Construction Of High-density SNP Genetic Map And Identification Of Candidate Genes For Quantitative Traits In Camellia Sinensis

As a perennial economic crop widely cultivated,tea plant(Camellia sinensis)has long growth periods and self-incompatibility,which leads to the inefficiency by using traditional breeding methods such as natural selection and cross-breeding.These problems limit the improvement and innovation of tea germplasm resources process.In this study,firstly we developed the high-throughput SNP molecular marker of tea plants to construct a higher density genetic map.We had chosen the F1 segregating population,consisted of 327 individuals,as mapped population,which were derived from an artificial hybridization between two clonal tea cultivars,C.sinensis cv.Longjing 43(LJ 43)and C.sinensis cv.Baihaozao(BHZ).LJ 43 was used as the female parent.The high-density genetic mapping could make map-clone easier.Furthermore,the potential genes for the target quantitative traits were explored by combining the studies of population phenotypic observation,QTL mapping and genomic alignment,etc.Our study provides a theoretical basis with regard to the application of molecular marker-assisted breeding techniques in the rapid selection of tea germplasm resources.The main results are as follows:1.Using the 2b-RAD sequencing technology,we established a standard library of the parents 'Longjing 43' and 'Baihaozao' using 5'-NNN-3' overhang,meanwhile,the restricted libraries of 327 progeny lines were established by using selective overhangs5'-NNA-3' and 5'-NNT-3'.A total of 871,736,246 high-quality reads were obtained from each sample library by Illumina sequencing.A total of 380,936 tags were obtained by high-quality reads clustering between two standard libraries of parents,and a total of46,932 polymorphic SNPs were screened out.We compared the progeny libraries with the parental reference libraries,and found that there were 13,446 SNP markers genotyped in more than 80% of the progeny lines.The ?2 test was performed on the selected SNP markers to evaluate the Mendelian separation ratio of the markers,and a total of 4463 SNP markers corresponding to the Mendelian segregation ratio(p ? 0.05)were obtained for genetic mapping.In order to ensure the framework of genetic map,483 SSR markers were added to the map.2.Based on the novel SNP developed in this study,the parental genetic maps were successfully constructed,respectively.The female genetic map contains 2470 markers distributed over 15 linkage groups covering the genome length of 1427.70 cM,while the male genetic map contains 2217 markers distributed over 15 linkage groups covering a genome length of 1710.63 cM.We integrated the female and male maps using the shared markers,a high-density map consisted of 3,800 SNPs and 417 SSRs,was successfully constructed.There were 15 linkage groups in the map,covering 1678.52 cM genome length,with only 0.40 cM marker spacing and the number of markers(Gap < 2 cM)reached at 98.19 %.The results indicated that firstly a population consisting of 327 progeies was used to construct genetic map,and the marker gaps of constructed genetic were reduced from more than 1 cM to 0.4 cM for the first time.3.In order to screen for candidate genes of anthocyanin-related,the OPC of mapped population were tested to perform the QTL analysis for the first time.The anthocyanin content of each sample was 980.20 ± 24.79 nmol / g,and the coefficient of variation reached at 31.89%.A significant positive correlation was observed between OPC and YSC.We detected 10 QTLs with an LOD from 4.55 to 30.94,constituting 6.30% to40.30% of the phenotypic variation based on the phenotypic data of OPC and YSC in the mapping population.Furthermore,3 clustered regions were formed by overlapping 9major QTLs.4.In this study,we collected phenotypic data of 18 parameters(TW,SL,SWI,MB,RW,LW,SW,LSW,RCR,LNCO,SNCO,RNCO,LNC,RNC,SNC,LSNC,TNC,LP)related to growth traits.The phenotyping data were tested by using the mapping population under the period of 2-years seedling,cutting and expanding.The first discoveries of 53 QTLs were detected in 13 Linkage groups with an LOD from 3.12 to6.58,constituting 4.80 % to 29.10% of the phenotypic variation.In addition,the QTLs overlapped to 8 clustersed region which distributed among the six linkage groups.5.The re-QTL analysis was performed by combing the genotyping data of high-density SNP genetic map with the phenotyping data of published quantitative traitsconsisting of YSC,catechin components,CAF,germination period and anthrax resistance.A total of 81 QTLs were detected in 15 linkage groups for above five quantitative traits,contained 35 novel QTLs.Among them,7 QTLs were stable major QTLs.6.Based on the results of marker alignment and annotation,3800 mapped SNP markers were located to the physical map of two reference genomes,respectively.There were 1558 and 1209 markers located on the scaffolds of the CSS and CSA reference genomes,respectively,with the selected condition of perfectly and unique match(PM_uniq).Among them,more than 70 % of the markers located in the intergenic region.A total of 9 candidate genes related to the quantitative traits(anthocyanin,germination period and growth potential)of tea plant were screened by the functional information of genes which labeled on both sides of the markers.Moreover,the candidate gene TEA009062.1 was preliminarily verified as an important relating to growth potential of tea plant by qRT-PCR technology.