Toxin-related Gene Mining Of β-ODAP With Development And Validation Of Molecular Markers In Grass Pea

Shan Li Dou(Lathyrus sativus L.),also known as grass pea,is an excellent and resilient crop that can be used as a grain,vegetable,feed,medicine,fertilizer,and horticulture.Therefore,it can play a very important role in coping with global climate change.Nonetheless,the non-protein amino acid(β-ODAP)existing in seeds and tissues,which can poison humans and animals with an excessive intake,limits the development and utilization of grass pea.In this study,on account of the inadequate research of ODAP content,toxin associated gene,the genetic map,and the molecular markers,we carried out ODAP concentrations identification of grass pea accessions,toxin related gene mining,as well as the development and validation of molecular marker,based on spectrophotometric method,high-performance liquid chromatography,multiple-omic analysis and GBS(genotyping-by-sequencing).The total results are as follows:1.Ten days after sowing,the ODAP concentrations of leaf in 137 grass pea accessions were tested,employing spectrophotometry.The results showed that the average value of ODAP in 137 accessions is 3.552%,and the frequency distribution of the ODAP content obeyed the normal distribution.Eventually,137 accessions were divided into 5 groups and 8 accessions with high and low toxins were selected respectively.Based on analysis of the ODAP concentrations of the seedling stage from 5 d to 20 d,the ODAP concentrations showed a trend from high to low.There was a significant positive correlation between seed traits(length,width,and 100-seed weight)and the ODAP by correlation analysis.2.Through the RNA sequencing of grass pea of S04(high concentrations)and S35(low concentrations)in leaf,cotyledon,and root,the transcriptome data containing an average of 73 million reads and 10.9 GB base were obtained.With removing some low-quality reads and adaptors,an average of 68 million clean reads was obtained with an average of 10.2 GB base and Q30 up to 93.17%.The number of DEGs in cotyledons of the two accessions was 9294 and 3genes related to β-ODAP were discoveried encoding CS(cysteine synthetase)and SAT(serine acetyl transferase).3.On account of non-targeted metabolome analysis,83 differential metabolites in 221 metabolites,including 35 positive ion and 48 negative ion,were identified between S04 and S35.Serine,intermediate metabolite of β-ODAP,was tested and identified.By the targeted test of S04 and S35,the ratio of β-ODAP content in leaf at 20 days after sowing was 2.106.4.Through the comparative analysis of non-targeted and targeted secondary metabolites combined with the metabolic pathways of Serine,it was determined as an important intermediate metabolite involved in the synthesis of β-ODAP.Through multi-omic analysis,3 unigenes including DN93539＿c1＿g7,DN99029＿c0＿g5,and DN95080＿c1＿g1 were associated with Serine metabolism expressed in the cotyledons of grass pea.With qRT-PCR verification,the expression level of the DN95080＿c1＿g1 gene at 5 days after sowing in leaf was 13.5 times of 20 days,which showed an obvious difference.It was confirmed that the DN95080＿c1＿g1 might participate in the β-ODAP biosynthetic process at the seedling stage.Based on RACE and BLAST analysis,the results showed that DN99029＿c0＿g5 and DN95080＿c1＿g1 encode amino acid permease and Serine hydroxymethyltransferase respectively,involving in transportation and anabolism of Serine.5.The GBS was applied to develop SNP markers and constructed a high-density genetic linkage map.Through library construction and sequencing,raw data processing,and aa×bb SNP loci calling,the high-density genetic linkage map was constructed including 3536 SNP markers,the total genetic distance reached 6975.67 c M,and the makers with gap distance less than 5 c M were 3205 SNPs,accounting for 90.6% in the total markers.On account of BLAST alignment with the grass pea draft genome,DN99029＿c0＿g5 and DN95080＿c1＿g1 are aligned to788.099-794.367 c M and 521.104-629.582 c M of LG1 and LG7 respectively.6.Through transcriptome sequencing analysis,3204 SSR markers were developed and 284 pairs of primers were randomly selected for verification,of which 87 pairs were polymorphic,and the average PIC value was 0.4158.Fifty KASP markers were designed and 40 SNP loci were successfully detected with polymorphism.The average PIC value of 40 markers was 0.2457.Although the PIC value of KASP markers was lower,the conversion rate from SNP to KASP was higher than SSR.