Aassociation Analysis Of Agronomic Traits With Molecular Marker And Clone HcDREB Transcription Factor Gene In H.compressa

Hemarthria compressa is a kind of perennial grass, with long growth period, fast growth, strong regeneration ability and well adaptability. So it is already widely used in animal husbandry in the south, and shows broad prospect of application in soil and water conservation and ecological management. There are rich wild H.compressa resources, which has the variety of ecological types. For that reason, it is very important to study its genetic diversity, colony genetic relationship, positioning of main gene for genetic improvement of H.compressa in theory and practice. In order to accelerate breeding process of H.compressa and explore the genetic diversity and genetic relationship among wild H.compressa resources, SRAP and EST-SSR molecular markers were used to study the genetic diversity and group structure among43H.compressa materials, and the association analysis method was used to detect the molecular marker associated with important agronomic traits.Low temperature, drought and saline-alkali are main limiting factors. Unlike single functional genes, the DREB transcription factors can regulate multiple stress-inducible genes expression simultaneously, and participate in physiological and biochemical process involved stress tolerance, result in increasing in stress resistance. The DREB transcription factor is very important to resistet to low temperature, drought and saline-alkali. As the warm-season grass, H.compressa is cold-tolerant, it can keep green in such4℃, and stop growing in winter. H.compressa can not withstand drought, and it will fail to grow well on drought soils. In the study, DREB-like gene was isolated and cloned from H.compressa and its function was analysed.The main results were as followed:1Trait differences and correlation analysis showed that:there were rich variation among43H.compressa clones in qualitative traits, such leaf colour, flowering phase, inflorescence shape and anther color. Similarityly, there were abundant variation among43H.compressa clones showed in stem, leaf, tiller, biomass and panicle characteristic. The degree of variation of every trait was different. The mean coefficient of variation was24.4%. Coefficient of variation of number of inflorescence per fertile stem is the largest at57.4%. It indicated that there were abundant variation within the species.2The result of genetic diversity showed that315PCR bands were detected in total of15pairs of SRAP primers, among which,294bands were polymorphic, accounting for93.2%of the total. The amplified bands and polymorphic bands per pair primer were averagely20.9and19.6, respectively. The polymorphic information content was from0.340to0.500.24H.compressa clones were identified through29special bands. A total of323fragments were amplificated with23pairs of EST-SSR primers of poaceae, among which,260fragments were polymorphic, accounting for80.4%of the total. The polymorphic information content was from0.354to0.500.20clones could be identified through34special bands. The Genetic similarity coefficient of SRAP and EST-SSR was0.640-0.949and0.690-0.913. The result showed that there were rich genetic variance, but there was genetic relationship among three varieties.3The different degrees of LD were detected among molecular marker. Genetic structure analysis showed that the H.compressa population was composed of four subpopulations based on SRAP molecular marker and five subpopulations based on EST-SSR molecular marker. The association analysis between two molecular markers and14agronomic traits was performed by using TASSEL GLM (general linear model) program.43SRAP locis and38EST-SSR locis associated with traits were screened respectively. Some locis were found to associate with a same trait. There were a few locis associated with two or more traits simultaneously, which might be the genetic reason of correlation among traits or pleiotropic phenomena.4A DREB2like genes from H.compressa under the low temperature stress were cloned by RT-PCR and RACE methods, named HcDREB2. Nucleotide sequence analysis indicated that the HcDREB had an ORF encoding264amino acids with predicted molecular weight of28.8kD and a isoelectric point of5.13. The putative protein contains a highly conserved AP2domain, a nuclear localization signal and an acidic activation domain. There were a specific motif AEIR between V14and E19in AP2/ERBP domain, which confirmed the HcDREB gene belong to DREB2subfimily. No significant sequence similarity with those of other known DREBs except for the conserved AP2DNA-binding domain.5The expression of the HcDREB2gene was induced by low temperature, dehydration and high-salt conditions. No significant changes in the HcDREB expression were observed under ABA stress, which showed that the expression of HcDREB was unindependent on ABA. Its expression was changed following the inducible time. The expression of HcDREB was highest after1h under4℃,20%PEG and250mM NaCl treatment, then the expression amount was lowest after4h.6To investigate the DNA-binding specificity in vivo activity, the yeast hybrid system was carried out, and the results confirmed that the AP2domain of HcDREB2could specifically interact with DRE cis-acting element and activate down-stream responsive genes.