Abstract
Pumpkin (Cucurbita spp.) is one of the major vegetable crops grown worldwide. The number of simple sequence repeat (SSR) markers in pumpkins lags far behind the requirement of genetic analysis. Transcriptomes generated from different genotypes provide valuable sequence resources for molecular marker development. In this study, transcriptomes from the roots of C. maxima and C. moschata inbred lines suffering salinity stress were separately de novo assembled. The assembled unigenes were aligned between the two genotypes to in silico screen the polymorphic SSR loci. A total of 328 putatively polymorphic SSR loci were identified. Of which, 211 loci that is suitable to the primer design were selected for polymorphic validation by polymerase chain reaction amplification and electrophoresis analysis. A total of 110 SSR markers exhibited polymorphism among 65 pumpkin varieties with the mean polymorphism information content value of 0.34. Cluster analysis showed that these SSR markers distinctly classified the pumpkin varieties into different groups according to their genetic backgrounds at species level. The SSR markers developed in this study provided valuable supplements to the available genetic tools of pumpkins.
Similar content being viewed by others
References
Blanca J, Canizares J, Roig C, Ziarsolo P, Nuez F, Pico B (2011) Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics 12. https://doi.org/10.1186/1471-2164-12-104
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics (Oxford, England) 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Chen H et al (2016) Development of gene-based SSR markers in rice bean (Vigna umbellata L.) based on transcriptome. Data Plos One 11. https://doi.org/10.1371/journal.pone.0151040
Diaz A et al (2015) Anchoring the consensus ICuGI genetic map to the melon (Cucumis melo L.) genome. Mol Breed 35. https://doi.org/10.1007/s11032-015-0381-7
Gong L, Stift G, Kofler R, Pachner M, Lelley T (2008) Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L. Theor Appl Genet 117:37–48. https://doi.org/10.1007/s00122-008-0750-2
Grabherr MG et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–U130. https://doi.org/10.1038/nbt.1883
Gupta PK, Balyan HS, Varshney RK, Gill KS (2013) Development and use of molecular markers for crop improvement. Plant Breed 132:431–432. https://doi.org/10.1111/pbr.12110
Haas BJ et al (2013) De novo transcript sequence reconstruction from RNA-Seq: reference generation and analysis with Trinity. Nat Protoc 8. https://doi.org/10.1038/nprot.2013.084
Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877. https://doi.org/10.1101/gr.9.9.868
Jewell E et al (2006) SSRPrimer and SSR taxonomy tree: biome SSR discovery. Nucleic Acids Res 34:W656–W659. https://doi.org/10.1093/nar/gkl083
Kalia RK, Rai MK, Kalia S, Singh R, Dhawan AK (2011) Microsatellite markers: an overview of the recent progress in plants. Euphytica 177:309–334. https://doi.org/10.1007/s10681-010-0286-9
Kong Q, Zhang G, Chen W, Zhang Z, Zou X (2012) Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper, Capsicum annuum (Solanaceae). Am J Bot 99
Kong Q et al (2014a) Genetic diversity of Cucurbita rootstock germplasm as assessed using simple sequence repeat markers. Sci Hortic 175:150–155. https://doi.org/10.1016/j.scienta.2014.06.009
Kong Q, Yuan J, Niu P, Xie J, Jiang W, Huang Y, Bie Z (2014b) Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon. PLoS One 9:e87197. https://doi.org/10.1371/journal.pone.0087197
Liu T, Zeng L, Zhu S, Chen X, Tang Q, Mei S, Tang S (2015) Large-scale development of expressed sequence tag-derived simple sequence repeat markers by deep transcriptome sequencing in garlic (Allium sativum L.). Mol Breed 35:204. https://doi.org/10.1007/s11032-015-0399-x
Mahato AK et al (2016) Leaf transcriptome sequencing for identifying genic-SSR markers and SNP heterozygosity in crossbred mango variety ‘Amrapali’ (Mangifera indica L.). PLoS One:11. https://doi.org/10.1371/journal.pone.0164325
R Core Team (2018). R: a language and environment for statistical computing. R foundation for statistical computing, Vienna, Austria. URL https://www.R-project.org/
Ren Y et al (2012) A high resolution genetic map anchoring scaffolds of the sequenced watermelon. Genome Plos One 7. https://doi.org/10.1371/journal.pone.0029453
