LcMYB4, an unknown function transcription factor gene from sheepgrass, as a positive regulator of chilling and freezing tolerance in transgenic Arabidopsis.

BACKGROUND: Sheepgrass (Leymus chinensis (Trin.) Tzvel) is a perennial forage grass that can survive extreme freezing winters (- 47.5 °C) in China. In this study, we isolated an unknown function MYB transcription factor gene, LcMYB4, from sheepgrass. However, the function of LcMYB4 and its homologous genes has not been studied in other plants. RESULTS: The expression of the LcMYB4 gene was upregulated in response to cold induction, and the LcMYB4 fusion protein was localized in the nucleus, with transcriptional activation activity. Biological function analysis showed that compared with WT plants, LcMYB4-overexpressing Arabidopsis presented significantly increased chilling and freezing tolerance as evidenced by increased germination rate, survival rate, and seed setting rate under conditions of low temperature stress. Furthermore, LcMYB4-overexpressing plants showed increased soluble sugar content, leaf chlorophyll content and superoxide dismutase activity but decreased malondialdehyde (MDA) under chilling stress. Moreover, the expression of the CBF1, KIN1, KIN2 and RCI2A genes were significantly upregulated in transgenic plants with chilling treatment. These results suggest that LcMYB4 overexpression increased the soluble sugar content and cold-inducible gene expression and alleviated oxidative damage and membrane damage, resulting in enhanced cold resistance in transgenic plants. Interestingly, our results showed that the LcMYB4 protein interacts with fructose-1,6-bisphosphate aldolase protein1 (LcFBA1) and that the expression of the LcFBA1 gene was also upregulated during cold induction in sheepgrass, similar to LcMYB4. CONCLUSION: Our findings suggest that LcMYB4 encodes MYB transcription factor that plays a positive regulatory role in cold stress.

A MYB-related transcription factor from sheepgrass, LcMYB2, promotes seed germination and root growth under drought stress.

BACKGROUND: Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. RESULTS: In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. CONCLUSIONS: Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.

bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy.

LcbHLH92, a pleiotropic gene from sheepgrass, negatively regulates anthocyanins/proanthocyandins and reduces seed dormancy in transgenic Arabidopsis.

LcFIN2, a novel chloroplast protein gene from sheepgrass, enhances tolerance to low temperature in Arabidopsis and rice.

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA-sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing-induced protein 2, encoded a chloroplast-targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non-photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal-related and photosynthesis-related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low-temperature tolerance in crops.

Transcriptomic Analysis Reveals a Comprehensive Calcium- and Phytohormone-Dominated Signaling Response in Leymus chinensis Self-Incompatibility.

Sheepgrass (Leymus chinensis (Trin.) Tzvel.) is an economically and ecologically important forage in the grass family. Self-incompatibility (SI) limits its seed production due to the low seed-setting rate after self-pollination. However, investigations into the molecular mechanisms of sheepgrass SI are lacking. Therefore, microscopic observation of pollen germination and pollen tube growth, as well as transcriptomic analyses of pistils after self- and cross-pollination, were performed. The results indicated that pollen tube growth was rapidly inhibited from 10 to 30 min after self-pollination and subsequently stopped but preceded normally after cross-pollination. Time course comparative transcriptomics revealed different transcriptome dynamics between self- and cross-pollination. A pool of SI-related signaling genes and pathways was generated, including genes related to calcium (Ca2+) signaling, protein phosphorylation, plant hormone, reactive oxygen species (ROS), nitric oxide (NO), cytoskeleton, and programmed cell death (PCD). A putative SI response molecular model in sheepgrass was presented. The model shows that SI may trigger a comprehensive calcium- and phytohormone-dominated signaling cascade and activate PCD, which may explain the rapid inhibition of self-pollen tube growth as observed by cytological analyses. These results provided new insight into the molecular mechanisms of sheepgrass (grass family) SI.

MADS-box family genes in sheepgrass and their involvement in abiotic stress responses.

