Genetic Improvement And Molecular Analysis Of Important Traits Of Capsicum Chinense

Capsicum spp.is native to South America,and has abundant germplasm resources.The growing area of pepper is more than 2 million hectares,ranking first among vegetables in China.C.chinense is an important cultivated species in the genus Capsicum.Due to its high spicy degree,strong heat resistance,and exceptional flavor,more and more attention has been given to the development and utilization of this species in China.In this study,we carried out molecular analysis of the important traits（in vitro regeneration ability,heterosis performance,and fruit color）in molecular breeding and genetically improved the fruit color,accumulating basic data for the important traits of C.chinense and serving the molecular breeding.The main results are as follows:1.In vitro regeneration characteristics of two C.chinense germplasmUsing two C.chinense germplasm（HNUCC22 and HNUCC16）,we detected the effects of explants and hormones on the in vitro regeneration of C.chinense.The best explant of C.chinense was a cotyledon with the petiole.The best placement method for explants is to place them abaxially.In this study,the best medium for the induction rate of adventitious buds was MS+3.0 mg/L 6-BA+0.1 mg/L IAA,and the highest induction rates were 48.06%and 37.33%,respectively.The best medium for the elongation was MS+3.0 mg/L 6-BA+1.0 mg/L IAA+4.0 mg/L GA₃,and the highest elongation rates were 66.78%and 64.67%,respectively.The best medium for the induction of rooting was 1/2MS+0.2 mg/L IAA,and the highest induction rates were83.08%and 89.88%,respectively.These results suggested that the two C.chinense germplasms had good regeneration ability in vitro.Further exploration indicated that there were significant differences in vitro regeneration ability between C.chinense（HNUCC22 and HNUCC16）and Capsicum baccatum（HNUCB81 and HNUCB226）.Comparative histology and comparative transcriptome analysis of the early stage of in vitro regeneration of the two cultivated species were carried out.Compared with C.baccatum,the callus of C.chinense produced vascular tissue for a little longer.Transcriptome analysis showed that there were significant changes in the expression of some key genes in plant hormone signal transduction and starch and sucrose metabolic pathways,especially GH3.6（BC332＿20033）and SAUR67（BC332＿29560）which were significantly up-regulated in C.baccatum.Moreover,it is suggested that WOX7 could play an important role in the early regeneration of pepper in vitro.2.Analysis of heterosis performance of C.chinenseA large number of SNPs were identified by re-sequencing two C.chinense germplasm（HNUCC22 and HNUCC16）,indicating that there are some genetic differences between the two germplasm,and their hybrids may have heterosis.There was no morphological difference between F₁generations produced by a reciprocal cross.Compared with their parents,the fruit weight,fruit length,fruit diameter and crown diameter of the F₁ generation at the flowering stage were significantly higher.The plant height of the F₁ generation in each period was significantly higher than that of the paternal,but there was no significant difference with the maternal,showing a dominant effect.Furthermore,comparative transcriptome analysis showed that 633（4.74%）additive genes and 12719（95.26%）non-additive genes were identified at the seedling stage and 800（13.69%）additive genes and 5042（86.31%）non-additive genes were identified during flowering.KEGG enrichment analysis showed that most of the different genes were mainly involved in plant hormone transduction pathway,including BC332＿21619、BC332＿18317、BC332＿12434、BC332＿25121,and novel.2052,which were overexpressed in both developmental stages.In addition,DE lnc RNAs,DE mi RNAs,and DE circ RNAs were identified by strand-specific transcriptome.DE lnc RNA targeting analysis showed that it was significantly enriched in photosynthesis,in which 13 genes such as psb K,atp B,psb B,psb I,atp E,psa B,pet B,atp A,psb F,psb T,pet A,atp H,and psa A were up-regulated in the F₁ generation.The DE mi RNA targeting analysis showed that CAB-10A and LHCP were significantly up-regulated in the photosynthetic antenna protein pathway in the F₁ generation.These results suggest that plant hormone signal transduction and photosynthesis may be involved in the regulation of heterosis in C.chinense.3.Genetic analysis and improvement of fruit color of C.chinenseGenetic analysis showed that the color of mature fruit（red and yellow）and immature fruit（dark green and light green）were controlled by a single locus,respectively.A high-density genetic map of 12 linkage groups containing 1347 bin markers was constructed by genotyping by sequencing.The total genetic distance was1206.37 c M,and the average genetic distance of adjacent markers was 0.9 c M.One gene locus related to immature fruit color was identified on chromosome 1,and one gene locus related to mature fruit color was identified on chromosome 6.The molecular markers corresponding to the two gene loci were binmarker19（34.61 c M）and binmaker849（48.01 c M）.The genomic regions of these two loci were further scanned and found that BC332＿00870,BC332＿00876,BC332＿17237,and BC332＿17250 are closely related to fruit color.Furthermore,two d CAPS markers and one KASP marker related to pepper fruit color were successfully developed and applied to molecular marker-assisted breeding.Early selection of fruit color was carried out on the hybrid progenies of two germplasm using molecular markers,and the germplasm with dark green immature fruit and red mature fruit,and light green immature fruit and yellow mature fruit were successfully cultivated.In conclusion,based on two C.chinense germplasm,the in vitro regeneration ability,heterosis performance,and fruit color were analyzed at the molecular level,and new germplasm were cultivated.The in vitro regeneration of pepper provides important conditions for genetic transformation.The C.chinense F₁ generation has certain heterosis.The candidate genes regulating heterosis were studied by transcriptome sequencing,which provides an important basis for further understanding the molecular mechanism of heterosis.Based on the genetic map,the candidate genes for fruit color traits were located,molecular markers were developed,and molecular marker-assisted breeding was carried out.These results accumulated important basic molecular data for the important traits of C.chinense and provided molecular markers and important germplasm for genetic improvement.