Dig Up Tall Fescue Plastid Genomes For The Identification of Morphotype-Specific DNA Variants

Background Tall fescue (Festuca arundinacea Schreb.) is an important cool-season perennial grass species. Hexaploid tall fescue has three distinct morphotypes used either as forage or turf purposes. Its chloroplast genome is conserved due to it being maternally inherited to the next generation progenies. To identify morphotype-specific DNA markers and the genetic variations, plastid genomes of all three tall fescue morphotypes, i.e., Continental cv. Texoma MaxQ II, Rhizomatous cv. Torpedo, and Mediterranean cv. Resolute, have been sequenced using Illumina MiSeq sequencing platform. Results The plastid genomes of Continental-, Rhizomatous-, and Mediterranean tall fescue were assembled into circular master molecules of 135,283 bp, 135,336 bp, and 135,324 bp, respectively. The tall fescue plastid genome of all morphotypes contained 77 protein-coding, 20 tRNAs, four rRNAs, two pseudo protein-coding, and three hypothetical protein-coding genes. We identified 630 SNPs and 124 InDels between Continental and Mediterranean, 62 SNPs and 20 InDels between Continental and Rhizomatous, and 635 SNPs and 123 InDels between Rhizomatous and Mediterranean tall fescue. Only four InDels in four genes (ccsA, rps18, accD, and ndhH-p) were identified, which discriminated Continental and Rhizomatous plastid genomes from the Mediterranean plastid genome. Here, we identified and reported eight InDel markers (NRITCHL18, NRITCHL35, NRITCHL43, NRITCHL65, NRITCHL72, NRITCHL101, NRITCHL104, and NRITCHL110) from the intergenic regions that can successfully discriminate tall fescue morphotypes. Divergence time estimation revealed that Mediterranean tall fescue evolved approximately 7.09 Mya, whereas the divergence between Continental- and Rhizomatous tall fescue occurred about 0.6 Mya. Conclusions To our knowledge, this is the first report of the assembled plastid genomes of Rhizomatous and Mediterranean tall fescue. Our results will help to identify tall fescue morphotypes at the time of pre-breeding and will contribute to the development of lawn and forage types of commercial varieties

Molecular Characterization of Jatropha curcas Resources and Identification of Population-Specific Markers

Jatropha curcas L. is cited as one of the best candidates for future oil and biodiesel production. It is widespread in many tropical and subtropical countries but has not yet received much genetic improvement. The objective of this study was to collect Jatropha germplasm and characterize it with molecular markers. A total of 64 genotypes, collected from seven geographic locations on two continents, were analyzed with 32 simple sequence repeat and two candidate gene-specific primers (ISPJ-1 gene and Curcin-P2 gene promoter). In general, markers were found to be highly conserved, and many (40%) were monomorphic in the studied populations. Polymorphic primers, which amplified population-specific fragments, were identified. The polymorphic information content of the polymorphic markers ranged from 0.03 to 0.47. Genetic similarity analysis identified two distinct groups at 0.73 DICE similarity coefficient. Group I included germplasm collected from the islands of Cuba and Cape Verde, and group II consisted of Brazil, Mozambique, and Senegal populations. Island genotypes were found to be very distinct compared to their mainland counterparts. Sequencing of monomorphic fragments identified single nucleotide polymorphism (SNP) between these two groups. High-resolution melting analysis of the SNP in the Jcps9 locus further confirmed the two gene pools. Sequencing of polymorphic fragments of the Jc03 locus identified a deletion in a (GT)4 repeat motif in the genotypes in group II. Several population-specific microsatellites and SNP markers have been recognized. The distinct Jatropha genotypes and the population-specific molecular markers identified in this study will be valuable resources in breeding programs

Isolation of intact chloroplast for sequencing plastid genomes of five festuca species

Isolation of good quality chloroplast DNA (cpDNA) is a challenge in different plant species, although several methods for isolation are known. Attempts were undertaken to isolate cpDNA from Festuca grass species by using available standard protocols; however, they failed due to difficulties separating intact chloroplasts from the polysaccharides, oleoresin, and contaminated nuclear DNA that are present in the crude homogenate. In this study, we present a quick and inexpensive protocol for isolating intact chloroplasts from seven grass varieties/accessions of five Festuca species using a single layer of 30% Percoll solution. This protocol was successful in isolating high quality cpDNA with the least amount of contamination of other DNA. We performed Illumina MiSeq paired-end sequencing (2 × 300 bp) using 200 ng of cpDNA of each variety/accession. Chloroplast genome mapping showed that 0.28%–11.37% were chloroplast reads, which covered 94%–96% of the reference plastid genomes of the closely related grass species. This improved method delivered high quality cpDNA from seven grass varieties/accessions of five Festuca species and could be useful for other grass species with similar genome complexity

Analysis of tall fescue ESTs representing different abiotic stresses, tissue types and developmental stages

