Genome-wide Association Analysis And Candidate Gene Associated With Low Temperature,Waterlogging And Combined Stresses During Soybean Germination

Soybean(Glycine max(L.)Merr.)is a major source of plant protein for human food and animal feed which originated in China.It has expanded into the tropics and high latitude cold regions with human migration,advances in agricultural science,evolution of cropping patterns and global warming,and is widely cultivated worldwide.Low temperature stress has become one of the major abiotic stresses limiting soybean growth and development in high latitude cold regions.In Manitoba,Canada,and northeastern China,soybean seeding is usually exposed to the combined stress of low temperature and waterlogging during the seeding period,which severely affects its emergence and yield potential.With challenging of global climate change and the intensifying consumer demand for soybeans,much work needs to be done to improve soybean’s tolerance to risk and yield.For example,evaluating the germplasm of soybean that is resistant to the combined stresses of low temperature and waterlogging,exploring the genes of soybean that are resistant to low temperature,waterlogging and combined stress,developing molecular markers for soybean that are resistant to low temperature,waterlogging and combined stress,and creating germplasm of soybean that is resistant to low temperature,waterlogging and combined stress.In this study,a natural population was constructed with soybean accessions from China(52 accessions)and Canada(60 accessions)as the main parts,while some soybean accessions from European regions and Russia were selected,totaling 187 test materials.Three temperature levels(10°C day/10°C night,14°C day/10°C night,20°C day/14°C night)and two soil moisture levels(normal moisture and impregnation stress),totaling six treatments,were set up to evaluate the changes in five germination traits(germination rate,germination time,germination index,germination uniformity,and coefficient of germination velocity)of soybeans under different treatments.The effects of low temperature,waterlogging and combined stress on soybean were explored by regression analysis.Germination traits of soybean seeds under combined stress were clustered to identify genotypes that maintained better germination ability under combined stress.Genome-wide analyses were performed on the germination trait dataset under low-temperature stress(10°C/10°C+normal moisture),the germination trait dataset under waterlogged stress(20°C/14°C+waterlogging),and the dataset of two treatments(10°C /10°C+waterlogging and 14°C/10°C+waterlogging)in the combined stress.QTNs significantly associated with five germination traits were localized and validated by phenotypic values to obtain QTN loci significantly affecting the phenotype.Finally,the validated QTNs were subjected to LD Block analysis,and candidate genes were extracted within the LD Block intervals and subjected to GO functional annotation and enrichment analysis,and the candidate genes were further screened according to the functional annotations.The main results derived from this study are as follows:1.Low temperature,waterlogging and combined stress negatively affected germination traits to varying degrees in soybean germination.The treatment with the greatest negative effect on germination rate was 20°C/14°C+waterlogging.The treatment with the greatest negative effect on germination time was 10°C/10°C+waterlogging.The treatments with the greatest negative effect on germination index were14°C/10°C+waterlogging and 20°C/14°C+waterlogging.The treatment with the greatest negative effect on germination uniformity was 10°C/10°C+waterlogging.The treatment with the greatest negative effect on germination velocity was 10°C/10°C+waterlogging.2.The variation in germination percentage,germination index and germination speed under different treatments was significantly(P<0.001)affected by temperature,moisture and their interaction.The variation in germination time was significantly(P<0.05)affected by temperature and its interaction with moisture.The variation in germination uniformity was significantly(P<0.05)affected by both temperature and the moisture conditions.3.Sixty-one tolerant soybean genotypes under combined stress conditions were screened.Among them,PI 507702 and PI 603147,which have the strongest germination ability,are unique materials for improving germination traits of soybean under combined stress of low temperature and waterlogging.4.Genome-wide association analysis of four germination datasets under low temperature,waterlogged,and their combined stresses(10°C/10°C+waterlogging and14°C/10°C+waterlogging)during the germination period detected 86,101,78,and 55 QTNs associated with germination traits,respectively.there was no overlapping of QTNs between a single stress and a combined stress,and there was no overlap of QTNs between the two combined stresses,indicating that combined stress has a specific genetic mechanism and cannot be inferred from the single stress analysis.5.Nine QTNs caused significant phenotypic changes in the low-temperature stress treatment,including two polycistronic loci,CM000848.3＿20935600 and CM000840.4＿5836071,which caused significant variation(P<0.05)in both soybean germination rate and germination index.Eight QTNs caused significant(P<0.05)phenotype changes in germination ability under waterlogged stress.Five QTNs caused significant(P<0.05)changes in phenotype under combined stress(10°C/10°C+waterlogging).Seven QTNs caused significant(P<0.05)phenotypic changes under combined stress(14°C/10°C+waterlogging).These QTNs can be used for further molecular marker development for selecting tolerance to low temperature,waterlogging and combined stresses during germination in soybean.6.Within the LD Block regions of significant QTNs,126 candidate genes were found related to seed germination under low temperature conditions,21 candidate genes related to germination under waterlogged conditions,46 candidate genes related to seed germination under combined stress(10℃/10℃+waterlogging)conditions and 13 candidate genes related to seed germination under combined stress(14℃/10℃+waterlogging)conditions.Based on the GO annotation and enrichment results,24,10,15 and 9 candidate genes were finally screened out,which were all directly or indirectly related to the stress defense mechanism or seed germination,and could be candidate genes for future gene cloning validation.In summary,the genome-wide association analysis soybean seed germination traits under low temperature,waterlogging,and combined stresses during the emergence period in this study has generated information for understanding the genetic basis of the target traits,as well as new knowledge for developing molecular marker-assisted selection for improving low temperature,waterlogging,and combined stress tolerance in soybean.