BACKGROUND: MADS-box genes are categorized into A, B, C, D and E classes and are involved in floral organ identity and flowering. Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important perennial forage grass and adapts well to many adverse environments. However, there are few studies on the molecular mechanisms of flower development in sheepgrass, especially studies on MADS-domain proteins. RESULTS: In this study, we cloned 11 MADS-box genes from sheepgrass (Leymus chinensis (Trin.) Tzvel), and phylogenetic analysis of the 11 genes with their homologs revealed that they are divided into nine subclades. Tissue-specific expression profile analysis showed that most of these MADS-box genes were highly expressed in floral organs. LcMADS1 and LcMADS3 showed higher expression in the stamen than in the other tissues, and LcMADS7 showed high expression in the stamen, glume, lemma and palea, while expression of LcMADS2, LcMADS9 and LcMADS11 was higher in vegetative organs than floral organs. Furthermore, yeast two-hybrid analyses showed that LcMADS2 interacted with LcMADS7 and LcMADS9. LcMADS3 interacted with LcMADS4, LcMADS7 and LcMADS10, while LcMADS1 could interact with only LcMADS7. Interestingly, the expression of LcMADS1 and LcMADS2 were significantly induced by cold, and LcMADS9 was significantly up-regulated by NaCl. CONCLUSION: Hence, we proposed that LcMADS1, LcMADS2, LcMADS3, LcMADS7 and LcMADS9 play a pivotal role in sheepgrass sexual reproduction and may be involved in abiotic stress responses, and our findings provide useful information for further exploration of the functions of this gene family in rice, wheat and other graminaceous cereals.

Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance.

Gibberellin stimulates regrowth after defoliation of sheepgrass (Leymus chinensis) by regulating expression of fructan-related genes.

Gibberellins (GAs) affect forage growth and development; however, it is largely unknown how GAs regulate the metabolism of fructan (an important polysaccharide reserve in many cereals) and the regrowth of forage plants after defoliation. To explore the mechanism of the responses of defoliated sheepgrass [Leymus chinensis (Trin.) Tzvel] to GA, we sprayed defoliated sheepgrass with GA3 and/or paclobutrazol (PAC; an inhibitor of GA biosynthesis) and analyzed the growth characteristics, carbohydrate contents, and transcript levels of genes related to GA metabolism, GA signal transduction, and fructan metabolism. The results showed that spraying exogenous GA3 onto defoliated sheepgrass promoted leaf and internode elongation, while spraying with PAC inhibited leaf and internode elongation, compared with the control. Spraying GA3 onto defoliated sheepgrass also altered the fructan content by extending the period of fructan utilization. At the transcriptional level, exogenous GA3 increased the transcript levels of genes related to GA metabolism in the sheath. Taken together, our results suggest that exogenous GA3 stimulates the regrowth of defoliated sheepgrass regrowth by regulating GA and fructan-related genes, and by promoting endogenous GA synthesis, fructan metabolism, and signaling.

Bovine serum albumin in saliva mediates grazing response in Leymus chinensis revealed by RNA sequencing.

BACKGROUND: Sheepgrass (Leymus chinensis) is an important perennial forage grass across the Eurasian Steppe and is adaptable to various environmental conditions, but little is known about its molecular mechanism responding to grazing and BSA deposition. Because it has a large genome, RNA sequencing is expensive and impractical except for the next-generation sequencing (NGS) technology. RESULTS: In this study, NGS technology was employed to characterize de novo the transcriptome of sheepgrass after defoliation and grazing treatments and to identify differentially expressed genes (DEGs) responding to grazing and BSA deposition. We assembled more than 47 M high-quality reads into 120,426 contigs from seven sequenced libraries. Based on the assembled transcriptome, we detected 2,002 DEGs responding to BSA deposition during grazing. Enrichment analysis of Gene ontology (GO), EuKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that the effects of grazing and BSA deposition involved more apoptosis and cell oxidative changes compared to defoliation. Analysis of DNA fragments, cell oxidative factors and the lengths of leaf scars after grazing provided physiological and morphological evidence that BSA deposition during grazing alters the oxidative and apoptotic status of cells. CONCLUSIONS: This research greatly enriches sheepgrass transcriptome resources and grazing-stress-related genes, helping us to better understand the molecular mechanism of grazing in sheepgrass. The grazing-stress-related genes and pathways will be a valuable resource for further gene-phenotype studies.

The large-scale investigation of gene expression in Leymus chinensis stigmas provides a valuable resource for understanding the mechanisms of poaceae self-incompatibility.

