Cloning Of VfSGR And Development And Application Of DCAPS Marker In Faba Bean

The quality and usage of legume crops are hot spots of research.As their high chlorophyll content,seeds with green cotyledons broadened the source of natural pigments in the food industry.Besides,green cotyledon trait is considered to be an excellent trait in the market and is welcomed by consumers.The senescence and death of plants are closely related to the degradation of chlorophyll.The degradation of chlorophyll leads to the fading of the green color of plant tissues and makes photosynthesis weakened.There is a series of stay-green mutants of legume crops,and chlorophyll degradation pathway of them is hinderedduring plant senescence and seed maturation.Thus,the leaves can remain green for a long time and the seeds have a green cotyledon phenotype.As a major cold-season legume,faba bean（Vicia faba L.）is widely planted in the world.However,so far,due to its large genome,multiple gene copies and unavailabe reference genome sequence,it is difficult to develop molecular markers for molecular breeding in faba bean.This study cloned a gene VfSGR that controled the color of cotyledons（yellow or green）,and developed a molecular marker closely linked and associated with the color of faba bean cotyledons.The main research contents are as follows:（1）VfSGR that controls the color of faba bean cotyledons has been cloned homologously.Blast alignment against the faba bean transcriptome sequence was conducted using the protein sequence encoded by the pea cotyledon color-related gene SGR as the query,and the sequence of the faba bean homologous gene VfSGR was obtained.Through amplifying and sequencing,the VfSGR was amplified and sequenced from yellow cotyledon faba bean HST and green cotyledon faba bean SNB7,and it was found that the gene consists of 3introns and 4 exons and encodes a putative protein with 259 amino acid residue.The CDS region of VfSGR was amplified using the cDNA of 11 yellow cotyledon faba bean accessions and 2 green cotyledon faba bean accessions as templates.Sequence alignment showed that there are 8 SNPs in VfSGR of these 13 faba bean accessions,and 2 SNPs of them caused changes of VfSGR coding Amino acid.One of the SNP mutations caused pre-stop codon in VfSGR of the green cotyledon faba bean,resulting in a protein containing170 amino acid residues.（2）Through the analysis of the function of VfSGR,it is found that VfSGR is related to the senescence and chlorophyll degradation of faba bean.Dark treatment was carried out using the yellow cotyledon faba bean HST and green cotyledon faba bean SNB7.It was found that whole-plant yellowing of HST plants was faster than that of SNB7,and the SPAD value（relative chlorophyll content）of the leaves of HST decreased faster than that of SNB7.q PCR results showed that the relative expression of VfSGR of SNB7 did not change much after the fourth day of dark treatment.However,the relative expression of VfSGR of HST continued to increase.Different tissues of SNB7and HST at the same developmental stage were analyzed for expression level.The results of q PCR showed that VfSGR was expressed in all tissues of faba bean.Among them,the gene highly expressed level in flowers and followed by old leaves.Expression of this gene in roots and stems was lower.And young leaves,seeds and pods showed very low expression of VfSGR.In flowers,old leaves,seeds and pods,the relative expression of VfSGR of HST was higher than that of SNB7.In young leaves,roots,and stems,the relative expression of VfSGR of SNB7 was higher than that of HST.VfSGR was transiently expressed in Nicotiana benthamiana by Agrobacterium infection,and the results showed that the leaves of N.benthamiana overexpressing VfSGR all turned yellow.Those leaves overexpressed VfSGR^-HSTturned yellow faster than which overexpressed VfSGR^-SNB7.The pre-stop mutation in VfSGR did not lead to the complete loss of gene function.These results indicated that VfSGR is a gene related to yellowing,senescence and chlorophyll degradation of broad bean plants.（3）We developed a dCAPS marker and verified the close linkage and association between the marker and the color traits of faba bean cotyledons.A dCAPS marker was designed according to the mutation in VfSGR that caused the pre-stop of amino acid coding.Results of amplification and restriction enzyme digestion showed that the PCR product of SNB7 could be digested by Age I and produced a band of76 bp.However,PCR product of HST could not be digested by Age I and then exhibited a band of 102 bp.In order to further verify the linkage between the marker and the color of faba bean cotyledons,an F₂population containing 87 plants was constructed by crossing between SNB7 and HST.It was found that the number of yellow cotyledons and green cotyledons accorded with the ratio of 3:1.These results indicated that the green cotyledon phenotype of faba beans was controlled by a recessive gene.Linkage analysis of the F₂population using the dCAPS marker showed that all of the green cotyledon plants produced SNB7-type bands,and the yellow cotyledon plants showed HST-type bands or heterozygous bands.This result indicated that the dCAPS marker was closely linked to the cotyledon color of faba bean.A natural population containing 191 faba bean accessions was genotyped using the dCAPS marker and the result showed that all green cotyledon faba beans got 76 bp bands,while all yellow cotyledon faba beans got 102 bp bands.This result indicated that the dCAPS marker was associated with the color of faba bean cotyledons.These results further clarify that VfSGR was a gene that controlled the color of faba bean cotyledons.The results obtained in this study,on one hand,preliminarily revealed the molecular genetic mechanism of the color of faba bean cotyledons.On the other hand,the dCAPS marker developed in this research,which was closely linked to the color of faba bean cotyledons,set up the foundation for the selection of new faba bean varieties with special commercial value through using molecular marker-assisted selection（MAS）.