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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Publications at this Location » Publication #345112
Title: Identification of single nucleotide polymorphism in Red Clover (Trifolium Pratense L.) using targeted genomic ampliconsequencing and RNA-seq

Author
item Li, Wenli
item Riday, Heathcliffe
item RIEHLE, CHRISTINA - University Of Wisconsin
item EDWARDS, ANDREA - University Of Wisconsin
item Dinkins, Randy

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/10/2019
Publication Date: 10/23/2019
Citation: Li, W., Riday, H., Riehle, C., Edwards, A., Dinkins, R.D. 2019. Identification of single nucleotide polymorphism in Red Clover (Trifolium Pratense L.) using targeted genomic ampliconsequencing and RNA-seq. Frontiers in Plant Science. 10. Article 1257. https://doi.org/10.3389/fpls.2019.01257.
DOI: https://doi.org/10.3389/fpls.2019.01257

Interpretive Summary: Genomic tools used in red clover have mainly relied on single sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often not easy to unambiguously map them using short reads generated by next-generation sequencing technology. Single-nucleotide polymorphisms (SNPs) have been successfully applied in genomics-assisted breeding in several agriculturally important species. Due to the increasing importance of legumes in forage production, there is a clear need to develop SNP-based markers for red clover that can be utilized in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in different red clover varieties using sequences generated by targeted amplicon amplification followed by sequencing. Then, using the same filtering stringency used in this pipeline, we identified a set of highly confident SNPs in red clover coding regions using RNA-sequencing data. SNPs identified in this study will contribute to further development of highly informative genomic tools for red clover breeding and other molecular studies.

Technical Abstract: Red clover (Trifolium pratense L.) is a diploid, naturally cross-pollinated, cool-season species. As a perennial forage legume, red clover is mostly cultivated in temperate regions worldwide. Being as red clover is a non-model crop species, genomic resources for it have been very scarce. So far, genomic tools used in red clover have mainly relied on single sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often not easy to unambiguously map them using short reads generated by next-generation sequencing technology. Single nucleotide polymorphisms (SNP) have been successfully applied in genomics-assisted breeding in several agriculturally important species. Due to the increasing importance of legumes in forage production, there is a clear need to develop SNP-based markers for red clover that can be utilized in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in different red clover varieties using sequences generated by targeted amplicon amplification followed by sequencing. Then, using the same filtering stringency used in this pipeline, we set out to identify a set of highly confident SNPs in red clover. In doing so, we identified coding-region SNPs using publicly available RNA sequencing data. A total of 67,994 SNPs were identified. Among these, 28% (19,116) of them are missense mutations in protein coding regions. Using Medicago truncatula as a reference, we annotated the genes affected by these missense mutations. Pathway analysis of these genes indicated that several biological processes are impacted by these mutations. Coding region SNPs generated from this study will contribute to further development of highly informative genomics tools for red clover breeding and other molecular studies.