BACKGROUND: Many Poaceae species show a gametophytic self-incompatibility (GSI) system, which is controlled by at least two independent and multiallelic loci, S and Z. Until currently, the gene products for S and Z were unknown. Grass SI plant stigmas discriminate between pollen grains that land on its surface and support compatible pollen tube growth and penetration into the stigma, whereas recognizing incompatible pollen and thus inhibiting pollination behaviors. Leymus chinensis (Trin.) Tzvel. (sheepgrass) is a Poaceae SI species. A comprehensive analysis of sheepgrass stigma transcriptome may provide valuable information for understanding the mechanism of pollen-stigma interactions and grass SI. RESULTS: The transcript abundance profiles of mature stigmas, mature ovaries and leaves were examined using high-throughput next generation sequencing technology. A comparative transcriptomic analysis of these tissues identified 1,025 specifically or preferentially expressed genes in sheepgrass stigmas. These genes contained a significant proportion of genes predicted to function in cell-cell communication and signal transduction. We identified 111 putative transcription factors (TFs) genes and the most abundant groups were MYB, C2H2, C3H, FAR1, MADS. Comparative analysis of the sheepgrass, rice and Arabidopsis stigma-specific or preferential datasets showed broad similarities and some differences in the proportion of genes in the Gene Ontology (GO) functional categories. Potential SI candidate genes identified in other grasses were also detected in the sheepgrass stigma-specific or preferential dataset. Quantitative real-time PCR experiments validated the expression pattern of stigma preferential genes including homologous grass SI candidate genes. CONCLUSIONS: This study represents the first large-scale investigation of gene expression in the stigmas of an SI grass species. We uncovered many notable genes that are potentially involved in pollen-stigma interactions and SI mechanisms, including genes encoding receptor-like protein kinases (RLK), CBL (calcineurin B-like proteins) interacting protein kinases, calcium-dependent protein kinase, expansins, pectinesterase, peroxidases and various transcription factors. The availability of a pool of stigma-specific or preferential genes for L. chinensis offers an opportunity to elucidate the mechanisms of SI in Poaceae.

LcWRKY5: an unknown function gene from sheepgrass improves drought tolerance in transgenic Arabidopsis.

KEY MESSAGE: The expression of LcWRKY5 was induced significantly by salinity, mannitol and cutting treatments. Arabidopsis- overexpressing LcWRKY5 greatly increased dehydration tolerance by regulating the expression of multiple stress-responsive genes. Based on the data of sheepgrass 454 high-throughout sequencing and expression analysis results, a drought-induced gene LcWRKY5 was isolated and cloned, and the biological role of the gene has not been reported until now. Bioinformatics analysis showed that LcWRKY5 contains one conserved WD domain and belongs to the group II WRKY protein family. LcWRKY5 shows high sequence identity with predicted or putative protein products of Hordeum vulgare, Aegilops tauschii, Triticum aestivum, Brachypodium distachyon, Oryza sativa, but it has low homology with WRKYs from dicotyledonous plants. Several drought-inducibility, fungal elicitor, MeJA-responsiveness, endosperm, light, anoxic specific inducibility, and circadian control elements were found in the promoter region of LcWRKY5. Tissue-specific expression patterns showed that LcWRKY5 is expressed in roots and leaves, without expression in other tissues. The expression of LcWRKY5 was induced significantly under salinity and mannitol stresses but was not notably changed under cold and Abscisic acid stress. The LcWRKY5 protein exhibits transcription activation activity in the yeast one-hybrid system. Overexpressing LcWRKY5 exhibited increased rates of cotyledon greening and plant survival in transgenic Arabidopsis compared with wild-type plants under drought stress, and the expression levels of DREB2A and RD29A in transgenic plants were enhanced under drought stress. These results indicated that LcWRKY5 may play an important role in drought-response networks through regulation of the DREB2A pathway. LcWRKY5 can be a candidate gene for engineering drought tolerance in other crops.

LcSAIN1, a novel salt-induced gene from sheepgrass, confers salt stress tolerance in transgenic Arabidopsis and rice.

