西瓜枯萎病生理小种1抗性QTL精细定位与InDel标记开发

【目的】通过QTL初定位检测栽培西瓜枯萎病生理小种1抗性的主效QTL,验证枯萎病抗性基因Fon-1的存在,结合亲本重测序信息开发紧密连锁易于检测的InDe1(insertion/deletion)分子标记,为西瓜枯萎病分子标记辅助育种提供技术支撑,并实现该QTL的精细定位,加速Fon-1的克隆和功能验证进程。【方法】以高抗枯萎病的栽培西瓜‘ZXG01478'和高感病的栽培西瓜‘14CB11'为亲本构建的F_2群体为试验材料,利用WinQTL cartographer 2.5软件基于复合区间作图法对枯萎病生理小种1抗性进行QTL定位。依据两个亲本材料进行高通量的重测序,并利用重测序信息,获取位于QTL置信区间的InDe1信息,利用自编的Per1程序提取参考基因组中插入缺失相应位置前后500 bp的序列,并利用Primer 5.0软件设计对应的InDe1引物对,开发InDe1标记。利用开发的InDe1标记进行精细定位(根据基因型鉴定结果找到群体中的交换单株,逐步缩小QTL区间)、图谱(利用JoinMap 4.0计算标记的连锁关系)和QTL的重新分析。并利用具有广泛代表性的130份不同抗性的西瓜种质资源的基因型和表型鉴定结果进行验证分析。【结果】F_2群体的病株率频率分布呈现明显的双峰分布且抗病和感病两种类型的株系分离比基本符合3:1的分离比(χ~2=0.52,P=0.47),表明西瓜枯萎病生理小种1抗性主要受一个主效QTL控制。F_2群体的QTL初定位在LG1鉴定到一个枯萎病抗性相关的主效QTL(fon1),其LOD峰值为26.05,解释80.18%的表型变异,置信区间对应的物理位置为1号染色体的193 333-2 775 577 bp。通过两个亲本重测序在QTL置信区间发现19个插入缺失片段大于20 bp的InDe1s,经引物设计与亲本筛选,获得理想引物12对,根据位于端点位置的插入缺失选取了6对InDe1引物利用F_2群体进行验证。初步的精细定位利用群体中的5个交换单株将QTL的置信区间锁定到InDe12_fon1的上游区域,连锁和QTL的重新分析结果显示新开发的一个分子标记InDe11_fon1出现在该QTL的峰值,LOD值为31.65,解释91.46%的表型变异。新开发的分子标记InDe11_fon1与已应用于枯萎病抗性研究的CAPS标记7716_fon在130份不同抗性的西瓜种质资源中的基因型鉴定结果完全一致,且基因型鉴定结果与田间抗病表型性状的符合率达70.8%。利用QTL峰值附近的SNP和InDe1标记与群体中的9个交换单株最终将fon1精细定位至246 kb的物理区间。【结论】主效QTL(fon1)验证了1号染色体上Fon-1的存在,并实现了精细定位。新开发的标记InDe11_fon1与Fon-1紧密连锁,且检测简单,成本低廉,能够更好的用于栽培西瓜枯萎病的分子标记辅助育种。

Fine-mapping of QTL and Development of InDel Markers for Fusarium oxysporum Race 1 Resistance in Watermelon

【Objective】In order to clone gene and provide technical supports for molecular assisted breeding for Fusarium oxysporum race 1 resistance in watermelon, combined QTL mapping and re-sequencing of parental lines, tightly linked InDel (insertion/deletion) markers were developed and finally the major QTL was fine mapped.【Method】Firstly, genome-wide QTL scanning for Fusarium oxysporum race 1 resistance in an F₂ segregating population derived from a cross between the cultivated resistance female parent ‘ZXG01478’ and the cultivated susceptible male parent ‘14CB11’ was performed using the composite interval mapping program of the WinQTL cartographer 2.5 software. Secondly, InDel markers were developed based on the InDel information on the QTL region by re-sequencing of parental lines. Finally, fine-mapping, genetic map and QTL analysis were re-performed using developed InDel markers. Moreover, a total of 130 watermelon germplasms with different Fusarium oxysporum race 1 resistances were used to perform validation analysis.【Result】In the F₂ population, the frequency distribution of susceptible plant rate deviated from normality and appeared to have a discontinuous bimodal pattern. Moreover, the ratio of resistance to susceptible to Fusarium oxysporum race 1 corresponded to the expected 3﹕1 segregation for a single-locus inheritance (c²=0.52，P=0.47). Preliminary QTL mapping only identified one QTL (fon1) for Fusarium oxysporum race 1 resistance on LG1, which showed peak LOD of 26.05 and could explain 80.18% of the phenotype variation. The confidence intervals of fon1 was 193 333-2 775 577 bp on chromosome 1 (physical map). A total of 19 InDels with length more than 20 bp were detected on QTL region by re-sequence analysis. Of these, 12 showed polymorphism between two parents. Six primer pairs were selected to genotype in the F₂ population. Using four recombinant lines in F₂ population, preliminary fine-mapping narrowed the QTL region to upstream of InDel2_fon1. QTL re-analysis showed that one of the new developed markers (InDel1_fon1) located on the peak QTL region, which showed peak LOD of 31.65 and could explain 91.46% of the phenotype variation. The genotype of InDel1_fon1 and 7716_fon were consistent with each other for all of the 130 watermelon germplasms, and there was a relative high coincidence rate (70.8%) of genotype and phenotype. Target QTL region was narrowed to physical distance of 246 kb using new developed InDel markers and flanking SNP markers. 【Conclusion】 Major QTL (fon1) confirmed the existence of Fusarium oxysporum race 1 resistance gene Fon-1 and was finely-mapped to a relative small region. One of the new developed markers, InDel1_fon1, was tightly linked to gene Fon-1, which could better applied in molecular assisted breeding for resistance to Fusarium wilt in cultivated watermelon.