<p>Abstract</p> <p>Background</p> <p>Tall fescue (<it>Festuca arundinacea </it>Schreb) is a major cool season forage and turf grass species grown in the temperate regions of the world. In this paper we report the generation of a tall fescue expressed sequence tag (EST) database developed from nine cDNA libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. The results of inter-library and library-specific <it>in silico </it>expression analyses of these ESTs are also reported.</p> <p>Results</p> <p>A total of 41,516 ESTs were generated from nine cDNA libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. The <it>Festuca </it>Gene Index (FaGI) has been established. To date, this represents the first publicly available tall fescue EST database. <it>In silico </it>gene expression studies using these ESTs were performed to understand stress responses in tall fescue. A large number of ESTs of known stress response gene were identified from stressed tissue libraries. These ESTs represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. Highly expressed ESTs representing genes of unknown functions were also identified in the stressed tissue libraries.</p> <p>Conclusion</p> <p>FaGI provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. Comparative genomic analyses between tall fescue and other grass species, including ryegrasses (<it>Lolium </it>sp.), meadow fescue (<it>F. pratensis</it>) and tetraploid fescue (<it>F. arundinacea var glaucescens</it>) will benefit from this database. These ESTs are an excellent resource for the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) PCR-based molecular markers.</p

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species

Isolation of good quality chloroplast DNA (cpDNA) is a challenge in different plant species, although several methods for isolation are known. Attempts were undertaken to isolate cpDNA from Festuca grass species by using available standard protocols; however, they failed due to difficulties separating intact chloroplasts from the polysaccharides, oleoresin, and contaminated nuclear DNA that are present in the crude homogenate. In this study, we present a quick and inexpensive protocol for isolating intact chloroplasts from seven grass varieties/accessions of five Festuca species using a single layer of 30% Percoll solution. This protocol was successful in isolating high quality cpDNA with the least amount of contamination of other DNA. We performed Illumina MiSeq paired-end sequencing (2 &times; 300 bp) using 200 ng of cpDNA of each variety/accession. Chloroplast genome mapping showed that 0.28%&ndash;11.37% were chloroplast reads, which covered 94%&ndash;96% of the reference plastid genomes of the closely related grass species. This improved method delivered high quality cpDNA from seven grass varieties/accessions of five Festuca species and could be useful for other grass species with similar genome complexity

Molecular discrimination of tall fescue morphotypes in association with <i>Festuca</i> relatives

<div><p>Tall fescue (<i>Festuca arundinacea</i> Schreb.) is an important cool-season perennial grass species used as forage and turf, and in conservation plantings. There are three morphotypes in hexaploid tall fescue: Continental, Mediterranean and Rhizomatous. This study was conducted to develop morphotype-specific molecular markers to distinguish Continental and Mediterranean tall fescues, and establish their relationships with other species of the <i>Festuca</i> genus for genomic inference. Chloroplast sequence variation and simple sequence repeat (SSR) polymorphism were explored in 12 genotypes of three tall fescue morphotypes and four <i>Festuca</i> species. Hypervariable chloroplast regions were retrieved by using 33 specifically designed primers followed by sequencing the PCR products. SSR polymorphism was studied using 144 tall fescue SSR primers. Four chloroplast (NFTCHL17, NFTCHL43, NFTCHL45 and NFTCHL48) and three SSR (nffa090, nffa204 and nffa338) markers were identified which can distinctly differentiate Continental and Mediterranean morphotypes. A primer pair, NFTCHL45, amplified a 47 bp deletion between the two morphotypes is being routinely used in the Noble Research Institute’s core facility for morphotype discrimination. Both chloroplast sequence variation and SSR diversity showed a close association between Rhizomatous and Continental morphotypes, while the Mediterranean morphotype was in a distant clade. <i>F</i>. <i>pratensis</i> and <i>F</i>. <i>arundinacea</i> var. <i>glaucescens</i>, the P and G1G2 genome donors, respectively, were grouped with the Continental clade, and <i>F</i>. <i>mairei</i> (M1M2 genome) grouped with the Mediterranean clade in chloroplast sequence variation, while both <i>F</i>. <i>pratensis</i> and <i>F</i>. <i>mairei</i> formed independent clade in SSR analysis. Age estimation based on chloroplast sequence variation indicated that the Continental and Mediterranean clades might have been colonized independently during 0.65 ± 0.06 and 0.96 ± 0.1 million years ago (Mya) respectively. The findings of the study will enhance tall fescue breeding for persistence and productivity.</p></div

Polymorphism and similarity among tall fescue morphotypes and related species in 20 kb chloroplast sequences.

<p>Polymorphism and similarity index was calculated between the two plant groups in each row.</p

Classification of tall fescue germplasms collected from Greece based on four selected morphotype-specific chloroplast primers.

<p>The horizontal scale at bottom shows the Nei and Li/dice coefficients.</p

Median network formation for age estimation of diverged groups of 12 <i>Festuca</i> varieties/accessions.

<p>Red circles are the hypothesized median vectors to build the network with maximum parsimony.</p

Morphotype determination of a tall fescue population grown in Oklahoma field with unknown origin using morphotype-specific NFTCHL45 chloroplast primer.

<p>Morphotype determination of a tall fescue population grown in Oklahoma field with unknown origin using morphotype-specific NFTCHL45 chloroplast primer.</p