Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing

BACKGROUND: Persimmon (Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros. In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. RESULTS: Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. CONCLUSIONS: Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions.

Genotyping-by-sequencing based single nucleotide polymorphisms enabled Kompetitive Allele Specific PCR marker development in mutant Rubus genotypes

Background: Rubus is an economically important fruit crop across the globe. Recently, several Rubus mutant genotypes with improved agronomic traits have been developed using gamma ray irradiation. This study investigated genetic diversity and variations in Rubus mutant genotypes using single nucleotide polymorphism (SNP) markers generated from genotyping-by-sequencing (GBS) analysis. A GBS library of 14 Rubus genotypes, consisting of seven boysenberry mutant lines, four blackberry mutant lines, and three original varieties, were sequenced on the Illumina Hiseq2000 platform. A set of SNPs were analyzed by Kompetitive Allele Specific PCR (KASP) assay in order to discriminate the Rubus genotypes. Results: A total of 50,831,040 (86.4%) reads of clean data were generated, and the trimmed length ranged from 116,380,840 to 509,806,521 bp, with an average of 228,087,333 bp per line. A total of 19,634 high-quality SNPs were detected, which contained 11,328 homozygous SNPs and 8306 heterozygous SNPs. A set of 1504 SNPs was used to perform a phylogenetic analysis, which showed that there were clear differences among the Rubus genotypes based on their origin. A total of 25 SNPs were used for the KASP assays, of which six KASP primer sets were successfully distinguished among the Rubus genotypes. Conclusions: This study demonstrated that the SNP and KASP method is an economically efficient tool for mutant screening in Rubus breeding programs.

misMM: An Integrated Pipeline for Misassembly Detection Using Genotyping-by-Sequencing and Its Validation with BAC End Library Sequences and Gene Synteny

As next-generation sequencing technologies have advanced, enormous amounts of whole-genome sequence information in various species have been released. However, it is still difficult to assemble the whole genome precisely, due to inherent limitations of short-read sequencing technologies. In particular, the complexities of plants are incomparable to those of microorganisms or animals because of whole-genome duplications, repeat insertions, and Numt insertions, etc. In this study, we describe a new method for detecting misassembly sequence regions of Brassica rapa with genotyping-by-sequencing, followed by MadMapper clustering. The misassembly candidate regions were cross-checked with BAC clone paired-ends library sequences that have been mapped to the reference genome. The results were further verified with gene synteny relations between Brassica rapa and Arabidopsis thaliana. We conclude that this method will help detect misassembly regions and be applicable to incompletely assembled reference genomes from a variety of species.