Molecular Identification Of Cultivars And Transcriptome Analysis Of Bracts In Bougainvillea

Plants in the woody genus Bougainvillea have been widely used for ornamental and greening purposes in global tropicsal and subtropical regions.In China,previous studies have focused on the pharmaceutical,silvicultural and chemical property aspects of Bougainvillea,but genetic studies remain very limited.Also,the origin and genetic background of Bougainvillea germplasm are mostly unclear,and cultivar homonyms and synonyms exist largely in China,indicating the necessity of reliable markers for cultivar identification in terms of research and application requirements.Meanwhile,bract color is the main trait of Bougainvillea for ornamental application,but its molecular mechanism is poor,for which the recently developed transcriptome sequencing technology provided a powerful tool.In this study,a number of molecular markers have been developed and utilized for cultivar identification in Bougainvillea,and some enzymes and genes related to the bract color were detected based on transcriptome sequencing.The results will provide useful molecular marker resources for genetic diversity analysis and cultivar identification and also lay a theoretical basis for understanding the formation of bract color in Bougainvillea.The results of this study are as follows:(1)Shuihong cultivar was used for transcriptome sequencing(RNS-seq).A total of 12.33 Gb data and 154085 non-redundant sequences with a total length of 14.13 Mb were obtained.The frequency of SSR was one SSR/2.34 kb and the detection rate of SSR was 39.13%.117 repetitive motifs of SSR types were included.The most abundant motif was A/T(71.67%),the second was AT/AT(6.65%),and the third was C/G(2.83%).Single nucleotide repeats accounted for 74.55% of the total SSR loci,followed by trinucleotide repeats(13.96%).The number of repeats of SSR motifs ranged from 5 to 25,with SSR loci repeated ten times being the most abundant(17.03%).The total length of repeats ranged from 10 to 302 bp,with an average of 18.1 bp.16 SSR polymorphic loci were developed.A total of 76 alleles were amplified in 18 cultivars.The average of alleles was 4.8,and the average polymorphic information was 0.544.(2)Genetic diversity and genetic relationship among cultivars were analyzed and the molecular fingerprints were established based on ISSR markers.161 bands were amplified by 11 ISSR primers in 131 cultivars,including 156 polymorphic bands with a polymorphic rate of 96.89%.Average observed alleles and effective alleles were 1.97 and 1.50,respectively.Nei’s and Shannon’s diversity indices were 0.29 and 0.45,respectively.Genetic diversity of 131 cultivars was low,with genetic distance ranged from 0.00 to 0.60 with an average of 0.365.Clustering analysis indicated that majority of the cultivars were clustered into one group,whereas some cultivars of the same species were grouped in different clusters or sub-clusters and certain cultivars from different species grouped in the same cluster.The highest discriminant rate was 80.92% with primer UBC841.All tested cultivars were distinguishable,and molecular fingerprints of cultivars could be uniquely established based on the combination of primers UBC841 and UBC876.(3)Genetic diversity and genetic relationship of the germplasm resources(131 cultivars)were analyzed,and the cultivar fingerprints were constructed.A total of 85 alleles were detected by 15 SSR loci in 131 cultivars,with an average of 5.60 alleles per locus.Average number of effective alleles,Shannon’s information index,observed heterozygosity and expected heterozygosity were 2.52,1.04,0.50 and 0.57 per locus,respectively.The genetic distance ranged from 0.00 to 0.60,with an average of 0.33.The genetic differentiation was low,and the genetic relationship was close.Clustering analysis result was in agreement with the result from ISSR.131 cultivar could be effectively identified with unique fingerprints by 11 SSR loci.(4)Four stages of bracts of Yunnan purple cultivar were sampled for transcriptome sequencing,and differentially expressed genes between two stages were obtained.Differentially expressed genes were determined with GO function annotations,with the most differentially expressed genes annotated to biological processes and the least to cellular components in each stage.The top 20 metabolic pathways in the most significant enrichment between two stages were obtained based on KEGG pathway.Flavonoid synthesis and betaine biosynthesis pathway were obtained,which related to the color of Yunnan purple bracts.11 enzymes(CHS,CHI,TC4 H,FLS,Caffel-Co A,F3’H,F3 H,Naringin,KCS11,CCo AOMT,SAM MSPI1 and CYP98A2)were involved in the flavonoid synthesis pathway,which were coded totally by 26 genes,and one enzyme(DOD)was involved in betaine biosynthesis pathway,which was coded by four genes.Ten genes were randomly selected from differentially expressed genes for qRT-PCR analysis.Five of them were identical with the results of sequencing,and the others were consistent with the transcriptome results in most stages.Thus,qRT-PCR results validated the reliability of sequencing data.