapping(CIM),and SSR loci were identi‐ fied by Microsatellite(MISA). 【Result】 A total of 423351627 reads were obtained from parent and offspring samples,of which 8130516 reads were from PPTC26-3-1-1-1-1-1 and 6621396 reads were from 1287. The average number of reads from 120 offspring samples of F2 generation was 34049987. A high-density genetic map of pepper was constructed,which contained 4671 SNP molecular markers distributed in 12 linked groups with a total map distance of 1567.53 cM and an average map distance of 0.34 cM. One QTL(CaR10.1)related to anthracnose resistance was detected. It was located in the physical range of 187930592-189766111 bp on chain group number 10. A total of 23 genes were located in the asso‐ ciated region. According to the gene function annotation,1 gene encoded the isoform X2 of the presumed late blight resis‐ tance protein homolog R1B-23,and 1 gene encoded PPR protein,which might be related to the resistance to anthracnose during the color transition stage of pepper fruit. SSR marker T482 was developed within the associated QTL interval,and verification in F2 population showed that the bands amplified by T482 primer in F2 population individual were consistent with phenotypic identification. In the F2 generation,53 strains had bands of resistant parents and 25 strains had bands of both resistant and susceptible parents. In F2 generation infected individuals,42 strains contained bands of infected parents. 【Conclusion】 A high-density genetic linkage map is constructed. The resistance to anthracnose during the color transition stage of pepper fruits is localized on chain group number 10,and an SSR molecular marker T482 is developed in the asso‐ ciated interval. The results can be used to identify the resistance to anthracnose of pepper fruit during color transition stage,and can be combined with needle inoculation method to improve the identification accuracy.

     

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