Development and utilization of molecular markers for resistance of peach (Prunus persica) rootstocks to southern root-knot nematodes

Abstract: 【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 Genome V2.0. The candidate genes were amplified by PCR in five resistant germplasm Nemaguard, Okinawa, 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 sequenced by agarose gel electrophoresis. The hybrid F2 population of disease-resistant germplasm Tsukuba 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 throughprevious 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 materials. 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 Okinawa 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, aggregated near the X axis, and the genotype is--/--. The detection results of KASP marker in F2 population 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 materials with target bands were resistant (A1), 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 conformity with separation phenomenon. The phenotypic survey of F2 population showed that the ratio of rootless nodules to rootless nodules was 147:53. Based on the phenotypic investigation results of F2 population 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 resistant materials was 66.5%, and the total coincidence rate was 52.0%. Among the three molecular markers 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 molecular marker developed by predecessors. The development of this marker improves the selection efficiency of resistant varieties and provides resources for accelerating molecular breeding.