Development of EST-SSR markers using transcriptome data in okra (Abelmoschus esculentus L.): a genetically orphan crop
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Abstract
Okra is an important vegetable crop with few genomic resources and only limited simple sequence repeat (SSR) markers. In the present investigation, we have performed RNA sequencing (RNA-Seq) from leaf samples of okra varieties Pusa Sawani and Pusa Bhindi-5. Out of the 106224 EST-SSRs identified, mononucleotide repeats were dominant with a frequency of 72.20% (71699) followed by trinucleotide 15% (14897) and dinucleotide 10.68% (10604) repeats. In total, 183 best primer pairs (156 di and 27 trinucleotides) were selected for validation in 12 okra accessions comprising wild and cultivated Abelmoschus species. Overall, 163 primer pairs produced expected PCR amplicons with an amplification efficiency of 89%. A total of 23 primer pairs showed a 14.1% polymorphism percentage with an average value of 0.32 polymorphic information content (PIC) and these polymorphic markers were used further for diversity analysis. The UPGMA dendrogram analysis separated the okra accessions into two main clusters. This study provided insight into the distribution and frequency of EST-SSRs in the okra transcriptome. The EST-SSR markers developed here will be a robust molecular tool for germplasm identification, genetic diversity analysis, and comparative mapping in okra and related species due to their high polymorphism and cross-transferability.
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References
Ahmed N., Nawaz S., Iqbal A., Mubin M., Butt A., Lightfoot D. A., Maekawa M. 2013. Extraction of high-quality intact dna from okra leaves despite their high mucilage. Biosci. Methods.,4: 4.http://dx.doi.org/10.5376/bm.2013.04.0004
Asadi A. A, Monfared S. R. 2014. Characterization of EST-SSR markers in durum wheat EST library and functional analysis of SSR-containing EST fragments. Mol Genet Genom.,289 (4): 625-640.http://dx.doi.org/10.1007/s00438-014-0839-z
Barman, P., Choudhary, A. K and Geeta, R. 2017. A modified protocol yields high-quality RNA from highly mucilaginous Dioscorea tubers. 3 Biotech, 7(2), 1-5.
Chen H., Liu L., Wang L., Wang S.,Somta P., Cheng X. 2015. Development and Validation of EST-SSR Markers from the Transcriptome of Adzuki Bean (Vignaangularis). PLoS One.,10 (7):e0131939. doi:10.1371/journal.pone.0131939
Cho Y. G., Ishii T.,Temnykh S., Chen X.,Lipovich L.,Mccouch S. R.,Cartinhour S. 2000. Diversity of microsatellites derived from genomic libraries and gen bank sequences in rice (Oryzasativa L.). Theo. Appl. Genet.,100 (5):713-722.
Das A., Yadav R. K.,Choudhary H.,Lata S., Singh S., Kumar., C and Talukdar A (2022) Population structure, gene flow and genetic diversity analyses based on agro-morphological traits and microsatellite markers within cultivated and wild germplasms of okra (Abelmoschusesculentus (L.) Moench.). Genet. Res. Cr.Evol.,69(2):771-791.http://dx.doi.org/10.1007/s10722-021-01263-9
Ellis J. R., Pashley C. H., Burke J. M., McCauley D. E. 2006. High genetic diversity in a rare and endangered sunflower as compared to a common congener. Mol. Eco.,15(9) 2345-2355.http://dx.doi.org/10.1111/j.1365-294X.2006.02937.x
Emebiri L. C. 2010. An EST-SSR marker tightly linked to the barley male sterility gene (msg6) located on chromosome 6H. J. Her.,101 (6):769-774.http://dx.doi.org/10.1093/jhered/esq083
Eujayl I.,Sorrells M. E., Baum M., Wolters P., Powell W. 2002. Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theo. Appl. Genet.,104 (2): 399-407.http://dx.doi.org/10.1007/s001220100738
Evanno G.,Regnaut S., Goudet J. 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Eco.,14 (8):2611-2620 http://dx doi.org/10.1111/j.1365-294X.2005.02553.x
Guo R., Landis J. B., Moore M. J.,Meng A., Jian S., Yao X., Wang H. 2017. Development and application of transcriptome-derived microsatellites in Actinidiaeriantha. Front. Pl. Sci.,8:1383.http://dx.doi.org/10.3389/fpls.2017.01383
Kalinowski S. T., Taper M. L., Marshall T. C. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Mol. eco.,16 (5): 1099-1106.http://dx.doi.org/10.1111/j.1365-294X.2007.03089.x
Kumar S., Parekh M. J.,Fougat R. S., Patel S. K., Patel C. B., Kumar M., Patel B. R. 2017. Assessment of genetic diversity among okra accessions using SSR markers. J. Plant. Biochem.Biot.,26:172–178
Lata S., Yadav R. K.,Tomar B. S. 2021. Genomic tools to accelerate improvement in okra (Abelmoschusesculentus). DOI: 10.5772/intechopen.97005
Lawson M. J., Zhang L. 2006. Distinct patterns of SSR distribution in the Arabidopsis thaliana and rice genomes. Geno.bio.,7(2): 1-11.
