The Development, Validation And Utilization Of EST-SSR Markers Based On The Transcriptome Data Of Bletilla Striata

Objective:To lay a solid foundation for functional gene mining,genetic diversity research and molecular-assisted selection(MAS)in B.striata.We firstly constructed a Unigenes library,and finished the development,validation and utilization of EST-SSR mrkers for this medicinal herb.Methods:Firstly,sequencing of the B.striata cDNA libraries was performed on the Illumina HiSeq~TMM 2000 platform,various tissues and organs of plants among the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing.After filtering,the clean reads were de novo assembled by using the Trinity package with default parameters.Further,MISA(MIcroSAtellite Identification Tool)and Primer 3.0 were employed for mining potential EST-SSR loci and designing primers(three primer pairs per marker loci),respectively.After that,a total of 100 EST-SSR primer pairs were randomly selected for synthesis and further validation by using 4 different landraces of B.striata.And to evaluate the transferability of polymorphic markers in validation test,six individuals from two relative orchids,Phalaenopsis and Dendrobium,with 3 individuals for each population,were amplified by the same PCR protocol.Finally,the screened monomorphic and polymorphic markers were used in validation test to analysis the genetic diversity of 23 B.striata individuals.Results:1.Construction of Unigenes Databases and Annotation AnalysisAfter filtering,a total of 106,054,784 paired-end clean reads were generated from the sequencing platform,and all clean sequencing data have been deposited in the NCBI database(assession number:SRR7058048).Further,127,261 unigenes were assembled by the Trinity package,with total length of 79,698,987 bp and average length of 612 bp.Further,a total of 67,494(51.86%)unigenes were annotated in at least one of Nr,NT,KO,Pfam,Swiss-Prot,KOG and GO databases.2.EST-SSRs Mining and Primer Pairs DevelopmentAs a result,a total of 18,335 EST-SSRs were identified in 15,433 unigenes by MISA online(except 778 compound EST-SSRs),the EST-SSR frequency in our study is 14.41%,and has a high distribution density of 4.35 kb per SSR loci.Based on the analysis,mono-repeats were the largest category and accounted for 49.78%(9,128 SSRs)of the unigenes,followed by di-(4,884 SSRs,26.64%),and tri-(4,166 SSRs,22.72%)repeats.Additionally,the quantitative statistics for mono-,di-,tri-and tetra-repeats showed that the most abundant motifs were A/T(8,996,98.55%),AG/CT(3,286,67.28%),AAG/CTT(1,020,24.48%).Based on the identified 18,335 EST-SSRs,a total of 25,935 EST-SSR markers were successfully designed by Primer 3.0.3.Availability and Cross Species Validation of EST-SSR Markers in B.striataTo evaluate the availability of designed markers,we randomly selected 100EST-SSR markers and amplified them in 4 different landraces of B.striata.The results showed that a total of 87 primer pairs were successfully amplified,of those,63yielded PCR products of the expected size and 25 primer pairs showed obvious polymorphism in this study.Cross-species amplification results showed that 2markers(ZYBS-1,ZYBS-52)were consistently amplified in both Phalaenopsis and Dendrobium,1 marker as ZYBS-14 only has bands in Phalaenopsis,and 2(ZYBS-18,ZYBS-60)were amplified only in Dendrobium.4.Study for Genomic DNA Isolation in B.striataAccording to previous genomic DNA isolation studies,CTAB method was finally improved to explore an efficient,economic and high-quality way for genomic DNA extracting from B.striata.By the contrast of UV and AGE results of DNA,which were isolated from leaves by improved CTAB,TIANGEN DNAsecure Plant kit,ZOMANBIO plant Genprep DNA kit and SDS method,the results all showed that the DNA quality and integrity of the improved CTAB method was significantly higher than others(A260/A280=1.82,A260/A230=2.09,C yield(?g/g)=160.68?g/g).And the EST-SSR amplification result also showed that the DNA extracted from the improved CTAB method could satisfy the EST-SSR related studies.In addition,the ITS2 tests showed that the DNA extracted by the improved CTAB method could be effectively amplified,but by SDS cannot be amplified.5.Optimization of EST-SSR-PCR Amplification System in B.striataTo optimize the SSR-PCR reaction system as a based approach for further studies,the response surface methodology was applied for optimizing the three key factors(concentrations of DNA template,primers and 2×PCR mix usage amountat three levels in SSR-PCR system,with an amplification volume of 10?L.We screened the optimized SSR-PCR reaction system that included:DNA template 1.60 ng/?L,primers 12?M,and 2×PCR mix 6.98?L.After the validation approaches,all the results showed that the optimized system was excellent applicability and stability.The SSR-PCR system was successfully established by this study,which could lay a solid experiment foundation for further related studies of SSR-PCR amplification in B.striata.6.Genetic Diversity Analysis of B.striata Based on Developed EST-SSR MarkersBased on the validation test results,52 primer pairs were selected and amplified in 23 lines of B.striata,6%non-PAGE results showed that a total of 25 markers had significant polymorphism,with 108 amplified locus,91 polymorphic locus and 1-8alleles.Moreover,the GS clustering results were highly consistent with PCoA,and 23lines were classified into three groups as expectably(Genetic structure analyses revealed that ZA(Numbered 8 to 15)and XW(Numbered 16 to 23)wild populations were the closest,while the ZY(Numbered 1 to 7)cultivar populations constituted a single cluster,respectively.).Conclusion:To the best of our knowledge,this research is the first study of the construction of unigenes database of global transcriptome of all developmental stages in B.striata by high sequencing technology.And successfully developed 25,935candidate EST-SSR primer pairs,which not only laid a reliable theoretical foundation for further studies in functional genomics,metabolomics,proteomics and molecular marker related studies.Our successful application of the developed markers in genetic diversity research of 23 lines,also laid a solid experimental foundation for the further studies of B.striata based on EST-SSR markers.