Junqin Zong

Systematic mining of salt-tolerant genes in halophyte-Zoysia matrella through cDNA expression library screening.

Though a large number of salt-tolerant genes were identified from Glycophyte in previous study, genes involved in salt-tolerance of halophyte were scarcely studied. In this report, an important halophyte turfgrass, Zoysia matrella, was used for systematic excavation of salt-tolerant genes using full-length cDNA expression library in yeast. Adopting the Gateway-compatible vector system, a high quality entry library was constructed, containing 3 × 10(6) clones with an average inserted fragments length of 1.64 kb representing a 100% full-length rate. The yeast expression library was screened in a salt-sensitive yeast mutant. The screening yielded dozens of salt-tolerant clones harboring 16 candidate salt-tolerant genes. Under salt-stress condition, these 16 genes exhibited different transcription levels. According to the results, we concluded that the salt-tolerance of Z. matrella might result from known genes involved in ion regulation, osmotic adjustment, as well as unknown pathway associated with protein folding and modification, RNA metabolism, and mitochondrial membrane translocase, etc. In addition, these results shall provide new insight for the future researches with respect to salt-tolerance.

Comparative transcriptome analysis provides new insights into erect and prostrate growth in bermudagrass (Cynodon dactylon L.)

Bermudagrass (Cynodon dactylon L.) is a prominent warm-season turf and forage grass species with multiple applications. In most C. dactylon cultivars and accessions, erect-growing stems (shoot) and prostrate-growing stems (stolon) often coexist. These two types of stems are both formed through tillering but grow in two directions with different tiller angles. Elucidating the mechanism of tiller angle regulation in bermudagrass could provide important clues to breed cultivars with different plant architectural features for diverse usage. In this study, we compared the stem internode transcriptome of two bermudagrass wild accessions with extremely different tiller angles and stem growth directions. A total of 2088 and 12,141 unigenes were preferentially expressed in prostrate-growing wild accession C792 and erect-growing wild accession C793, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology-based Annotation System (KOBAS) analyses further indicated that light- and gravity-responsive genes were enriched in accession C792, whereas lignin synthesis-related genes were enriched in accession C793, which well explains the difference in lignification of vascular bundles and mechanical tissues in the two accessions. These results not only expand our understanding of the genetic control of tiller angle and stem growth direction in bermudagrass but also provide insight for future molecular breeding of C. dactylon and other turfgrass species with different plant architectures.

Genetic variation of salinity tolerance in Chinese natural bermudagrass (Cynodon dactylon (L.) Pers.) germplasm resources

Bermudagrass (Cynodon dactylon (L.) Pers.) is a widely used turfgrass in tropical, subtropical and warm-temperate regions in the world. It is a salinity-tolerant plant, but much genetic variation exists among its genotypes. Genetic diversity has been shown to exist from a morphologic level to a molecular level among Chinese natural bermudagrass germplasm resources; however, the extent of variation in their salinity tolerance has remained unclear. We conducted greenhouse hydroponic and sand culture experiments to evaluate the variation in salinity tolerance of natural bermudagrass germplasm resources obtained from their main areas of distribution in China and treated at a 33 dS m−1 salinity level for three weeks. Compared with non-saline controls, salinity stress significantly decreased shoot clipping weight (46.5% and 44.2%) and increased leaf firing (41.1% and 37.6%) in hydroponic and sand culture experiments, respectively, across all genotypes. However, significant genetic variations in relative leaf firing percentage (RLF) and relative shoot weight (RSW) were found among genotypes in both experiments, and their coefficients of variation ranged from 25.5% to 41.6%, indicating that considerable variation exists in the salinity tolerance of Chinese natural bermudagrass germplasm resources. Shoot Na+ concentrations increased under salinity stress for all genotypes in both experiments. However, significant genetic variations were also found in shoot Na+ concentrations. Salinity tolerance in bermudagrass genotypes was found to be significantly negatively correlated with shoot Na+ concentrations in both experiments. Using a cluster analysis of RLF and RSW data, all genotypes were classified into four groups with different salinity tolerances. The results of our evaluation indicate that there is much potential for improving salinity tolerance among Chinese natural bermudagrass cultivars.

Growth and physiological responses of two phenotypically distinct accessions of centipedegrass (Eremochloa ophiuroides (Munro) Hack.) to salt stress

Salinity is one of the major abiotic environmental stress factors affecting plant growth and development. Centipedegrass (Eremochloa ophiuroides [Munro)] Hack.) is an important warm-season turfgrass species with low turf maintenance requirements, but is sensitive to salinity stress. To explore salt tolerant germplasms in centipedegrass and better understand the growth and physiological responses of centipedegrass to salinity, we conducted anatomic observation and phytochemical quantification, examined growth parameters, and investigated photosynthetic machinery and antioxidant system in two phenotypically distinct centipedegrass accessions under NaCl salt stress. The morphophenotypical difference of the stems in the two accessions mainly depends on whether or not a thickened epidermal horny layer with purple colour was formed, which was caused by anthocyanin accumulation in the tissue. Successive salinity treatment was found to result in an inhibition of leaf growth, a marked decrease in photosynthesis, chlorophyll contents, and the maximal photochemical efficiency of PSII (Fv/Fm). Under the same treatment, purple-stem accession (E092) showed a lower degree of inhibition or decrease than green-stem one (E092-1). With the exception of malondialdehyde level, both proline content and antioxidant enzymes were upregulated to a greater extent in E092 following exposure to salinity condition. Meanwhile, significant enhancements of anthocyanin accumulation and total protein synthesis were detected in E092 after salt treatment, but not in E092-1. These results demonstrated that E092 favor better accumulation of anthocyanins under salinity condition, which contribute to salt tolerance by adjusting physiological functions and osmotic balance, and better maintenance of high turf quality. Hence, genetic phenotype can be utilized as a key indicator in E. ophiuroides breeding for salt-tolerance.

