Genetic mapping and analysis of candidate genes regulating watermelon rind stripe

Abstract:【Objective】Watermelon is an important crop in China, which plays a great role in increasing farmers’income and meeting people's growing life needs. As one of the most important appearance traits of Cucurbitaceae crops, the rind stripe is one of the important traits that breeders, growers and consumers may concern, and has important research value. Several regulation genes in Cucurbitaceae crops have been discovered in recent years, which are of great significance for molecular marker- assisted breeding and for understanding their regulation mechanism. In this study, genes that regulate watermelon rind stripe types were mapped and markers to aid molecular breeding were developed.【Methods】 F1, BC1P1, BC1P2 and F2 populations were constructed by crossing the inbred line H2 (rind pencil stripe,male) and inbred line H9 (netted stripe, female) to map the gene(s) controlling the rind stripe types of watermelon. All materials were grown in greenhouses in Xinxiang Experimental Base of Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. From F2 segregation population, 7 plants for rind pencil trait and 13 plants for netted trait were selected. DNA was extracted by Cetyltriethylammnonium Bromide (CTAB) method, and two DNA mixing pools (dominant pool and recessive pool) were constructed separately. The constructed two DNA mixing pools of F2 population and two parent DNA mixing pools were used for database construction and sequencing, and the whole genome resequencing data were obtained. Using Burrows-Wheeler-Alignment Tool (BWA) (http://bio-bwa.sourceforge.net/), reads were aligned to Watermelon reference genome [Watermelon (97103) v2]. The promising SNP loci were detected by Sequence Alignment/mapping (SAMtools) (http://www.htslib.org/). According to the initial positioning candidate interval, insertion-deletion (InDel) markers with good polymorphism were designed and selected to distinguish the genotype of F2 single plant by polyacrylamide gel electrophoresis. The candidate interval was further narrowed. Candidate genes were selected based on the functional annotations and sequences. Using non-synonymous mutations of candidate genes, Derived Cleaved Amplified Polymorphic Sequences (dCAPS) and InDel markers were designed. Linkage relationship between these markers and target gene was verified by the F2 populations and natural population materials.【Results】Genetic analysis indicated that the separation ratio of rind pencil stripe to netted stripe was 3∶1 (c2 = 0.13＜c2 0.05 = 3.841). The separation ratio of rind pencil stripe to netted stripe in BC1P1 population was 1∶1 (c2 = 0.16＜c2 0.05 = 3.841). The BC1P2 population were all rind pencil stripe. The genetic analysis showed that the rind pencil stripe was dominant to netted stripe and was controlled by a pair of dominant gene ClRs (Citrullus lanatus rind stripe). The resequencing and association analysis indicated the ClRs gene was preliminarily located in the interval of 24.3-29.4 Mb on chromosome 6. In order to further narrow the target interval, the polymorphic InDel markers were developed and screened in the initial interval, and all 368 F2 individual plants were used to fine-map the ClRs gene. Finally, 21 markers were used to locate the ClRs gene between the markers Indel-128 and Indel-124, with genetic distances of 1.5 cm and 3.0 cm, respectively, within the region of 28 252 905 bp-28 558 579 bp on chromosome 6. There were 35 genes in this region. According to the functional annotation, four genes (Cla97C06G126560, Cla97C06G126680, Cla97C06G126710 and Cla97C06G126770) have their functions related to the development of rind stripe. By cloning the CDS sequences of the four genes, the CDS sequences encoding the Myb transcription factor Cla97C06G126680 were identical in the two parents, and the other three genes all had non-synonymous mutation sites. Two non-synonymous mutations caused by single base substitution were detected in the coding region of Cla97C06G126560 gene; three non- synonymous mutations caused by single base substitution were detected in the coding region of Cla97C06G126710 gene; there were 8 non-synonymous mutation sites caused by single base substitution and 1 tribase insertion deletion site in the coding region of Cla97C06G126770 gene. Using nonsynonymous mutations of three genes, dCAPS and InDel markers were designed. Linkage relationship between these markers and target gene was verified by the F2 populations and the other 20 watermelon materials. By identifying the genotypes of these mutation sites in F2 population and natural population materials, it was shown that the non-synonymous mutation sites of the three genes were co-segregated with the target trait. The developed molecular marker InDel-93 can be used for molecular assisted breeding.【Conclusion】F1, BC1P1, BC1P2 and F2 populations were constructed by crossing the inbred line H2 (rind pencil stripe, male) and inbred line H9 (netted pencil stripe, female) to map the gene (s) controlling the rind pencil stripe types of watermelon. The regulatory genes of watermelon rind stripe were mapped to the 305.7 kb interval on chromosome 6. No recombination progeny was screened out after further expanding the F2 population, which perhaps induced by the recombination inhibition in this chromosome region. According to the functional annotation, four genes (Cla97C06G126560, Cla97C06G126680, Cla97C06G126710 and Cla97C06G126770) probably have functions related to the development of rind stripe. We also developed an InDel-93 marker with high recognition and obvious difference, which can be used in molecular assisted breeding.