Haidong Yan

Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions

Background Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied. Methodology/Principal Findings In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments. Conclusions/Significance These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.

Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Switchgrass Under Various Abiotic Stress Conditions

Quantitative real-time reverse transcriptase PCR (qRT-PCR) is a sensitive and powerful technique for measuring differential gene expression; however, changes in gene expression induced by abiotic stresses are complex and multifaceted. Therefore, a set of stably expressed reference genes for data normalization is required. Switchgrass (Panicum virgatum L.) is a prime candidate crop for bioenergy production. The expression stability of reference genes in switchgrass, especially under different experimental conditions, is largely unknown. In order to identify the most suitable reference genes for abiotic stress studies in switchgrass, we evaluated 14 candidate genes for their expression stability under drought, high salinity, cold, heat, and waterlogging treatments using the Delta Ct, geNorm, BestKeeper, and NormFinder approaches. Validation of reference genes indicated that the best reference genes should be selected based on the stress treatment. Actin 2 (ACT2), carotenoid-binding protein 20 (CBP20), and Tubulin (TUB) were found to have the highest expression stability to study drought stress. 18S ribosomal RNA1 (18S rRNA1), ACT2, and TUB were the most stably expressed genes under salt stress. Ubiquitin-conjugating enzyme (UBC), TUB, and cyclophilin2 (CYP2) were the most suitable reference genes across cold treatments. Likewise, 18S rRNA1, UBC, and TUB were good reference genes for studying heat stress, while ACT2, 18S rRNA1, and ubiquitin3 (UBQ3) were the top three reference genes under waterlogging treatment. Considering that reference gene expression may vary across switchgrass tissues, ACT2 and 18S ribosomal RNA2 (18S rRNA2) were shown to be the most stably expressed genes in switchgrass leaves and roots, respectively. The highly ranked reference genes that were identified in this study were shown to be capable of detecting subtle differences in the expression rates of other genes. These differences may have been missed if a less suitable reference gene was used.

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

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.

Identifying differentially expressed genes under heat stress and developing molecular markers in orchardgrass (Dactylis glomerata L.) through transcriptome analysis

Orchardgrass (Dactylis glomerata L.) is a long‐lived, cool‐season forage grass that is commonly used for hay production. Despite its economic importance, orchardgrass genome remains relatively unexplored. In this study, we used Illumina RNA sequencing to identify gene‐associated molecular markers, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), as well as heat stress‐induced differentially expressed genes (DEGs) in two orchardgrass genotypes, ‘Baoxing’ (heat resistant) and ‘01998’ (heat susceptible). Approximately 163 million high‐quality trimmed reads were generated from 207 million raw reads using the Illumina HiSeq 2000 platform. A total of 126 846 unigenes were obtained after de novo assembly of the trimmed reads, and 40 078 unigenes were identified as coding sequences (CDSs). Based on the assembled unigenes, 669 300 high‐quality SNPs, including 416 099 transitions and 257 736 transversions, were contained in 75 875 unigenes. In addition, a total of 8475 microsatellites were detected in 7764 unigenes. When placed under heat stress, the total number of DEGs in ‘Baoxing’ (3527) was higher than in ‘01998’ (2649), indicating that in comparison with heat‐susceptible ‘01998’, heat‐resistant ‘Baoxing’ seems to have more unigenes that respond to heat stress. The high‐throughput transcriptome sequencing of orchardgrass under heat stress provides useful information for gene identification and for the development of SNP and SSR molecular markers. The comparison of DEGs under different periods of heat stress allowed us to identify a wealth of candidate DEGs that can be further analysed in order to determine the genetic mechanisms underlying heat tolerance in orchardgrass.

Molecular diversity and population structure of the forage grass Hemarthria compressa (Poaceae) in south China based on SRAP markers.

Hemarthria compressa is one of the most important and widely utilized forage crops in south China, owing to its high forage yield and capability of adaptation to hot and humid conditions. We examined the population structure and genetic variation within and among 12 populations of H. compressa in south China using sequence-related amplified polymorphism (SRAP) markers. High genetic diversity was found in these samples [percentage polymorphic bands (PPB) = 82.21%, Shannons diversity index (I) = 0.352]. However, there was relatively low level of genetic diversity at the population level (PPB = 29.17%, I = 0.155). A high degree of genetic differentiation among populations was detected based on other measures and molecular markers (Neis genetic diversity analysis: G(ST) = 54.19%; AMOVA analysis: F(ST) = 53.35%). The SRAP markers were found to be more efficient than ISSR markers for evaluating population diversity. Based on these findings, we propose changes in sampling strategies for appraising and utilizing the genetic resources of this species.

Genetic Diversity and Association of EST-SSR and SCoT Markers with Rust Traits in Orchardgrass (Dactylis glomerata L.)

