Establishment Of High-throughput Whole-Seedling Phenotyping System And Genome-wide Association Analysis Of Salt Tolerance In Soybean

Soybean(Glycine max)is an important cash crop,and its yield is significantly affected by salt stress.It is important to identify key genes that regulate soybean salt tolerance and to develop salt-tolerant cultivars.The association analysis of phenome and genome data in population is an emerging strategy to clarify complex genetic traits.However,the acquisition of accurate high-throughput phenotypic data is currently a bottleneck.Existing phenotyping platforms also cannot fully meet the needs of identification of salt tolerance of soybean population in terms of throughput and environmental controllability.This paper proposed the WinRoots system,a low-cost and high-throughput plant soil cultivation and phenotyping system that can provide uniform,controlled soil stress conditions for plant culture and accurately quantify the whole-plant phenome,including roots phenotype.We collected the phenomic data in a soybean germplasm population of 399 cultivars.By genome resequencing,10,975,191 SNPs were obtained in the wholegenome range of the population.Using GWAS,ten new candidate salt tolerance related chromosomal regions were identified,which include 35 candidate genes.The main results include:1.Establishment of high-throughput whole-seedling phenotyping systemWe established WinRoots,a high-throughput plant cultivation and phenotyping system that is based on a soil matrix and offers root visibility,with a cultivation density of 180 soybean seedlings per square meter and a cultivation throughput of 2,160 individual plants per batch.The system integrates RGB and hyperspectral imaging modules to collect digital data at a speed of up to 800 whole plant RGB images,720 canopies hyperspectral images or 81 isolated leaves hyperspectral images per hour,realizing high-density and high-throughput plant soil cultivation and rapid collection of the digital phenotype of the whole plant including the roots.Using the WinRoots system,a total of 820 seedlings of 12 soybean germplasm were used as research materials,and the primary root length,dry and fresh weight of the aboveground part and leaf area of soybean were used as indicators.The WinRoots system was evaluated through experiments of position effect,soil conductivity and batch effect.The results show that the system can provide a highly controlled and uniform soil salt stress environment for high-throughput plant cultivation,and the collected phenotypic data is accurate and reliable.The variation of three phenotypic indicators,including primary root length,fresh weight,and percentage of moisture,of 660 seedlings from 11 soybean cultivars under the control and three salt stress treatments was further analyzed.It was found that the phenotypic indicators under 175 mM NaCl treatment not only showed a significant downward trend of salt stress,but also had a larger variation range than other concentration conditions,thus determining the salt stress conditions suitable for the identification of salt tolerance phenotypes of soybean populations in the WinRoots system.2.GWAS of salt tolerance in soybean germplasm populationUsing WinRoots,germination box and field experiments,we obtained 32 sets of measured data based on images,developmental and physiological characteristics in the soybean germplasm population of 399 cultivars.Phenotypic indicators,such as primary root length,leaf area,fresh weight of shoot without cotyledon,and emergence rate in saline field,were further analyzed using significant difference analysis,variation analysis,normality test,and normality transformation.As a result,17 sets of salt tolerance phenotypic data were obtained to be directly used in GWAS.In addition,the results of multi-phenotype correlation analysis illustrated the complexity of salt tolerance phenotypes and the necessity of multi-phenotype research.10,975,191 SNP markers were obtained by genome sequencing of the above soybean germplasm population.The genetic characteristics of the population were analyzed using phylogenetic tree,LD analysis,population structure and genetic relationship analysis.The results showed that the population was suitable for GWAS.This conclusion was further demonstrated with GWAS of two traits reported before.On this basis,using the 17 sets of soybean salt tolerance related data combined with optimized whole-genome data including 589,608 SNPs,we conducted GWAS based on the MLM model and identified 3 significantly associated regions.The regions were further verified using multi-model GWAS and 7 other associated regions were found.Finally,using association analysis with full SNP markers in single chromosome and homology comparison,we determined the boundaries of the 3 candidate regions and annoted the 35 candidate genes.In conclusion,WinRoots,the whole-seedling phenotyping system,realized the highthroughput cultivation of soybean germplasm population under controllable soil salt stress and the acquisition of multi-level salt tolerance phenotypic data.The multiphenotypic data used in GWAS lead to the identification of 10 new salt tolerance related candidate chromosomal regions and 35 candidate genes.These results provide the basis for clarifying the genetic mechanism of salt tolerance in soybean and salt tolerant soybean breeding.