The Key Gene Excavation And Association Analysis With Salinity Tolerance In Perennial Ryegrass

Soil salinization is a major factor limiting agricultural production.Soil salinization becomes increasingly severe due to climate change and low-quality water irrigation.The harm of soil salinization is mainly due to sodium chloride accumulation in soil,as a result,excessive Na⁺ uptake occurs to the roots.When large amounts of sodium ions accumulate in plant cytoplasm,they can be toxic to plants.The effects of salt stress for plant are mainly shown in two aspects.Firstly,salt stress could cause osmotic stress,which reduces the plant growth.Secondly,Na⁺ accumulation could cause accelerate aging of the old leaves.K⁺ is an indispensable nutrient element for plants.Due to the similarity of K⁺ and Na⁺ hydration radius and chemical properties,Na⁺ and K⁺ often compete at the binding site of the transporter under salt stress.Therefore,K⁺ and Na⁺ transportation plays a key role in plant resistance to salt stress.To reduce the adverse effects of soil salinization on plants,it wuld be very useful to study the physiological and molecular mechanisms of plant responses to salt stress.There are many Na⁺ and K⁺ transporters in plants.It is necessary to explore and analyze the gene encoding these transporters expression and their regulatory network for salt tolerance.Perennial ryegrass is a widely cultivated cool-season turf and forage grass around the world.This sepcies had high forage quality,quick revcovery from traffic stress,and strong resistacne to distance and insects.Native to Europe,Asia and Northern Africa,perennial ryegrass shows diverse ecotypes and variations of stress tolerance.Moreover,it is a only diploid grass among popular cool-season turf and forage grasses,thus provding a good model for gene identification and gene-trait association analysis for stess tolerance.However,it is not clear how the key genes regulate the salinity tolerance of perennial ryegrass.Therefore,the objective of this study was perform homologous cloning,association analysis and real-time quantitative PCR to excavate the key genes and gene regulation mechanisms of salt tolerance in perennial ryegrass.We obtained the following results:1.All eight accessions were classified into tolerant?T?,moderate?M?and sensitive?S?groups based on the growth traits and nutrient elements accumulation under 300 mM NaCl for 10 days,including salt tolerance group?T?has two varieties,medium resistance to salt group?M?has four varieties,salt sensitive group?S?there are two varieties.The growth of eight accessions were all affected by salt stress.Among them,the Na⁺ content in overground parts of tolerant group?T?was lower than that of moderate group?M?and sensitive group?S?,and the relatively high K⁺ /Na⁺ specific C content and the relative balance between Ca²⁺and Mg²⁺could be maintained.However,the change of microelements was more complex under 300 mM NaCl.2.We obtained the outer rectifying K⁺ channel protein gene and inner rectifying K⁺ channel protein gene LpAKT1 partial sequence with lengths of 558 bp and 407 bp,respectively,from perennial ryegrass.LpSKOR and LpAKT1 encodes 186 amino acids,respectively.Both LpSKOR and LpAKT1 mainly expressed in root and induced by salinity.3.Full cDNA sequence of LpHKT1;4,LpHKT1;5,LpHKT2;1 were obtained from perennial ryegrass.LpHKT1;4 mainly expressed in leaf and root;LpHKT1;5mainly expressed in root xylem parenchyma;LpHKT2;1 mainly expressed in root.These three HKT genes were induced by salinity.4.Three population structure groups were identified with SNP markers.Six SNP sites detected from candidate gene of LpHKT1;5 were associated with K⁺ /Na⁺ ratio under salinity and reduction of K⁺ concentration under control compared with salinity?R-K⁺ ?.5.Na⁺ transport-related genes HKT1;5,HKT2;1,SOS1 and NHX1 could coordinate to regulate salt tolerance of perennial ryegrass.The salt-tolerant materials showed better t salt tolerance through stronger capacity of Na⁺ regionalizationIn summary,these results revealed that the physiological responses of different perennial ryegrass accessions to salt stress were significantly different.In the process of salt stress resistance,the candidate gene LpHKT1.5 played an important role in maintaining K⁺ and Na⁺ homeostasis together with other genes.It laid a foundation for further revealing the salt tolerance mechanism of perennial ryegrass.