Leonarduzzi C.,Spanu I.,Labriola M., González-Martínez S. C.,Piotti A.,Vendramin G. G. 2016. Development and characterization of three highly informative EST-SSR multiplexes for Pinushalepensis and their transferability to other Mediterranean pines. Pl. Mol. Bio. Rep.,34 (5):993-1002.http://dx.doi.org/10.1007/s11105-016-0980-4
Metzgar D.,Bytof J and Wills C. 2000. Selection against frameshift mutations limits microsatellite expansion in coding DNA. Geno. Res.,10 (1):72-80.
National Horticulture Board., data 2019-20 (second advance estimates). http://nhb.gov.in/StatisticsViewer.aspx?enc=MWoUJibk35dW2g36TUJWAoZqESmAYFi7h2irlsmjlINTcFl1rG/kLbq8ZQbWUvuM
Peakall R.,Smouse P. E. 2006. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol. Eco. Notes.,6(1):288-295. http://dx.doi.org/10.1111/j.1471-8286.2005.01155.x
Perrier X.,Jacquemoud-Collet J. P. 2009.DARwin software: Dissimilarity Analysis and Representation for Windows (version 5.0. 157).
Ravishankar K. V.,Muthaiah G.,Mottaiyan P.,Gundale., S. K (2018). Identification of novel microsatellite markers in okra (Abelmoschusesculentus (L.) Moench) through next-generation sequencing and their utilization in the analysis of genetic relatedness studies and cross-species transferability. J. gen., 97(1):39-47.
Schafleitner R., Kumar S., Lin C. Y.,Hegde SG., Ebert A. 2013. The okra (Abelmoschusesculentus) transcriptome as a source for gene sequence information and molecular markers for diversity analysis. Gene.,517(1): 27-36.http://dx.doi.org/10.1016/j.gene.2012.12.098
Scott K. D.,Eggler P., Seaton G.,Rossetto M., Ablett E. M., Lee L. S., Henry R. J. 2000. Analysis of SSRs derived from grape ESTs. Theo. Appl. Gen., 100(5):723-726.http://dx.doi.org/10.1007/s001220051344
Strickler S. R.,Bombarely A., Mueller L. A. 2012. Designing a transcriptome next‐generation sequencing project for a nonmodel plant species. American J. Bot., 99(2) 257-266.
Wei L., Li S., Liu S., He A., Wang D., Wang J., Wu X. 2014. Transcriptome analysis of Houttuyniacordata by Illumina paired-end RNA sequencing and SSR marker discovery. PloS one 9(1) e84105.http://dx.doi.org/10.1371/journal.pone.0084105
You F. M.,Huo N.,Gu Y. Q., Luo M. C., Ma Y.,Hane D., Anderson O. D (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC bio.Info., 9(1):1-13.