Development of a core SNP arrays based on the KASP method for molecular breeding of rice

The development and utilization of genetic markers play a pivotal role in marker-assisted breeding of rice cultivars during pyramiding of valuable genes. Among molecular markers, SNPs have become the most promising due to their wide distribution within genomes and suitability for high -throughput automated genotyping. Although metadata of SNPs have been identified via next generation sequencing in rice, a large gap between the development of SNP markers and the application in breeding still exists. To promote the application of SNP markers based on the KASP (Kompetitive Allele-Specific PCR) method in rice breeding, a set of core SNP arrays was built via the screening of SNP databases and literature resources based on the KASP method.

Five hundred and ninety six SNPs classified into eight subsets including quality control, indica-indica variation, highly polymorphic, functional genes, key genes targeting sites, gene cloned region, important trait associated and gap filling sites were chosen to design KASP primers and 565 out of them were successfully designed, and the assay design success rate was 94.8%. Finally, 467 out of the 565 successfully-designed SNPs can display diversity at the loci were used to develop a set of core SNP arrays. To evaluate the application value of the core SNP markers in rice breeding, 481 rice germplasms were genotyped with three functional KASP markers designed from the sequences of GBSSI, SSIIa, and Badh2 from the core SNP arrays for estimation of their grain quality performance. Eighteen rice lines, including Xiangwanxian 13, Basmati 370, Ruanhua A, and PR 33319-9-1-1-5-3-5-4-1, harbor all three favorable alleles. The core KASP arrays were also used for rice germplasm assessment, genetic diversity and population evaluation. Four hundred and eighty-one rice germplasms were divided into 3 groups: POP1, POP2 and POP3. POP1 and POP2 were indica rice subgroups consisting of 263 and 186 rice germplasms, respectively. POP3 was a japonica rice subgroup consisting of 32 rice germplasms. The average F_ST value for the three subgroups was 0.3501; the F_ST value of POP1 and POP3 was the largest (0.5482), while that of POP1 and POP2 was the smallest (0.0721). The results showed that the genetic distance between the japonica and indica rice subspecies was large, indicating that the core SNP markers were effective at discriminating the population structure of the germplasms. Finally, the core KASP arrays were used for association analysis with milled grain traits. A total of 31 KASP markers were significantly associated (P < 0.01) with ML and the LWR. Among the 31 markers, 13 were developed based on cloned genes or on identified loci related to yield traits. Notably, several KASP markers associated with grain quality were also found to be associated with brown planthopper resistance or green leafhopper resistance simultaneously.

The core KASP arrays developed in our study were efficient and versatile for rice germplasm assessment, genetic diversity and population evaluation and are valuable for promoting SNP molecular breeding in rice. Our study demonstrated that useful assays combined with molecular breeding can be exploited for important economic trait improvement in rice breeding.