[目的]南方根结线虫是威胁桃产业绿色发展的地下主要害虫,开发抗性分子标记,对抗性砧木分子育种具有重要意义.[方法]根据前人对桃砧木抗根结线虫的定位结果,在GDR网站peach genome V2.0查询定位区间的候选基因.在5个抗病、5个感病种质中扩增候选基因,并通过DNAMAN、IGV等软件对候选基因进行序列差异分析,开发抗南方根结线虫KASP分子标记,在抗性种质筑波3号与感性种质哈露红的杂交F2群体中对该分子标记进行验证,并与前人开发的SCAR和35bpindel抗南方根结线虫分子标记的准确性进行比较.[结果]KASP标记结果将基因型划分为3种,分别为抗性纯合型(A),抗性杂合型(B),感性纯合型(C),A∶B∶C=42∶94∶64,与表型符合率为88.5％;SCAR标记检测结果划分为2种,分别为抗病型(A1)和感病型(C1),A1∶C1=135∶65,与表型符合率为87.0％;35 bp indel分子标记分为3种类型,分别为抗性纯合型(A2),抗性杂合型(B2),感性纯合型(C2),A2∶B2∶C2=1∶154∶45,与表型符合率为52.0％.[结论]本研究中开发的KASP标记提高了分子标记选择准确率,对抗南方根结线虫分子育种具有重要意义.
Development and utilization of molecular markers for resistance of peach(Prunus persica)rootstocks to southern root-knot nematodes
[Objective]Meloidogyne incognita is an underground pest threatening the development of peach industry.It is of great significance to develop molecular markers for the resistance to the pest for breeding new rootstocks.[Methods]According to the mapping results of peach rootstocks published by the predecessors,the candidate genes in the mapping interval were queried in GDR website Peach Ge-nome V2.0.The candidate genes were amplified by PCR in five resistant germplasm Nemaguard,Oki-nawa,Tsukuba 2,Tsukuba 3 and Shouxingtao 1,and five susceptible germplasm Bailey,Kashi 1,Kashi 2,Harrow Blood and Siberian C.The target fragments of PCR products were purified,ligated and se-quenced by agarose gel electrophoresis.The hybrid F2 population of disease-resistant germplasm Tsuku-ba 3 and susceptible germplasm Harrow Blood was inoculated with M.incognita,and the phenotypes of the population were investigated three months later to verify the accuracy of the KASP marker,and compared with the accuracy of molecular markers developed by the predecessors for the resistance to Meloidogyne incognita in SCAR and 35 bp indel.[Results]Six candidate genes were found through previous studies,namely Prupe.2G055500,Prupe.2G055600,Prupe.2G055700,Prupe.2G055800,Prupe.2G055900 and Prupe.2G056000.Through sequence comparison,it was found that there were regular variations in the resistant and susceptible varieties of the gene Prupe.2G055500,and there was a 2 bp indel variation(Pp02:6 601 310 bp,G→GAT)in its intron,and at insertion existed in the resistant varieties,but not in the susceptible varieties.In addition,using IGV software,with v2.0.a1 version as the reference genome,the same results were found in 10 resequencing data of peach germplasm materi-als.A molecular marker for genotyping was developed by using the above mutation sites.Five resistant germplasm and five susceptible germplasm were detected by this marker.The results showed that FAM and HEX fluorescence signals were simultaneously detected in the resistant germplasm Nemaguard,and the signal point was red,and the genotype was AT/--;The signal points of resistant germplasm Oki-nawa Tsukuba2 Tsukuba3 Shouxingtao 1 are green,aggregated near the Y axis,and the genotype is AT/AT;The signals of sensitive germplasm Bailey Kashi 1 Kashi2 Harrow Blood Siberian C are blue,ag-gregated near the X axis,and the genotype is--/--.The detection results of KASP marker in F2 popula-tion divided the genotypes into three types,green fluorescence was homozygous for resistance(A),red fluorescence was heterozygous for resistance(B),blue fluorescence was homozygous for sensibility(C),and A∶B∶C=42∶94∶64 was close to 1∶2∶1,which was consistent with separation of mendelian law.The detection results of SCAR markers in F2 population were also divided into two types.The ma-terials with target bands were resistant(Al),and the materials without target bands were sensitive(C1),and A1∶C1=135∶65,which did not conform to separation phenomenon.Three bands can be amplified by 35 bp indel marker in F2 population.Taking Hong Gen Gan Su Tao 1 as control,one band in the same position is classified as A2,two corresponding bands in the same position are classified as B2,and no band in the same position is classified as C2,and A2∶B2∶C2=1∶154∶45,which is not in confor-mity with separation phenomenon.The phenotypic survey of F2 population showed that the ratio of root-less nodules to rootless nodules was 147:53.Based on the phenotypic investigation results of F2 popula-tion resistance to Meloidogyne incognita,the selection efficiency of three markers was evaluated.The results showed that the selection coincidence rate of KASP for resistant materials was 95.6％,that of susceptible materials was 73.4％,and the total coincidence rate was 88.5％.The selection coincidence rate of SCAR marked as resistant material is 94.8％,that of scar marked as sensitive material is 70.8％,and the total coincidence rate is 87.0％.The selection coincidence rate of 35 bp indel marked as resis-tant materials was 66.5％,and the total coincidence rate was 52.0％.Among the three molecular mark-ers for resistance to Meloidogyne incognita,the correct rate of KASP molecular marker was the highest,followed by SCAR marker,and the correct rate of 35 bp indel marker was the lowest.[Conclusion]Based on the mapping results of resistance genes of cultivated peaches to Meloidogyne incognita,this study developed a KASP molecular marker,which was verified in the F2 population.It was found that the KASP molecular marker developed in this study had the highest accuracy compared with the molec-ular marker developed by predecessors.The development of this marker improves the selection efficien-cy of resistant varieties and provides resources for accelerating molecular breeding.
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