Sablok G et al (2016) PlantFuncSSR: integrating first and next generation transcriptomics for mining of SSR-functional domains markers. Front Plant Sci 7. https://doi.org/10.3389/fpls.2016.00878
Sim S-C, Hong J-H, Kwon Y-S (2015) DNA profiling of commercial pumpkin cultivars using simple sequence repeat polymorphisms. Hortic Environ Biotechnol 56:811–820. https://doi.org/10.1007/s13580-015-0123-0
Sun H et al (2017) Karyotype stability and unbiased fractionation in the paleo-allotetraploid Cucurbita genomes. Mol Plant 10:1293–1306. https://doi.org/10.1016/j.molp.2017.09.003
Tsukazaki H et al (2015) Development of transcriptome shotgun assembly-derived markers in bunching onion (Allium fistulosum). Mol Breed 35:55. https://doi.org/10.1007/s11032-015-0265-x
Vieira MLC, Santini L, Diniz AL, Munhoz CF (2016) Microsatellite markers: what they mean and why they are so useful. Genet Mol Biol. https://doi.org/10.1590/1678-4685-gmb-2016-0027
Vukosavljev M, Esselink GD, van’t Westende WPC, Cox P, Visser RGF, Arens P, Smulders MJM (2015) Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals. Mol Ecol Resour 15:17–27. https://doi.org/10.1111/1755-0998.12289
Wu H-B et al (2014a) Large-scale development of EST-SSR markers in sponge gourd via transcriptome sequencing. Mol Breed 34:1903–1915. https://doi.org/10.1007/s11032-014-0148-6
Wu TQ et al (2014b) The first Illumina-based de novo transcriptome sequencing and analysis of pumpkin (Cucurbita moschata Duch.) and SSR marker development. Mol Breed 34:1437–1447. https://doi.org/10.1007/s11032-014-0128-x
Xanthopoulou A et al (2017) De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers. Gene 622:50–66. https://doi.org/10.1016/j.gene.2017.04.035
Xiang X, Zhang Z, Wang Z, Zhang X, Wu G (2015) Transcriptome sequencing and development of EST-SSR markers in Pinus dabeshanensis, an endangered conifer endemic to China. Mol Breed 35:158. https://doi.org/10.1007/s11032-015-0351-0
Xiang C et al (2018) A high-density EST-SSR-based genetic map and QTL analysis of dwarf trait in Cucurbita pepo L. Int J Mol Sci 19:3140
Xie J, Lei B, Niu M, Huang Y, Kong Q, Bie Z (2015) High throughput sequencing of small RNAs in the two Cucurbita germplasm with different sodium accumulation patterns identifies novel microRNAs involved in salt stress response. PLoS One 10:e0127412. https://doi.org/10.1371/journal.pone.0127412
Xu Y, Guo S-R, Shu S, Ren Y, Sun J (2017) Construction of a genetic linkage map of rootstock-used pumpkin using SSR markers and QTL analysis for cold tolerance. Sci Hortic 220:107–113. https://doi.org/10.1016/j.scienta.2017.03.051
Zhai L, Xu L, Wang Y, Cheng H, Chen Y, Gong Y, Liu L (2014) Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.). Mol Breed 33:611–624. https://doi.org/10.1007/s11032-013-9978-x
Zhang W et al (2011) Construction of a high density integrated genetic map for cucumber (Cucumis sativus L.). Theor Appl Genet 124:249–259
Zhang Q, Yu ED, Medina A (2012) Development of advanced interspecific-bridge lines among Cucurbita pepo, C. maxima, and C. moschata. Hortscience 47:452–458
Zhong R et al (2016) SSR marker development from peanut gynophore transcriptome sequencing. Plant Breed 135:111–117. https://doi.org/10.1111/pbr.12336
Zhong Y-J, Zhou YY, Li JX, Yu T, Wu TQ, Luo JN, Luo SB, Huang HX (2017) A high-density linkage map and QTL mapping of fruit-related traits in pumpkin (Cucurbita moschata Duch.). Sci Rep 7. https://doi.org/10.1038/s41598-017-13216-3
Funding
This work was supported by the National Natural Science Foundation of China (31471894) and Da Bei Nong Group Promoted Project for Young Scholar of HZAU (Grant No. 2017DBN020).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key Message
Transcriptomes from the roots of two pumpkin inbred lines suffering salinity stress were separately de novo assembled. A total of 328 putatively polymorphic SSR loci were identified by aligning the assembled unigenes. Of which, 110 SSR markers were validated to be polymorphic by PCR amplification and electrophoresis analysis.
Rights and permissions
About this article
Cite this article
Kong, Q., Liu, Y., Xie, J. et al. Development of Simple Sequence Repeat Markers from De Novo Assembled Transcriptomes of Pumpkins. Plant Mol Biol Rep 38, 130–136 (2020). https://doi.org/10.1007/s11105-019-01189-8
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11105-019-01189-8