A Study Of Genetics Breeding, Seed Multiplication And The Transcriptome Of Low Coumarin Sweetclover (Melilotus Spp.)

Sweetclover?Melilotus spp.?is a widely distributed herbage belonging to the legume family.Members of the Melilotus genus have strong adaptation and good tolerance to drought,cold and infertile soils.M.officinalis and M.albus are the two most cultivated species in this genus which have potential to be used as good forage.However,high coumarin concentration in native cultivars is a major limiting factor in the use of Melilotus species as forage.This study reports on an investigation focused on M.officinalis and M.albus to;a)characterize adaptability and coumarin content among germplasm accessions,b)estimate quantitative genetic parameters for key traits based on half-sib family analysis,c)transcriptomic analysis of coumarin biosynthesis,d)plant density and phosphorus fertilizer effects on seed yield.These results will provide theoretical foundation and materials to the genetics breeding for low coumarin Melilotus,and also contribute to a better understanding of the coumarin biosynthesis.The main results were as follows:1.The adaptability and coumarin content of 101 introduced Melilotus accessions were evaluated at Yuzhong,Gansu Province,and a total of 95 accessions were successfully established.Significant?P<0.05?genotypic variation was observed for the traits:plant height?PH?,dry matter yield?DMY?,leaf stem ratio?LSR?,coumarin content?Cou?and powdery mildew resistance?PMR?.The agronomy and quality traits of 19 elite biennial accessions were also evaluated.There was significant?P<0.05?genotypic variation for PH,DMY,acid detergent lignin?ADL?and Cou.In this study,the application of principal component analysis?PCA?and cluster analysis enabled identification of 5 accession groups.2.A quantitative genetic analysis was conducted on biomass,yield-related traits and coumarin data collected from first generation?F₁?half-sib?HS?families of the two Melilotus species evaluated at two locations:Yuzhong and Linze,Gansu Province.A mixed linear model was used for the analyses all traits using the REML algorithm,the results showed that significant?P<0.05?genotypic variation among the HS families for all 7 traits was observed.There was also significant?P<0.05?genotype-by-environment interaction for most traits such as DMY,PH,stem number?SN?,stem diameter?SD?and spring vigour?SV?.Pattern analysis helped identify eleven M.officinalis members and eight M.albus members with high expression of DMY and low expression of Cou.Smith-Hazel?SH?selection index was used to predict expression of the traits DMY,PH,SN and LA?Taken as Cou in this study?.The resulting expected genetic gain based on the SH index,for the individual traits DW,PH,SN and LA were 10.9%,3.5%,6.4%and-1.0%,respectively.3.The genes of five M.albus near isogenic lines?NILs?that had different coumarin content were analyzed by RNA-Seq.A total of 104358 unigenes,including 2095 differential expressed genes?DEGs?were obtained.Gene expression pattern analysis suggested similar gene expression trends in the genotypes with the similar coumarin content.The KEGG analysis of DEGs was used to identify 111 unigenes,with several DEGs among them possibly being related to coumarin synthesis pathways;Unigenes encoding hexokinase,abscisic acid receptor,phenylalanine ammonia-lyase?PAL?and peroxidases,particularly showed association with the coumarin content.A gene expression pathway of coumarin biosynthesis for M.albus was proposed preliminarily,and unigenes likely associated with coumarin biosynthesis was validated by qRT-PCR.4.Field trials were conducted at Hexi Corridor,Gansu Province,during two years to study the effects of different within-row spacing?30,45,60 cm?and phosphorus fertilizer rates?0,40,80 and 120 kg/hm² P₂O₅?on seed yield and yield components of two M.officinalis cultivars:Tianshui and Norgold.Results showed that actual seed yield of two cultivars was affected significantly?P<0.01?by within-row spacing,fertilizer rate and their interactions.The 60 cm within-row spacing and fertilizer rate of 80 kg/hm² P₂O₅ produced the highest mean seed yield over 2 years for cultivar Tianshui,giving 1234 kg/hm².The highest mean seed yield for cultivar Norgold was obtained from applications of 80 P₂O₅ kg/hm² and 45 cm within-row spacing,resulting in yields of 1520 kg/hm².Path coefficient analysis indicated that the number of fertile shoots had the greatest influence on seed yield among the seed yield components.