Previously, we identified >1,500 genes that were induced by high salt stress in sheepgrass (Leymus chinensis, Gramineae: Triticeae) when comparing the changes in their transcription levels in response to high salt stress by next-generation sequencing. Among the identified genes, a gene of unknown function (designated as Leymus chinensis salt-induced 1, LcSAIN1) showed a high sequence identity to its homologs from wheat, Hordeum vulgare and Oryza sativa, but LcSAIN1 and its homologs produce hypothetical proteins with no conserved functional domains. Transcription of the LcSAIN1 gene was up-regulated by various stresses. The overexpression of LcSAIN1 in Arabidopsis and rice increased the greening rate of cotyledons, the fresh weight, root elongation, plant height and the plant survival rate when compared with control plants and conferred a tolerance against salt stress. Subcellular localization analysis indicated that LcSAIN1 is localized predominantly in the nucleus. Our results show that the LcSAIN1 gene might play an important positive modulation role in increasing the expression of transcription factors (MYB2 and DREB2A) and functional genes (P5CS and RAB18) in transgenic plants under salt stress and that it augments stress tolerance through the accumulation of compatible solutes (proline and soluble sugar) and the alleviation of changes in reactive oxygen species. The LcSAIN1 gene could be a potential resource for engineering salinity tolerance in important crop species.

Responses of biomass accumulation and nutrient utilization along a phosphorus supply gradient in Leymus chinensis.

Phosphorus (P) deficiencies are widespread in calcareous soils. The poor availability of nitrogen (N) and P in soils often restricts crop growth. However, the effects of P addition on plant growth and plant nutrient transport changes during the establishment of Leymus chinensis fields in Xinjiang are not clear. We investigated the responses of Leymus chinensis biomass and nutrient absorption and utilization to changes in soil N and P by adding P (0, 15.3, 30.6, and 45.9 kg P ha-1 year-1) with basally applied N fertilizer (150 kg N ha-1 year-1). The results showed that (a) Principal component analysis (PCA) of biomass, nutrient accumulation, soil available P, and soil available N during the different periods of Leymus chinensis growth showed that their cumulative contributions during the jointing and harvest periods reached 95.4% and 88%, respectively. (b) Phosphorus use efficiency (PUE) increased with the increase of P fertilizer gradient and then decreased and the maximum PUE was 13.14% under moderate P addition. The accumulation of biomass and nutrients in Leymus chinensis can be effectively improved by the addition of P fertilizer at 30.6 kg ha-1. Different P additions either moderately promoted or excessively inhibited Leymus chinensis growth and nutrient utilization.

[Application of next generation sequencing techniques in plant transcriptome].

With the development of DNA sequencing techniques, the next-generation sequencing (NGS) techniques with the characteristics of high-throughput and low cost have become the first choice for more and more researchers to carry out the biological researches. Among the next-generation sequencing techniques, the 454 sequencing platform is the first commercially available and relatively mature one and widely used in various fields of biological research. Taking 454 sequencing platform as an example, we illustrate the advantages and disadvantages of NGS technical principles, review their applications in plant transcriptome, and outlook their future development and applications in plant research field.

Ectopic Expression of a Salt-Inducible Gene, LcSAIN3, from Sheepgrass Improves Seed Germination and Seedling Growth under Salt Stress in Arabidopsis.

Sheepgrass is a perennial native grass species in China, and it can tolerate high levels of salt stress with an aggressive and vigorous rhizome system. Many salt-stress-responsive genes have been identified in sheepgrass. In this study, we report the cloning and characterization of a novel salt-induced gene, LcSAIN3 (Leymus chinensis salt-induced 3), from sheepgrass. Expression analysis confirmed that LcSAIN3 was induced by PEG, ABA, and salt treatments, and the expression of LcSAIN3 was significantly increased in salt-tolerant germplasms under salt treatment. Subcellular localization analysis indicated that the GFP-LcSAIN3 protein was mainly localized in the chloroplasts. The heterologous expression of LcSAIN3 in Arabidopsis increased the seed germination rate of transgenic plants under salt, ABA, and mannitol treatments. The seedling survival rate, plant height, and fresh weight of the transgenic plants were higher than those of WT plants under salt stress. The overexpression of LcSAIN3 caused a relatively high accumulation of free proline, enhanced SOD activity, and led to the upregulation of several stress-responsive genes such as AtRD26, AtRD29B, AtSOS1, and AtP5CS1. These results suggest that LcSAIN3 could be a potential target for molecular breeding to improve plants' salt tolerance.

A comparative proteomic analysis of rice seedlings under various high-temperature stresses.

