Genetic Analysis And Fine Mapping For Fruit Bitterness Gene In Cucumis Sativus L.

Cucumber is one of the important vegetable crops in China. Fruit bitterness can cause big loss in cucumber production. Cucurbitacin is the chemical substance causing bitterness in cucumber. It's important to study the related genes in cucurbitacin biosynthesis and the molecular mechanism of fruit bitterness using modern molecular biology technology. Developing tightly linked molecular markers and fine mapping for fruit bitterness genes is significant in making clear the development mechanism of fruit bitterness by efficient technique and it will provide academic guidance to the breeding work of non-bitterness cucumber.Cucumber bitterness include foliage bitterness and fruit bitterness. It was reported Bi and Bi-2 were the genes controlling foliage bitterness in cucumber, where as Bt and Bt-2 were the genes controlling fruit bitterness. Former studies about cucumber bitterness focused on genetic analysis, gene linkage, environment effect, varieties bred, and so on. There were only a few reports conducted molecular-biology studies for bitterness genes in cucumber. And most of them focused on foliage bitterness Bi gene. Up to the present, no studies were found with regard to SSR markers and genetic mapping for fruit bitterness gene Bt and Bt-2.The forlige and fruit of 46GBt, a purified inbred line in our lab, are all bitter. But the genetic relations between the fruit bitterness gene in 46GBt and Bt and Bt-2 were not make sure. 46GBt, PI173889 (BiBiBtBt), LJ90430 (BiBiBt-2Bt-2), 931, 9110Gt and 9930 were used as experimental materials in this study. Genetic populations were constructed to explore the heredity and genetic relations of Bt and Bt-2 with other genes. SSR, SNP, InDel marker tightly linked to Bt and Bt-2 genes were developed. Genetic mapping was conducted for Bt and Bt-2 genes and the candidate genes for Bt-2 were also predicted. The main conclusions were as follows:1. Genetic analysis was conducted using P1, P2, F1, F2, BC1P1 and BC1P1 derived from two combinations 46GBt×LJ90430 and 46GBt×PI173889. It was confirmed that the fruit bitterness gene in 46GBt is Bt-2, the same as LJ90430. Fruit bitterness character is controlled by one dominate gene Bt-2, and fruit neck bitterness character is controlled by one dominate gene Bt. When the Bt-2 and Bt are presented at the same time, there is dominate and recessive epistasis between these two genes, the Bt-2 gene have dominate epistasis on the Bt gene, the Bt-2 gene masks the expression of the Bt gene, while the bt gene have recessive epistasis on the bt-2 gene, the phenotype of plants with homozygous bi is the same as Bt-2Bt-2 genotype presented, and the fruit is bitter.2. The segregation and linkage analysis showed that there were no linkages between Bt-2 and F gene, Bt-2 and Dg gene, B -2 and Fsd gene, Bt-2 and D gene in 46GBt. And there were no linkages between Bt and W gene, Bt and Fsd gene in PI173889.3. Genetic and SSR analysis were conducted on fruit bitterness character in 9110Gt×9930 RILs population. Two loci controlling fruit bitterness were detected. They were located on Chromosome 5 (Chr.5) and Chr.6 respectively. The lous located on Chr.6 was linked to Bi locus tightly. These results explained the cause for bitter fruit presenting in the offspring of two fruit non-bitter parental lines.4. SSR technology was adopted to analyze a F2 populations including 158 individuals derived from the combination of PI173889×931. A SSR linkage for Bt gene consisting four SSR markers was constructed. Two SSR markers named SSR00116 and SSR00398 were screened linked to Bt gene with the genetic distance of 20 and 33 cM respectively. Compared with the integrated genetic and cytogenetic map, the Bt gene was mapped on Chr.5.5. A F2 population including 184 individuals derived from two cucumber inbred lines 46GBt and 931 was used to construct a linkage of SSR markers to fruit bitterness gene (Bt-2) in cucumber (cucumis sativus L.). Eight SSR markers were mapped on the linkage. The flanking markers SSR10795 and SSR07081 were linked to the Bt-2 gene with genetic distances of 0.8 and 2.5cM, respectively. The linkage was compared with the integrated genetic and cytogenetic map, and the Bt-2 gene was mapped to the short arm of cucumber chr.5 within the region of 3.3cM.6. Based on primary mapping of Bt-2 gene and re-sequence of two parental lines, Bioinformatics was employed to develop new SSR and InDel markers in the primary mapping region of Bt-2 gene. SSR analysis and InDel analysis were conducted on 1315 individuals of F2 population using these new SSR and InDel primer. Then the Bt-2 gene was fine mapped to chromosome 5 within the region of 1.5cM. SSR02118 and SSR15564 were the flanking markers linked to the Bt-2 gene with genetic distances of 1.1 and 0.4cM. The physical distance of the Bt-2 gene fine mapping region was 300Kb based on the whole genome sequence of cucumber, and there are 31 candidate genes in this region. Among of these candidate genes, we speculated the candidate gene Csa012474 belonging to a kind of transport protein was the most related gene to the Bt-2 gene.7. The veracity of SSR10795 and SSR07081 was tested using another F2 population with 60 individuals, and the accuracy rate for the two markers was 91.6%. The veracity of Bt-InDel-1 was also tested, and the accuracy rate for this marker was 94.8%. Taken together, we identified two SSR markers closely linked to the Bt-2 gene which will play an important role in marker-assisted selection (MAS) for non-bitter cucumber breeding program.