RNA-Seq of Japanese barley cultivars

Genome sequences enable us to genotype genome wide polymorphisms among closely related genotypes. In barley, a highly-assembled genome sequences of six-row barley cv. Morex was available in 2017. Using this resource, we can map various markers physically on the genome. To construct new markers, whole genome resequencing is one of the best procedures. However, because of the large genome size and the high proportions of repetitive elements on the genome sequences, whole genome resequencing of multiple barley genotypes is not a practical solution. While SNP arrays were developed based on the limited donor genotypes, their application to the genotyping of barley germplasm other than these donor genotypes was less effective. Even though genotype by sequencing is the more effective method to detect random polymorphisms, frequent deficient alleles occurred in the alignment of sequenced genotypes. Here, we developed a de novo genotyping procedure using RNA-Seq in the germplasm used in Japanese barley breeding programs. By using RNAs from seedling shoot, seedling root and immature spike samples, we mapped NGS data on the transcribed regions corresponding to ~590 Mb of the total genome sequences (~ 4.8 Gbp). From 150 samples from 108 cultivars, we could detect more than 10,998 genome wide sequence polymorphisms whose minor allele frequencies were more than 0.1 both (?) in two-row and six-row barley. To evaluate the efficiency of polymorphism detection of this method, we conducted an amplicon sequencing using IonS5 platform on the 387 polymorphic sites. While more than 85% of SNPs evaluated were validated by the amplicon sequencing, half of indel/multiple alleles showed different or more diverged polymorphisms. These results demonstrated that our RNA-Seq based polymorphism detection could generate genome-wide markers even in the closely-related barley genotypes, such as genotypes from germplasm in Japanese breeding programs.