To understand the responses of rice seedlings to different high-temperature stresses, seven-day-old rice seedlings were exposed to different high temperatures for 48 h, and the maximal quantum yield of PS II photochemistry measurements, ascorbate peroxidase activity assays and proteomic analyses in leaf tissue were performed. The results showed that when rice seedlings were exposed to high temperatures at 35 degrees C, 40 degrees C and 45 degrees C, the maximal quantum yield of photosystem II photochemistry, the activity of ascorbate peroxidase and the proteome changed greater at higher temperature. The proteomics analysis showed that proteins such as lignification-related proteins were regulated by high temperature and distinct proteins related to protection were up-regulated at different high temperatures. All the results indicated that different strategies were adopted at different levels of high temperature: the higher the temperature, the more protection machineries were involved. At 35 degrees C, some protective mechanisms were activated to maintain the photosynthetic capability. At 40 degrees C, antioxidative pathways were also active. When rice seedlings encountered high-temperature stress at 45 degrees C, in addition to those induced at 35 degrees C and 40 degrees C, heat shock proteins were effectively induced.

Major energy plants and their potential for bioenergy development in China.

China is rich in energy plant resources. In this article, 64 plant species are identified as potential energy plants in China. The energy plant species include 38 oilseed crops, 5 starch-producing crops, 3 sugar-producing crops and 18 species for lignocellulosic biomass. The species were evaluated on the basis of their production capacity and their resistance to salt, drought, and/or low temperature stress. Ten plant species have high production and/or stress resistance and can be potentially developed as the candidate energy plants. Of these, four species could be the primary energy plants in China: Barbados nut (Jatropha curcas L.), Jerusalem artichoke (Helianthus tuberosus L.), sweet sorghum (Sorghum bicolor L.) and Chinese silvergrass (Miscanthus sinensis Anderss.). We discuss the use of biotechnological techniques such as genome sequencing, molecular markers, and genetic transformation to improve energy plants. These techniques are being used to develop new cultivars and to analyze and manipulate genetic variation to improve attributes of energy plants in China.

Enhanced drought tolerance in transgenic Leymus chinensis plants with constitutively expressed wheat TaLEA3.

Leymus chinensis is an important grassland perennial grass. However, its drought tolerance requires to be improved. LEA (late embryogenesis abundant) genes are believed to confer resistance to drought and water deficiency. Using Agrobacterium-mediated transformation, a wheat LEA gene, TaLEA(3), was integrated into L. chinensis. The transgenic lines showed enhanced growth ability under drought stress during which transgenic lines had increased the relative water content, leaf water potential, relative average growth rate, but decreased the malondialdehyde content compared with the non-transgenic plant. Thus, transgenic breeding is an efficient approach to enhance drought tolerance in L. chinensis.

Germination characteristics among different sheepgrass (Leymus chinensis) germplasm during the seed development and after-ripening stages.

Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important forage grass in the Eurasian steppe. However, little information is available concerning its seed morphological features and germination characteristics during seed development and after-ripening among different germplasm. To clarify the appropriate seed harvest time and the effects of germplasm, seed development and after-ripening on seed germination, 20 germplasm of sheepgrass were selected. Moreover, the seed morphological and physical changes as well as the seed germination and dormancy characteristics of sheepgrass during seed development stages were analyzed using a seven-d gradient of day after pollination (DAP). The results indicated that the seed water content decreased significantly during 35-42 DAP and that the highest seed germination rate of most germplasm was observed at 35-42 DAP. Thus, 35-42 DAP may be the best time to harvest sheepgrass to obtain the maximum seed germination rate and avoid seed shattering. Furthermore, our results indicated that there were six types of germination patterns, including germplasm with increasing germination rates in the developing seed, such as S19 and S13, and germplasm that maintained a consistently low germination rate, such as S10. Moreover, we compared the seed germination rate of eight germplasm during seed development in both 2016 and 2017, and the results indicated that the seed germination patterns of the eight germplasm were highly consistent between the two consecutive years, suggesting that germplasm rather than year is the major factor in determining germination during seed development. The effect of after-ripening on seed germination was different among the germplasm where four types of germination patterns were revealed for 10 germplasm and resulted in various dormancy features. A two-factor ANOVA analysis suggested that the germplasm of the sheepgrass has a large influence on seed germination, whether during seed development or after-ripening. Thus, these findings lay the foundation for future studies on seed dormancy and germination and may guide the breeding of new cultivars of sheepgrass with better germination performance.