Development and characterization of KASP makers based on a whole genome in a hybrid population of Shine Muscat × Suffolk Red

【Objective】Grape is a perennial twining vine of the genus Vitis in the family Vitaceae. Crossbreeding is a conventional and main method of grape breeding. Crossbreeding can produce favorable genetic benefits, and the offsprings can have the excellent characteristics of both parents.The development of modern molecular biology techniques has made it possible to use molecular markers closely related to specific traits for parent selection or pre- selection of hybrid offspring, thereby improving breeding efficiency. The study aimed to develop Kompetitive Allele Specific PCR (KASP) markers for grapevine leaf and fruit traits, and provide technical support for marker-assisted breeding in grapevine. 【Methods】Using Shine Muscat (Vitis vinifera × V. labrusca) and Suffolk Red (V. vinifera×V. labrusca) and their 150 hybrid progeny as materials, we collected leaves and mature fruits, and performed whole genome resequencing of PE150 using the Illumina platform, and analyzed the genome of Shine Muscat and Suffolk Red through population SNP detection, core SNP screening and KASP marker- assisted breeding. The trait association analysis was carried out by combining phenotypic data. The young leaves of Shine Muscat and Suffolk Red and their 150 hybrids were used to extract DNA from the young leaves on the shoots by conventional CTAB method, and the quality and concentration of DNA were detected. The crape leaf samples were collected from July to August in 2023—2024. The leaf length and leaf width were measured by vernier caliper, and the leaf area was calculated. The weight of single fruit was measured by electronic balance, and the transverse and longitudinal diameters of fruit were measured by vernier caliper. The hardness of the fruit was measured by CT-3 texture analyzer, and the central position of the fruit was placed directly below the probe. The contents of total phenols and flavonoids were determined by Cao Jiankang's method. The sugar composition and content in the fruit were determined according to the method of Komatsu et al. by liquid chromatography. Whole-genome resequencing data were processed through quality control, reference genome alignment, variant detection to identify SNP and develop core KASP makers.【Results】The group genome resequencing of Shine Muscat and Suffolk Red and their 150 hybrid offsprings was performed by sequencing platform, and a total of 819 Gb high-quality bases were obtained. Among them, the high-quality bases obtained by Shine Muscat were 10.25 Gb, the high-quality bases obtained by Suffolk Red were 10.02 Gb, and the total number of high-quality bases obtained by 150 hybrids was 799 Gb. The comparison rates for the parents and hybrid offspring were 97.79%, 96.79%, and 97.25%, respectively. A total of 432 926 SNPs were identified. Through principal component analysis, the maximum principal component was 10.39% . It can be seen that no principal component can clearly distinguish the hybrid offspring.The SNPS were filtered, and 74 core SNPs were attained. The missing rates of core SNPs ranged from 0.01 to 0.055; most of the minimal allele frequency (mAF) ranged from 0.15 to 0.19; most of polymorphism information content (PIC) ranged from 0.260 to 0.295; and the SNP heterozygosity rate ranged from 0.275 to 0.300. The 19 KASP makers from 43 samples were develop. The heterozygosity rate of 0.295- 0.300 was 73.7%, the missing rate of 0.048-0.055 was 52.6%, the genetic diversity indices ranged from 0.250 to 0.287 and PIC ranged from 0.260 to 0.295. The KEGG enrichment analysis for KASP makers revealed that flavone and flavonol biosynthesi was the most significant pathway. The others were fructose and mannose metabolism, pentose and glucuronic acid conversion, and plant-pathogen interaction. These pathways could provide reference for the study of fructose, glucose content and total phenol, flavonoid content and other related traits.The phenotypic and physiological indexes of the 11 traits related to the leaves and fruits of the parents and hybrids were analyzed by SPSS software. The results showed that the parents had differences in leaf area, fruit vertical diameter, grain weight, hardness, fructose, glucose and other traits. The variation range of its hybrid offspring was large, and the trait separation was obvious. The coefficient of variation of the leaf area was the largest, followed by the total phenol and grain weight, and the lowest was fructose. Through the Shapiro-Wilk test of the traits of the hybrid offspring, the leaf length and width, fruit grain weight, fructose and glucose all showed normal distribution. The correlation analysis between 43 phenotypic traits and KASP genotypes identified 14 makers associated with the leaf and fruit characteristics.【Conclusion】Through whole-genome resequencing of Shine Muscat, Suffolk Red and their 150 hybrid progenies, a total of 432 926 SNP loci were detected. After filtering based on depth, completeness and genetic parameters, 74 core SNP loci were obtained, and 19 KASP markers were successfully developed. Combined with association analysis of the leaf and fruit traits, 14 functional markers were screened out, among them 4 were related to the leaf morphology, 2 were associated with the fruit size, 4 were involved in the fructose/glucose metabolism, and 3 were related to the total phenol/flavonoid synthesis. The KEGG analysis showed that the genes where these markers were located were significantly enriched in the flavonoid and flavonol biosynthesis pathways (P＜0.01), and this would provide molecular targets for the study of the fruit color and quality regulation. The KASP markers developed in this study could be used for early trait selection in grape breeding. The 14 KASP markers developed in this study could be used for molecular selection of the grape leaf and fruit traits, and the associated pathways such as flavonoid synthesis would provide a theoretical reference for quality breeding.