Xinquan Zhang

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.

Exogenous Spermidine Improves Seed Germination of White Clover under Water Stress via Involvement in Starch Metabolism, Antioxidant Defenses and Relevant Gene Expression

This study was designed to determine the effect of exogenous spermidine (Spd) (30 μM) on white clover seed germination under water stress induced by polyethylene glycol 6000. Use of seed priming with Spd improved seed germination percentage, germination vigor, germination index, root viability and length, and shortened mean germination time under different water stress conditions. Seedling fresh weight and dry weight also increased significantly in Spd-treated seeds compared with control (seeds primed with distilled water). Improved starch metabolism was considered a possible reason for this seed invigoration, since seeds primed with Spd had significantly increased α-amylase/β-amylase activities, reducing sugar, fructose and glucose content and transcript level of β-amylase gene but not transcript level of α-amylase gene. In addition, the physiological effects of exogenous Spd on improving seeds’ tolerance to water deficit during germination were reflected by lower lipid peroxidation levels, better cell membrane stability and significant higher seed vigour index in seedlings. Enhanced antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase), ascorbate-glutathione cycle (ASC-GSH cycle) and transcript level of genes encoding antioxidant enzymes induced by exogenous Spd may be one of the critical reasons behind acquired drought tolerance through scavenging of reactive oxygen species (ROS) in water-stressed white clover seeds. The results indicate that Spd plays an important function as a stress-protective compound or physiological activator.

Transcriptional Profiles of Drought-Related Genes in Modulating Metabolic Processes and Antioxidant Defenses in Lolium multiflorum.

Drought is a major environmental stress that limits growth and development of cool-season annual grasses. Drought transcriptional profiles of resistant and susceptible lines were studied to understand the molecular mechanisms of drought tolerance in annual ryegrass (Lolium multiflorum L.). A total of 4718 genes exhibited significantly differential expression in two L. multiflorum lines. Additionally, up-regulated genes associated with drought response in the resistant lines were compared with susceptible lines. Gene ontology enrichment and pathway analyses revealed that genes partially encoding drought-responsive proteins as key regulators were significantly involved in carbon metabolism, lipid metabolism, and signal transduction. Comparable gene expression was used to identify the genes that contribute to the high drought tolerance in resistant lines of annual ryegrass. Moreover, we proposed the hypothesis that short-term drought have a beneficial effect on oxidation stress, which may be ascribed to a direct effect on the drought tolerance of annual ryegrass. Evidence suggests that some of the genes encoding antioxidants (HPTs, GGT, AP, 6-PGD, and G6PDH) function as antioxidant in lipid metabolism and signal transduction pathways, which have indispensable and promoting roles in drought resistance. This study provides the first transcriptome data on the induction of drought-related gene expression in annual ryegrass, especially via modulation of metabolic homeostasis, signal transduction, and antioxidant defenses to improve drought tolerance response to short-term drought stress.

Polyamine regulates tolerance to water stress in leaves of white clover associated with antioxidant defense and dehydrin genes via involvement in calcium messenger system and hydrogen peroxide signaling.

Endogenous polyamine (PA) may play a critical role in tolerance to water stress in plants acting as a signaling molecule activator. Water stress caused increases in endogenous PA content in leaves, including putrescine (Put), spermidine (Spd), and spermine (Spm). Exogenous application of Spd could induce the instantaneous H2O2 burst and accumulation of cytosolic free Ca2+, and activate NADPH oxidase and CDPK gene expression in cells. To a great extent, PA biosynthetic inhibitor reduced the water stress-induced H2O2 accumulation, free cytosolic Ca2+ release, antioxidant enzyme activities and genes expression leading to aggravate water stress-induced oxidative damage, while these suppressing effects were alleviated by the addition of exogenous Spd, indicating PA was involved in water stress-induced H2O2 and cytosolic free Ca2+ production as well as stress tolerance. Dehydrin genes (Y2SK, Y2K, and SK2) were showed to be highly responsive to exogenous Spd. PA-induced antioxidant defense and dehydrin genes expression could be blocked by the scavenger of H2O2 and the inhibitors of H2O2 generation or Ca2+ channels blockers, a calmodulin antagonist, as well as the inhibitor of CDPK. These findings suggested that PA regulated tolerance to water stress in white clover associated with antioxidant defenses and dehydrins via involvement in the calcium messenger system and H2O2 signaling pathways. PA-induced H2O2 production required Ca2+ release, while PA-induced Ca2+ release was also essential for H2O2 production, suggesting an interaction between PA-induced H2O2 and Ca2+ signaling.

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.

Diversity comparison and phylogenetic relationships of cocksfoot (Dactylis glomerata L.) germplasm as revealed by SSR markers

Phylogenetic relationships among and within 16 accessions collected from China, Russia, Kazakhstan, India and Japan were investigated using SSR markers. The 21 SSR primer pairs generated a total of 143 polymorphic alleles, with an average of 6.8 alleles per locus, and a range of 3-13 alleles. The average genetic diversity, as measured by the polymorphic information content (PIC), was 0.33. The average polymorphic rate (P) for this species was 90.7%, suggesting a high degree of genetic diversity. Analysis of molecular variation (AMOVA) revealed larger genetic variation within accessions (65.75%) and geographical regions (75.58%) rather than between them, while among ploidy level variance contributed only 2.62%. A high degree of genetic diversity within tetraploid group was detected based on the polymorphic rate (P = 94.23%) and shannon’ s information index of diversity (I = 0.4300) when compared with diploid group (P = 91.25%, I = 0.3922). The values of the genetic identity (GI) among accessions ranged fro...

Construction of high-density genetic linkage map and identification of flowering-time QTLs in orchardgrass using SSRs and SLAF-seq

Orchardgrass (Dactylis glomerata L.) is one of the most economically important perennial, cool-season forage species grown and pastured worldwide. High-density genetic linkage mapping is a valuable and effective method for exploring complex quantitative traits. In this study, we developed 447,177 markers based on SLAF-seq and used them to perform a comparative genomics analysis. Perennial ryegrass sequences were the most similar (5.02%) to orchardgrass sequences. A high-density linkage map of orchardgrass was constructed using 2,467 SLAF markers and 43 SSRs, which were distributed on seven linkage groups spanning 715.77 cM. The average distance between adjacent markers was 0.37 cM. Based on phenotyping in four environments, 11 potentially significant quantitative trait loci (QTLs) for two target traits–heading date (HD) and flowering time (FT)–were identified and positioned on linkage groups LG1, LG3, and LG5. Significant QTLs explained 8.20–27.00% of the total phenotypic variation, with the LOD ranging from 3.85–12.21. Marker167780 and Marker139469 were associated with FT and HD at the same location (Ya’an) over two different years. The utility of SLAF markers for rapid generation of genetic maps and QTL analysis has been demonstrated for heading date and flowering time in a global forage grass.

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.