西瓜果皮覆纹调控基因的定位与候选基因分析

Genetic mapping and analysis of candidate genes regulating watermelon rind stripe

【目的】精细定位调控西瓜果皮覆纹类型基因,开发分子标记。【方法】以选育的果皮覆齿条西瓜材料H2和覆网纹西瓜材料H9的2个高代自交系为亲本,构建F_(1)、BC_(1)P_(1)、BC_(1)P_(2)和F_(2)群体。利用两亲本和F_(2)群体中的果皮覆齿条单株和覆网纹单株分别构建2个亲本DNA混池和2个F_(2)混池,开展全基因组混池重测序分析。基于重测序数据,初步定位候选基因,之后在初定位区间内开发和筛选多态性InDel标记,利用F_(2)分离群体对候选基因进行进一步定位,筛选连锁标记,并分析区间内候选基因的序列。【结果】西瓜果皮覆纹性状是由1对显性基因控制的,西瓜果皮覆齿条对网纹为显性。通过混池重测序分析,初步将候选基因定位在6号染色体24.3~29.4 Mb的区间。在初定位区间内开发和筛选多态性InDel标记,利用全部368个F_(2)单株将ClRs(Citrullus lanatus rind stripe)基因定位在28252905~28558579 bp约305.7 kb的区间内,该区间内有35个基因,根据功能注释发现有4个基因(Cla97C06G126560、Cla97C06G126680、Cla97C06G126710和Cla97C06G126770)的功能可能与果皮覆纹相关。通过克隆4个基因的CDS序列,发现Cla97C06G126680的CDS序列在两亲本中没有差异位点,其余3个基因均存在非同义突变位点。通过在F_(2)群体和自然群体材料中对这些突变位点的基因型进行鉴定,发现3个基因的非同义突变位点与目标性状是共分离的。【结论】将西瓜果皮覆纹调控基因定位在6号染色体305.7 kb区间内并开发了可用于辅助分子育种的标记InDel-93,Cla97C06G126560、Cla97C06G126680、Cla97C06G126710、Cla97C06G126770可能是西瓜果皮覆纹候选调控基因。

【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】F_(1), BC_(1)P_(1), BC_(1)P_(2) and F_(2) 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 F_(2) 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 F_(2) 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 F_(2) 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 F_(2) populations and natural population materials.【Results】Genetic analysis indicated that the separation ratio of rind pencil stripe to netted stripe was 3∶1(χ^(2)= 0.13<χ^(2) _(0.05)= 3.841). The separation ratio of rind pencil stripe to netted stripe in BC_(1)P_(1) population was 1∶1(χ^(2)= 0.16<χ^(2) _(0.05)= 3.841). The BC_(1)P_(2) 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 Cl Rs(Citrullus lanatus rind stripe). The resequencing and association analysis indicated the Cl Rs 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 Findividual plants were used to fine-map the Cl Rs gene. Finally, 21 markers were used to locate the Cl Rs 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 F_(2) populations and the other 20 watermelon materials. By identifying the genotypes of these mutation sites in F_(2) 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】F_(1), BC_(1)P_(1), BC_(1)P_(2) and F_(2) 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 F_(2) 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.