Transcriptome Analysis Of Flowering Chinese Cabbage Under Heat Stress And The Development Of SSR Markers

Flowering Chinese cabbage is a Brassica vegetable that originated in South China and could be planted in all seasons.However,in summer and autumn,the growth of flowering Chinese cabbage is often suffered from heat stress in different degrees,and results in a decline in yield and quality.In recent years,the research on heat tolerance of flowering Chinese cabbage has received more attention.The previous research on heat tolerance in flowering Chinese cabbage mainly focused on agronomic traits,physiological and biochemical characteristics and few on the responsive molecular mechanism.In the current study,RNA-seq technique was used to analyze the transcriptomes of two heat-resistant and two heat-sensitive flowering Chinese cabbages genotypes under heat stress and control conditions.The molecular mechanism response to heat stress in flowering Chinese cabbage was dissected by analysis of differentially expressed genes.Based on the transcriptome data,EST-SSR markers were developed and these markers could provide the basis for marker-assisted selection breeding in flowering Chinese cabbage.The main findings are as follows:1.The leaves transcriptomes of the four flowering Chinese cabbage genotypes grown under both control and heat stress conditions were analyzed by using RNA-seq technique.2.95×10~8 high-quality reads with 125 bp were obtained with total data of36.9 Gb.Using Trinity software to de novo assemble flowering Chinese cabbage transcriptome,a total of 48,975 unigenes were obtained.The average length and N50of unigenes were 779 bp and 1,317 bp,respectively.All assembled unigenes were aligned to public databases,and 37,494(76.56%),21,994(44.91%),23,267(47.51%)and 7,825(15.98%)of unigenes were matched to the NR,Uniprot,GO and COG databases,respectively.2.Comparative analysis of transcriptomes of four flowering Chinese cabbage genotypes under control and heat stress conditions showed that up-regulated differentially expressed genes(DEGs)were more than down-regulated DEGs in four genotypes,and common DEGs in two heat-resist genotypes were more than DEGs in two heat-sensitive genotypes.There were 42 DEGs with same expression patterns identified in all genotypes,these genes mainly involving cell membrane and protein stability,signal transduction and gene activation,and could constitute the common heat stress responsive mechanism in flowering Chinese cabbage.There were 132common DEGs with same expressed patterns(130 up-regulated and 2 down-regulated)only in both two heat-resist genotypes,these genes mainly involving signaling,active oxygen scavenging and metabolic process,and could constitute the specifically heat stress responsive mechanism in heat-resist genotypes.3.A total of 8,165 EST-SSR loci were identified in de novo assemblies,and 98%SSR loci were the motif repeats of mononucleotide,dinucleotide and trinucleotide.According to EST-SSR loci,4,912 primer pairs of SSR markers were designed,and170 primer pairs were randomly selected to verify their usability.The results showed96 primer pairs could produce clear amplicons,and 48 of them were polymorphic.A genetic diversity analysis for 34 flowering Chinese cabbage genotypes was conducted by using the 48 polymorphic EST-SSRs,and these genotypes were classified into 3clusters,indicating the novel developed EST-SSRs can be effectively applied in the studies of flowering Chinese cabbage.