QTL Mapping Of Green Curd Trait In Cauliflower And Analysis Of Candidate Genes

Cauliflower(Brassica oleracea var.Botrytis)is one of the most popular vegetable crops in the world.Curd color is an important trait because consumers have preferences for vegetables with colors,which can promote health.Green curd cauliflower has great potential in the market,but the genetic mechanism underlying green curd is very limited.Fine mapping of QTLs that control green curd not only helps to develop molecular markers that are closely linked to the trait,which can be used for marker-assisted selection in breeding program,but also lays a solid foundation for map-based cloning of target gene that controls green curd,which will promote the unveiling of the mechanism underpinning ectopic development of chloroplasts in green curd.In this study,QTL-seq was applied to rapidly map the QTLs underlying green curd in F₂population,which was obtained by crossing white curd cultivar Stovepipe and green curd variety ACX800.Then the SNPs in the identified QTL region were converted into CAPS markers to verify these QTLs by traditional QTL mapping.For each verified QTL,more CAPS markers were designed and fine mapping was performed in another large F₂population to narrow down the QTL interval.Based on genomic annotations,the genes in the interval were preliminarily analyzed,and candidate genes were determined based on comprehensive information from four aspects.The differences in expression level and subcellular localization of candidate genes between white curd and green curd were analyzed.Eventually,two best candidate genes were transformed into the Arabidopsis ap1/cal1 double mutant,and a cauliflower regeneration system was established.The results are as follows:1.QTL mapping of green curd traitBy applying QTL-seq,two QTLs were identified,one was located on chromosome 5(13.4 Mb?39.8 Mb)and the other on chromosome 7(41.6 Mb?47.0 Mb),which were named Gr5.1 and Gr7.1 respectively.Eight CAPS markers were developed within each QTL interval,which was verified by traditional QTL mapping.The results showed that Gr5.1 is the major QTL controlling the green curd phenotype.QTL fine mapping was conducted within a second F₂ population containing 1291individuals.The result showed that Gr5.1 was located between another two flanking markers A3585 and A3609,with a genetic distance of 0.3 c M and a corresponding genomic interval of 236 Kb.This region contains 35 genes,9 of which are located in the nucleus or chloroplast,and their coding region contains nonsynonymous mutations.2.Expression analysis of candidate genesThe q PCR analysis of 9 candidate genes between white and green curd parent,as well as other white and green curd varieties showed that the expression level of MKS1(MAP Kinase Substrate 1)gene,LOC106295954,was significantly different between white and green curd(p<0.01),and the fold change was the largest among 9 candidate genes.Another gene UMPK,LOC106295953,which encodes UMP kinase,is predicted to be located in chloroplast.Homologous UMPK genes in Arabidopsis and rice are involved in chloroplast biosynthesis and development.Therefore,these two genes were identified as best candidate genes.3.Sequence analysis of candidate genesAlignment of green curd and white curd UMP kinase showed that one amino acid Tyr was changed to Cys at position 10 and a new Gly was inserted at position 315.For MKS1protein,there were two amino acid changes from Gln to Leu and Cys to Ser at position 15and 207,respectively.There were more than 100 SNPs and 8 deletions in the green curd MKS1 promoter.The longest deletion was 521 bp,which contains a 117 bp LTR/Gypsy retroposon.Promoter regulatory element analysis showed that two cis-acting elements,O2-site and TCA-element,only existed in green curd and were involved in zein metabolism regulation and salicylic acid response,respectively.4.Genetic transformation of candidate genesSubcellular location analysis by transient expression of UMPK and MKS1 gene in tobacco showed that both green and white curd UMP kinases were localized in the chloroplast,while both MKS1 proteins were localized in the nucleus.Besides,both genes were introduced into Arabidopsis ap1/cal1 double mutant by flower dip method and seeds from transgenic plants have been harvested.Moreover,by selecting the cotyledon of a white curd commercial variety Absolute F1 as explant,regenerated plants were obtained,which laid a solid foundation for further transformation of target gene.