| Pepper is cultivated worldwidely as an important vegetable and economic crop.Molecular biology research of pepper is relatively backward.And the number of available molecular markers is limited.In pepper,it is of great practical value to develop large-scale number of molecular markers and construct high density intraspecific complete genetic linkage maps.Haploid culture technology has important application value in pepper breeding,and microspore culture is the most important method to obtain pepper haploid.While the genotype dependent microspore embryogenesis has become a bottleneck restricting the application of the technology.Based on pepper genome information,large-scale of molelular markers were developped,and two high density complete genetic linkage map were constructed combined with SLAF-seq technology and RIL population.The microspore embryogenesis was used as a phehotypic trait to study the causes of different microspore embryogenesis genetically and mine candidate genes regulating microspore embryogenesis in pepper.The results of the study are as follows.1.The non-redundant Capsicum species expressed sequence tag(EST)database from the National Center for Biotechnology Information(NCBI)was used in this study to develop a total of 902 usable EST-SSR markers.Additionally,177,587 SSR loci were identified based on the pepper genomic information,including 9,182 SSR loci 500bp both upstream and downstream of coding regions.Another 4,497 stable and reliable insertion/deletion(InDel)loci were also developed.2.From 9182 SSR and 4497 InDel loci,3,356 pairs of genomic SSR primers and 1,400 pairs of InDel primers that were evenly distributed in 12 chromosomes were selected.A high-density intraspecific genetic map of C.annuum was constructed using the F10-generation recombinant inbred line(RIL)of parents PM702 and FS871 as the mapping population.Eventually,a high-density complete genetic intraspecific linkage map of C.annuumcontaining 12 linkage groups and 708 molecular markers with a length of 1260.00 cM and an average map distance of 1.78 cM was produced.This intraspecific,high-density,complete genetic linkage map of C.annuum contains the largest number of SSR and InDel markers and the highest amount of saturation so far.3.A high-density complete genetic intraspecific linkage map of C.annuum was developed using specific-locus amplified fragment sequencing(SLAF-seq),based on 142 recombinant inbred lines(RILs)derived from an intraspecific cross between PM702 and FS871.The map contained 9,328 SLAFmarkers on 12 linkage groups(LGs),and spanned a total genetic distance of 2,009.69 centimorgan(cM)with an average distance of 0.22 cM.The sequencing depth for the map was 72.39-fold in the male parent,57.04-fold in the female parent,and 15.65-fold in offspring.4.The ability of microspore embryogenesis was proved to be a quantitative trait through the statistical analysis on the data of microspore embryogenesis of 142 RIL lines.On the basis of the genetic map developed using SLAF-seq,the QTL of microspore embryogenesis was analyzed.There were 7 QTLs identified on LG2,LG6,LG8,LG 11 and LG 12 seperately,with the peak value of LOD 3.51-6.66 and the phenotypic variance explanation of 4.76-48.30%.One major QTL,named Me2.2,was identified in the116.5-118.5 cM section of LG2 with corresponding physical section 166621667-167038758 bp.A total of 24 candidate genes were predicted.On the basis of the current function annotation,these candidate genes were related to the initiation of cell division and the growth,symmetric or asymmetric cell division,and so on.The study will provide a large number of practical molecular markers for pepper breeding,and provide theoretical basis and practical information for the establishment of efficient and stable haploid culture technology in pepper. |
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