Development of HRM Markers Based on SNPs Identified from Next Generation Resequencing of Susceptible and Resistant Parents to Gummy Stem Blight in Watermelon

Abstract

Watermelon (Citrullus lanatus) is an economically important vegetable crop all over the world, which has functional compounds such as lycopene and citrulline. Gummy stem blight caused by Didymella bryoniae is one of the most devastative diseases in watermelon. Single nucleotide polymorphisms (SNPs), which are genetic variations occurring between individuals with respect to a single base, were often used to construct genetic linkage maps and develop molecular markers linked to a variety of horticultural traits and resistance to several diseases. In this study, we developed high-resolution melting (HRM) markers based on SNPs generated from NGS resequencing of two parents in watermelon. Plant materials were C. lanatus '920533' (female and susceptible parent), C. amarus 'PI 189225' (male and resistant parent), and their F1 and F2 progenies. A total of 13.6 Gbp ('920533') and 13.1 Gbp ('PI 189225') of genomic sequences were obtained using NGS analysis. A total of 6.09 million SNPs between '920533' and 'PI 189225' were detected, and 354,860 SNPs were identified as potential HRM primer sets. From these, a total of 330 primer sets for HRM analysis were designed. As a result, a total of 61 HRM markers that have polymorphic melting curves were developed. These HRM markers can be used for the construction of SNP-based linkage maps and for the analysis of quantitative trait loci (QTLs) related to gummy stem blight resistance.

QTL Mapping for Gummy Stem Blight Resistance in Watermelon (Citrullus spp.)

“…The mapped sequences were used to detect SNPs between ‘920533’ and ‘PI 189225’. A total of 6 million raw SNPs were obtained between the parental lines ( Table S2 ) [ 35 ].…”

“…Among the total SNPs obtained, 5 million SNPs were selected as homozygous SNPs ( Table S2 ) [ 35 ]. Subsequently, 4.3 million SNPs were filtered for A/G, A/C, T/G, and T/C combinations, excluding A/T and G/C.…”

“…Furthermore, around 2 million SNPs whose flanking sequence (single copy) was found only once in the whole genome were chosen using the basic local alignment search tool nucleotide (BLASTN). Finally, 354,860 SNPs located on the genic region were further selected, and then 888 HRM markers that were evenly distributed on the watermelon genome were developed ( Table S2 ) [ 35 ]. Polymorphisms of these HRM markers were tested using two parental lines, the F 1 plants and 13 randomly selected plants of the F 2 population.…”

“…D. bryoniae can infect the watermelon plants at any stage and on all parts of the plant, including the stem, foliar and fruits. Disease resistance to GSB normally assessed through spraying the inoculum with spore suspension at four to six leaf stage of watermelon seedlings [ 28 , 29 , 35 ]. Pathogenicity, aggressiveness and environmental factors such as temperature and humidity can influence the resistance evaluation [ 28 ].…”

“…The homozygous susceptible ‘920533’ and resistant ‘PI 189225’ parental lines were sequenced through next-generation sequencing by the bioinformatics company Macrogen (Seoul, Korea) [ 35 ]. The sequencing libraries were prepared according to the manufacturer’s instructions using TruSeq DNA Nano Sample Preparation Kit (Illumina Inc., San Diego, CA, USA).…”