Orchardgrass (Dactylis glomerata L.), is a well-known perennial forage species; however, rust diseases have caused a noticeable reduction in the quality and production of orchardgrass. In this study, genetic diversity was assessed and the marker-trait associations for rust were examined using 18 EST-SSR and 21 SCoT markers in 75 orchardgrass accessions. A high level of genetic diversity was detected in orchardgrass with an average genetic diversity index of 0.369. For the EST-SSR and SCoT markers, 164 and 289 total bands were obtained, of which 148 (90.24%) and 272 (94.12%) were polymorphic, respectively. Results from an AMOVA analysis showed that more genetic variance existed within populations (87.57%) than among populations (12.43%). Using a parameter marker index, the efficiencies of the EST-SSR and SCoT markers were compared to show that SCoTs have higher marker efficiency (8.07) than EST-SSRs (4.82). The results of a UPGMA cluster analysis and a STRUCTURE analysis were both correlated with the geographic distribution of the orchardgrass accessions. Linkage disequilibrium analysis revealed an average r2 of 0.1627 across all band pairs, indicating a high extent of linkage disequilibrium in the material. An association analysis between the rust trait and 410 bands from the EST-SSR and SCoT markers using TASSEL software revealed 20 band panels were associated with the rust trait in both 2011 and 2012. The 20 bands obtained from association analysis could be used in breeding programs for lineage selection to prevent great losses of orchardgrass caused by rust, and provide valuable information for further association mapping using this collection of orchardgrass.

Genetic Variability and Population Structure of the Potential Bioenergy Crop Miscanthus sinensis (Poaceae) in Southwest China Based on SRAP Markers

The genus Miscanthus has great potential as a biofuel feedstock because of its high biomass, good burning quality, environmental tolerance, and good adaptability to marginal land. In this study, the genetic diversity and the relationship of 24 different natural Miscanthus sinensis populations collected from Southwestern China were analyzed by using 33 pairs of Sequence Related Amplified Polymorphism (SRAP) primers. A total of 688 bands were detected with 646 polymorphic bands, an average of 19.58 polymorphic bands per primer pair. The average percentage of polymorphic loci (P), gene diversity (H), and Shannon’s diversity index (I) among the 24 populations are 70.59%, 0.2589, and 0.3836, respectively. The mean value of total gene diversity (HT) was 0.3373 ± 0.0221, while the allelic diversity within populations (HS) was 0.2589 ± 0.0136 and the allelic diversity among populations (DST) was 0.0784. The mean genetic differentiation coefficient (Gst = 0.2326) estimated from the detected 688 loci indicated that there was 76.74% genetic differentiation within the populations, which is consistent with the results from Analysis of Molecular Variance (AMOVA) analysis. Based upon population structure and phylogenetic analysis, five groups were formed and a special population with mixed ancestry was inferred indicating that human-mediated dispersal may have had a significant effect on population structure of M. sinensis. Evaluating the genetic structure and genetic diversity at morphological and molecular levels of the wild M. sinensis in Southwest China is critical to further utilize the wild M. sinensis germplasm in the breeding program. The results in this study will facilitate the biofuel feedstock breeding program and germplasm conservation.

Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses.

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses.

Genome-wide survey of switchgrass NACs family provides new insights into motif and structure arrangements and reveals stress-related and tissue-specific NACs

NAC proteins comprise of a plant-specific transcription factor (TF) family and play important roles in plant development and stress responses. Switchgrass (Panicum virgatum) is the prime candidate and model bioenergy grass across the world. Excavating agronomically valuable genes is important for switchgrass molecular breeding. In this study, a total of 251 switchgrass NAC (PvNACs) family genes clustered into 19 subgroups were analyzed, and those potentially involved in stress response or tissue-specific expression patterns were pinpointed. Specifically, 27 PvNACs were considered as abiotic stress-related including four membrane-associated ones. Among 40 tissue-specific PvNACs expression patterns eight factors were identified that might be relevant for lignin biosynthesis and/or secondary cell wall formation. Conserved functional domains and motifs were also identified among the PvNACs and potential association between these motifs and their predicted functions were proposed, that might encourage experimental studies to use PvNACs as possible targets to improve biomass production and abiotic stress tolerance.

Genome-Wide Analysis of the PvHsp20 Family in Switchgrass: Motif, Genomic Organization, and Identification of Stress or Developmental-Related Hsp20s

Hsp20 proteins exist in all plant species and represent the most abundant small heat shock proteins (sHSPs) in plants. Hsp20s were known as chaperones maintaining cellular homeostasis during heat or other kinds of abiotic stresses. The objective of this study was to understand the phylogenetic relationship, genomic organization, diversification of motif modules, genome localization, expression profiles, and interaction networks of switchgrass (Panicum virgatum L.) Hsp20s (PvHsp20s). A total of 63 PvHsp20s were identified with their consensus as well as unique ACD motifs and gene structures analyzed. Most PvHsp20s (87%) were responsive to heat and other kinds of abiotic stresses. When under optimum growth condition, 38 of them displayed relative higher expression levels in inflorescence and seeds, suggesting their protective roles in the stress-sensitive reproductive organs. An in silico analysis of interaction network of PvHsp20 proteins further revealed potential interactive proteins, including stress-inducible ones in the network. Furthermore, PvHsp20 genes unevenly distributed in two sets of homeologous chromosomes, and only segmental duplication was found among the paralogous gene pairs, reflecting that the allotetraploidization of switchgrass allowed the accumulation of PvHsp20s that in turn facilitated its successful adaptation in hot and dry plateaus of North America. The present results provided an insight into PvHsp20s with an emphasis on the uniqueness of this gene family in switchgrass. Such information shall also be useful in functional studies of PvHsp20 genes and molecular breeding of switchgrass.