| Chinese cabbage(Brassica rapa L.ssp.Pekinensis,AA)is one of the most important vegetable crops in China,belonging to Brassica rapa species of Brassica genus in Cruciferae family.Leafy head is the main edible part of Chinese cabbage,lamina and leafy head development traits are related to the yield and quality of Chinese cabbage directly.In recent years,a lot of researches have been performed about QTL mapping for traits related to lamina and leafy head development in Chinese cabbage.However,the QTL mapping were mainly focused on leaf length,width,head height and weight,and traditional molecular markers such as SSR and InDel were usually used to identify above QTL.With this method,the density of genetic map is much lower,and the detected QTL region was mostly greater than 5 cM.Therefore,the functional genes controlled lamina and leafy head development of Chinese cabbage cannot be identified.The whole genome information showed that lamina polarity development genes and auxin responsive factors are related to the morphological development of Chinese cabbage,however there was not any published reports about the identification of candidate genes related to leafy heading type.That's why,it was necessary to construct high density genetic map by high-throughput sequencing technology,to identify QTL and candidate genes for traits related to lamina and leafy head development,which could provide the references for the discovery of candidate genes that directly affect the development of lamina and leafy head.In this research,the SSRs were identified based on the whole genome sequences information of Chinese cabbage.The specific SSR markers related to leaf adaxial-abaxial polarity development genes were developed and their variation characteristics in sequence were analyzed;the Chinese cabbage F2 population(CCF2)derived from parents of overlapping heading '14Q-141' and curling outwards heading '14Q-279',and another F2 populations(PCCCF2)produced from parents of Pak choi 'PC-101' and heading Chinese cabbage 'CC-48' were used as research materials to analyze the genetic variation in 19 lamina and 10 leafy head-related traits.By using WGS(Whole Genome Resequencing)and GBS(Genotyping-by-Sequencing)high density genetic maps were constructed,the QTL and candidate genes for traits related to lamina and leafy head development were further identified combining with bulk segregant analysis(BSA)technology.Furthermore,we verified the functions of BrARF3.1 gene screened above,and analyzed the transcriptional regulatory patterns of overexpressed transgenic plants.The purpose of this research was to detect main chromosomal regions that regulate traits related to lamina and leafy head development in Chinese cabbage,to explore the candidate genes that co-localized with QTLs,and to reveal the expression regulation patterns of main candidate genes.This will establish the theoretical foundation for analyzing the molecular mechanism of lamina and leafy head development in Chinese cabbage.It is helpful to breed new varieties of Chinese cabbage with different heading types directionally.The main results are listed as followed.1.A total of 173,892 SSRs were detected in the whole genome level,and the main form of SSR loci was P type(perfect repeat motifs).The density of mono-to trinucleotide repeat motifs is much higher than tetra-to hexanucleotide repeat motifs,and the dominant repeat motifs were A/T.In total 341 SSRs were detected based on 41 leaf adaxial-abaxial polarity development genes in Chinese Cabbage.Using CCF2 population as material,one genetic map including 150 markers(23 specific SSR primers and 127 random SSR,SNP and InDel primers)distributed across 10 linkage groups was constructed,with a total length of 1747.57 cM.2.Using CCF2 population,combing with WGS method one high density genetic map including 2,904 SNPs was constructed,with a total length of 1,399.44 cM and average distance 0.48 cM between markers.A total of 11 QTLs controlling 6 lamina development related traits and 23 QTLs controlling 7 leafy head development related traits were detected in year 2016 and 2017,which distributed across 10 linkage groups.One major QTL controlling lamina development related traits were detected,which controlling petiole area of head leaf,the phenotypic variability interpretation rate is 14.5%;6 major QTLs controlling leafy head development related traits were detected,which controlling leafy head overlap type,head height and head weight,the phenotypic variability interpretation rate is 10.7%to 17.5%.The QTL controlled leafy head overlap type were co-localized in 6.44?7.14 Mb on A04 chromosome by QTL analysis in 2 years.And 11 candidate genes related to leaf and leafy head development related traits were detected within 8 QTL regions,containing 5 candidate genes co-localized with QTL of leaf development related traits,which is BrAS1,BrROT4,BrCycD3;3,BrKRP2 and BrAN3;6 candidate genes co-localized with QTLs of leafy head development related traits,which is BrPKL,BrPFL2,BrGA20OX3,BrPHV,BrCOWl and BrCycD3;1.3.Using PCCCF2 population,combing with GBS method one high density genetic map including 3,194 SNPs was constructed,with a total length of 1,522.89 cM and average distance 0.48 cM between markers.In total 15 QTLs controlling 6 lamina development related traits and 13 QTLs controlling 4 leafy head development related trait were detected,which distributed across 5 linkage groups A04?A05?A06?A08?A09.And 4 major QTLs controlling lamina development related traits were detected,which controlling the whole plant area,plant height,outer leaf length and outer leaf petiole length,the phenotypic variability interpretation rate is 14.5%to 21.6%;5 major QTLs controlling leafy head development related traits were detected,which controlling leafy head overlap type,head degree,head weight and total weight,the phenotypic variability interpretation rate is 11.0%to 17.6%.21 candidate genes related to leaf and leafy head development related traits were detected in 12 QTL regions,containing 10 candidate genes co-localized with QTL of leaf development related traits,15 candidate genes co-localized with QTL of leafy head development related traits,4 candidate genes both control leaf and leafy head development related traits,which is BrARF3,BrYABl,BrHST and BrANT1.9 candidate genes related to leafy head overlap type were detected by bulk segregate analysis.Candidate genes BrARF3,BrARL and BrANT1 which control leafy head overlap type were both detected in bulk segregate and QTL analysis.4.By QTL analysis in CCF2 population and bulk segregate analysis in PCCCF2 population,the QTL controlled leafy head overlap type were both corresponding to the candidate gene BrGA20OX3,which regulate the biosynthesis of gibberellin.5.The gene BrARF3.1 in Pak choi and in Chinese cabbage were cloned in this study.CDS sequences revealed a mutation C/G at 1563 bp,resulting in amino acid change from Gln(CCBrARF3.1)to His(PCBrARF3.1).Overexpressed genes PCBrARF3.1 and CCBrARF3.1 were transferred to Pak choi respectively,the transgenic positive plants showed the heading trait.The transcriptome analyses showed that there were evident differential expressions in 9 candidate genes related to leafy heading development between mutant and wild type,expression of candidate genes BrAS2,BrREV,BrSGS3,BrAS1,BrARF3 and BrAGO1 was up-regulated,and expression of BrRDR6,BrKRP2 and BrARGOS was down-regulated in transgenic plants. |
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