Effects of low nitrogen nutrition on plant growth characteristics and nitrogen accumulation in Chinese natural bermudagrass (Cynodon dactylon (L.) Pers.) germplasm resources

ABSTRACT Bermudagrass (Cynodon dactylon (L.) Pers.) is a widely used warm-season turfgrass and has the highest nitrogen (N) fertilization requirements among turfgrasses. Wild ecotype found in N-poor habitats can potentially tolerant N limiting conditions for reducing N inputs in the turf industry. However, the extent of variation in their low N tolerance has remained unclear. We conducted hydroponic experiments to evaluate the variation on growth, N accumulation, and partitioning of wild bermudagrass from China pre-core collection under normal (5 mM) and limited (0.05 mM) N levels in two experiments. The results showed that low N nutrition significantly decreased clipping growth, while had no effects on verdure and root growth; in addition, the magnitude of clippings N accumulation reduction was higher than verdure and root across all accessions. However, significant genetic variations in all studied traits except for root N content in experiment 2 were found among accessions. Using a cluster analysis, 56 accessions in experiment 1 and 12 accessions in experiment 2 were classified into four groups and two groups separately. The accessions with superior low N tolerance had improved growth and accumulated more N in the verdure and roots; it is interesting that other accessions with low N tolerance had different tolerance mechanisms and transferred more N nutrition from the roots to the clippings to maintain better turf quality. The different molds in low N tolerance suggest that there is much potential for improving low N tolerance among Chinese natural bermudagrass accessions.

De novo assembly and comparative transcriptome analysis reveals genes potentially involved in tissue-color changes in centipedegrass (Eremochloa ophiuroides [Munro] Hack.).

Turf color is the most important characteristics of visual quality for a turfgrass species with high ornamental value and wide application prospects. Centipedegrass is a well-adapted warm-season turfgrass species in tropical, subtropical and temperate regions, possessing many outstanding properties including uniform green color. However, quite a few centipedegrass accessions or cultivars produce stolons and spike tissues with red-purple color, thereby decreasing their aesthetic value. A research focus in centipedegrass is to develop high-quality cultivars with uniform green color. To explore the major genes associated with the color changes in certain organs/tissues contributes to understand the molecular mechanisms of the same tissues having different phenotypic characteristics. In the present study, two phenotypically distinct centipedegrass accessions, E092 being a wild-type (WT) with red-purple stolons and spike tissues and E092-1 being a mutant (MT) with uniform green stolons and spike tissues, were used. Using the Illumina sequencing platform, approximately 401.7 million high-quality paired-end reads were obtained. After de novo assembly and quantitative assessment, 352,513 transcript sequences corresponding to 293,033 unigenes were generated with an average length of 735 bp. A total of 145,032 (49.49%) unigenes were annotated by alignment with public protein databases. Of these unigenes, 329 differentially expressed genes (DEGs) were identified between WT and MT stolons, with 156 up-regulated and 173 down-regulated; and 829 DEGs were detected between WT and MT spike tissues, including 497 up-regulated and 332 down-regulated. The expression profile of 10 randomly selected DEGs was confirmed with RT-qPCR. Candidate genes involved in the flavonoid biosynthesis were identified showing significant transcript changes between WT and MT organs/tissues. And transcript abundances of these flavonoid biosynthetic pathway-related genes were positively correlated with the accumulation of total anthocyanin in respective organs/tissues. This assembled transcriptome of centipedegrass can be served as a global description of expressed genes of above-ground organs/tissues and provide more molecular resources for future functional characterization analysis of genomics in warm-season turfgrass. Identified genes related to centipedegrass organ/tissue changes will contribute to molecular improvement of turf quality through genetic manipulation.

High-throughput SSR marker development and its application in a centipedegrass (Eremochloa ophiuroides (Munro) Hack.) genetic diversity analysis

Centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is a perennial, warm-season C4 grass species that shows great potential for use as a low-maintenance turfgrass species in tropical and subtropical regions. However, limited genetic and genomic information is available for this species, which has impeded systematic studies on the enhancement of its turf quality and resistance against biotic and abiotic stress. In this study, Illumina HiSeq high-throughput sequencing technology was performed to generate centipedegrass transcriptome sequences. A total of 352,513 assembled sequences were used to search for simple sequence repeat (SSR) loci, and 64,470 SSR loci were detected in 47,638 SSR containing sequences. The tri-nucleotides were the most frequent repeat motif, followed by di-nucleotides, tetra-nucleotides hexnucleotides, and pentanucleotides. A total of 48,061 primer pairs were successfully designed in the flanking sequences of the SSRs, and 100 sets of primers were randomly selected for the initial validation in four centipedegrass accessions. In total, 56 (56.0%) of the 100 primer pairs tested successfully amplified alleles from all four centipedegrass accessions, while 50 were identified as polymorphic markers and were then used to assess the level of genetic diversity among 43 centipedegrass core collections. The genetic diversity analysis exhibited that the number of alleles (Na) per locus ranged from 3 to 13, and the observed heterozygosity (Ho) ranged from 0.17 to 0.83. The polymorphism information content (PIC) value of the markers ranged from 0.15 to 0.78, and the genetic distances (coefficient Nei72) between the accessions varied from 0.07 to 0.48. The UPGMA-based dendrogram clustered all 43 core collections into two main groups and six subgroups, which further validated the effectiveness of these newly developed SSR markers. Hence, these newly developed SSR markers will be valuable and potentially useful for future genetic and genomic studies of E. ophiuroides.