De novo Transcriptome Analysis and Molecular Marker Development of Two Hemarthria Species

Abstract

Hemarthria R. Br. is an important genus of perennial forage grasses that is widely used in subtropical and tropical regions. Hemarthria grasses have made remarkable contributions to the development of animal husbandry and agro-ecosystem maintenance; however, there is currently a lack of comprehensive genomic data available for these species. In this study, we used Illumina high-throughput deep sequencing to characterize of two agriculturally important Hemarthria materials, H. compressa “Yaan” and H. altissima “1110.” Sequencing runs that used each of four normalized RNA samples from the leaves or roots of the two materials yielded more than 24 million high-quality reads. After de novo assembly, 137,142 and 77,150 unigenes were obtained for “Yaan” and “1110,” respectively. In addition, a total of 86,731 “Yaan” and 48,645 “1110” unigenes were successfully annotated. After consolidating the unigenes for both materials, 42,646 high-quality SNPs were identified in 10,880 unigenes and 10,888 SSRs were identified in 8330 unigenes. To validate the identified markers, high quality PCR primers were designed for both SNPs and SSRs. We randomly tested 16 of the SNP primers and 54 of the SSR primers and found that the majority of these primers successfully amplified the desired PCR product. In addition, high cross-species transferability (61.11–87.04%) of SSR markers was achieved for four other Poaceae species. The amount of RNA sequencing data that was generated for these two Hemarthria species greatly increases the amount of genomic information available for Hemarthria and the SSR and SNP markers identified in this study will facilitate further advancements in genetic and molecular studies of the Hemarthria genus.

Development of SSR Markers Based on Transcriptome Sequencing and Association Analysis with Drought Tolerance in Perennial Grass Miscanthus from China

“…However, the amplification of repeats other than tri-nucleotide is prevented in coding DNA, resulting in frame-shift mutations (Metzgar et al, 2000), which could be a possible explanation for the abundance of tri-nucleotide repeats. In the other hand, the AG/TC of di-nucleotide SSRs and CCG/GGC of tri-nucleotide SSRs were reported to be the largest number of motif in Hemarthria and grain crops (Temnykh et al, 2000; Kantety et al, 2002; Huang et al, 2016). The inverse GA/CT motif can describe multiple codons when transcribed into mRNA.…”

Phylogenetics and diversity analysis of Pennisetum species using Hemarthria EST‐SSR markers

“…Although the application of these markers to Hemarthria enabled the identification of 241 different bands (Huang et al., ), the PA and PIC values determined in the case of Pennisetum (100.0000% and 0.8430%, respectively) were higher than those observed in this genus (95.6006% and 0.7230%).…”

“…A total of 49 Hemarthria EST‐SSR primers (Huang et al., ), which were developed by our research team and synthesized by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China), were screened and selected to genotype the 35 Pennisetum accessions. Polymerase chain reaction (PCR) was performed in a final reaction volume of 15 μl containing 30 ng template, 0.4 μM each of forward and reverse primers, 7.5 μl PCR mix (1.5 mM Mg 2+ included, Tiangen), 1.5 U Taq polymerase and ddH 2 O.…”

Influence of radiation on Hemarthria compressa's genetic variations

“…The EST‐SSR primers were obtained by the H. compressa 's transcriptome sequence development from our group's previous study (Huang et al . ). In this experiment, the dose of 90 Gy of 60 Co‐ γ was used to irradiate forage type H. compressa cv.…”

“…Meanwhile, the labor-and cost-effective (Sorrells 2005) features of EST-SSR being more reproducible and less prone to null alleles (Kim et al 2008;Sullivan et al 2013) make it ideal for genetic study where genotyping errors may strongly affect final estimates (Leonarduzzi et al 2016). The EST-SSR primers were obtained by the H. compressa's transcriptome sequence development from our group's previous study (Huang et al 2016). In this experiment, the dose of 90 Gy of 60 Co-c was used to irradiate forage type H. compressa cv.…”