Fine-mapping and identification of the gene Cla019481 for patches at hilum on testa of watermelon seeds

Run-Bu LV¹Dan ZHOU²Hong-Yuan LIU³Yu-Fu Zhang¹Cheng-Yuan XIE¹Xiao-Yu ZHANG¹Dong-Ye LI¹Shuang-Wu MA²Ji-Feng CHEN^1*

• ¹School of Life Sciences, Zhengzhou University, Zhengzhou, China
• ²Chinese Academy of Agricultural Sciences Zhengzhou Fruit Research Institute, Zhengzhou, China
• ³National Supercomputing Center in Zhengzhou, Zhengzhou University, Zhengzhou, China

The trait of patches at seed hilum is a valuable phenotypic marker for breeding new cultivars and identifying watermelon germplasm resources, though the candidate gene regulating this trait remains unknown. In this study, the F1 generation seeds (patches at hilum) derived from a cross between the female parent (no patches at hilum), and the male parent (with patches at hilum), and a back cross (BC) population was generated by crossing F1 with the female parent. The segregation ratio of patches and without patches trait is conformed to the expected 1:1 Mendelian ratio in BC population. The restriction-site associated DNA sequencing was performed on BC population to construct high-density genetic map. It revealed a major quantitative trait locus (QTL) on chromosome 3 with 5,375,019–5,784,364bp, harboring 35 annotated genes from Cla019451 to Cla019485, governing the stably inherited trait of patches at hilum on the testa of watermelon seeds. The reliable derived cleaved amplified polymorphic sequence (dCAPS) marker was developed within the interval, demonstrating perfect genotype-phenotype co-segregation. Consequently, the target QTL was delimited to a 40-kb region on chromosome 3, and in the physical distance of 40-kb, it contains candidate gene Cla019481 for patches at hilum. Insertions/deletions (Indel) and single nucleotide polymorphisms (SNPs) analyses indicated that Cla019481 was the top candidate responsible gene for the presence or absence of patches at hilum. Based on the dCAPS marker development for SNP genotype identification and human visible phenotype classification in different groups of watermelon accessions, there was no phenotypic inconsistency with genotype in materials of absence patches at hilum. In other words, the genotype showed without patches at hilum then the phenotype showed the same in applied materials. Gene expression validation experiments using the material of with/without patches at hilum and the method of quantitative RT-PCR (qRT-PCR) revealed a positive correlation. Elevated Cla019481 expression coincided with progressive darkening of hilum pigmentation during the three seed development processes (8, 18, 25 days after flowering). The verification test results demonstrate that: Cla019481 expression critically regulates the patches formation at hilum. The gene Cla019481 plays a significant role in the trait of patches at hilum on watermelon seeds.