Genetic Analysis And QTL Mapping Of Tillering In Purple-caitai

Purple-caitai?Brassica campestris L.ssp.chinensis var.purpurea Hort.2n=2x=20?is one of the subspecies of Brassicaceae.It originated from the Yangtze River basin in China.Its flower stems are tender,juicy and bright-colored.Rich in anthocyanin which is beneficial to human body,it is deeply loved by consumers.In the base of its stem,there are multiple lateral branches similar to those of the grass family,which is an important factor for the yield.Therefore,studying the genetic regularity of the tiller traits and cloning of related genes in purple-caitai is of great practical significance for the high yield breeding of purple-caitai.In this study,the self-incompatibility line L-100 of purple-caitai witch show more tillering was used as the female parent and the male-inbred line L-101 of cai-xin witch show less branch as the male parent to construct four generations of F₁,F₂,BCP1,and BCP2.Six generations of joint genetic analysis were performed on the tiller traits.At the same time,the QTL-seq was combined with the traditional QTL mapping method,and the F2 segregation generation was used to locate the QTLs controlling the bifurcation.The main results are as follows:1.The genetic application of tillering in purple-caitai:Through the joint analysis of tillers traits of the six generations,it can be concluded that the tiller traits are consistent with the D model,that is,one pair of additive-dominant-major genes plus additive-dominant-epitasis polygenes control.In the three isolates population of BCP1,BCP2,and F₂theheritability of major gene were estimated to be21.63%,1.17%,36.1%,respectively,while the polygene was 67.13%,13.56%,and36.8%,respectively.The polygene heritability of all generations was significantly higher than that of major genes,show that the trait of tillering were dominated by multiple genes.In the three isolates population,environment variance accounted for about 35.8%of the phenotypic variation.These results indicated that tillering of purple-caitai was obviously affected by environment factors and suitable for selection in early generation.2.The QTL mapping of tillering trait:Selecting individual showing extreme phenotypes in F2 population construct extreme pools and subjected to whole-genome sequencing.Through bioinformatics analysis,it was found that there was a significant SNP imbalance within the range of 18Mb-25Mb of A07 chromosome,so it was inferred that A0718Mb-24Mb range was a QTL locus for the number of tillering.The traditional QTL positioning method was used to verify the accuracy of QTL-seq analysis.Through the analysis and comparison of the sequencing results between the two parents,a total of 236 pairs of InDel and CAPS markers were designed.The markers were screened by the two parents and two extreme pools,and finally 11 pairs of polymorphic markers were obtained.Using the screened 11 pairs polymorphism markers in the QTL analysis of 185 F2 individuals,the results showed that QTLs controlling tillers existed between the markers InA0740 and InA0788,explaining16%of the phenotypic variation.The two markers correspond to 18.91Mb and21.1Mb physical positions respectively,which coincided with the QTL-seq results.The locus was preliminary located at A0718.91Mb-21.1Mb.3.Candidate gene prediction of tiller traits in purple-caitai:By literature review and gene annotation,6 candidate genes were obtained in the target region.After that,we analyzed the relative expression levels of the six genes in each part of the two parents.The results showed that,at the axillary,the relative expression of Bra004212,the homologue gene of TCP1,in female parents was 16.1 times less than that of the parents with fewer tillering,and the relative expressions of Bra004117,the homologue gene of TSF,were different between the two parents in three part.The proportion of more-tillering parents were 16.29,3.56,and 10.59 times that of the less-tillering parents.TCP1,a member of the TCP transcription factors,was active in the cotyledonary petioles and the distal part of the expanding leaves,as well as the midrib region and the petiole.And associated with the elongation of leaves and stems.Bra004117 is the paralog of TSF,which is one of the four floral pathway integrator genes and play dominant role in the promotion of lateral shoot development in short-day condition.Bra004212 and Bra004117 can be speculated as candidate functionary genes at this site.To further investigate candidate genes,the CDS sequences of Bra004212 and Bra004117 were obtained using TA cloning.MegAlign software was used to analyze the difference between the two genes in the two parents.The results showed that there were two single-base mutations in Bra004212 between the two parents,resulting in two amino acid changes.There was only one SNP mutation in the CDS sequence between Bra004117's parents,and the mutation was synonymous and did not cause amino acid sequence change.4.The construction and selection of the F_2:3 segregation population.In order to further clone the genes of interest related to tiller traits,we need to construct a F_2:3population firstly.Using the linkage markers InA0740 and InA07234 to screen and identify 230 individuals of the F2 segregating population.The individual plant whose genotype were heterozygous were self-pollination to getF_2:3population.During the full flowering period,the tiller traits were investigated.According to the number of tillers,they were divided into three grades.The segregation ratio was selected to be3:1 and the final population was 106-216.