Selection Of EMS-mutagcnized Materials And Mechanism Of Flower Color Mutation In Mengnong Sainfoin

Mengnong sainfoin was a multifunctional forage grass.It could beautify the environment and act as a nectar plant.With the increase of using times,its productivity and cold resistance were gradually decreasing.So it was urgent to research about purification and variety substitution of mengnong sainfoin.In our study,EMS was used to induce the imbibed seeds of mengnong sainfoin to determine median lethal dose(LD50),and this dose was used to construct the M1 generation mutant population.Based on this,we constructed M2 and M3 generation mutant populations of mengnong sainfoin.The content of the research about the mutant population of mengnong sainfoin was as follows.First,SSR molecular markers were used to analyze the genetic diversity of different mutant populations to determining the DNA polymorphism at the molecular level.Second,based on the SPAD and the plant height we divided M1,M2 and CK into three groups,respectively.We mensurated the diurnal dynamics of photosynthetic characteristics and chlorophyll content of different species in each group.The purpose was to screen the parent material of high-yield breeding of sainfoin.The last,a light-colored flower mutant was found in the M1 generation mutant population.Through the study of the phenotype,structure,composition and transcriptome of different flower colors for the further development and utilization of sainfoin to provide the theoretical basis.The main results were as follows:(1)The increase of EMS intensity led to the germination ability and vigor decrease of Mengnong sainfoin seeds.And 0.9%EMS treatment for 18 hours was LD50 for imbibed seeds of Mengnong sainfoin,which was also the appropriate dose to establish mutation population.In the mutant population of Mengnong sainfoin,there were many trait variations such as leaf color,leaf quantity,flower color,and inflorescence branching.The multi-leaf variation rate which was 30%in the M1 generation was highest.(2)The seeds induced by EMS and then germinated under low temperature stress got M1 plants.The main physiological and biochemical indexes of M1 plants significantly changed in seedling stage and overwintering stage,and the root structure also changed significantly during the overwintering stage.The above research results show that this methad could improve the cold resistance of Mengnong sainfoin.M2 mutant of Mengnong sainfoin(SPAD:25.28～48.84,plant height:80～107cm)could be used as a parent material for high-yield breeding which had the highest photosynthetic rate(5.76μmol·CO2·m-2·s-1)and higher water use efficiency(2.33 μmol·mmol-1).And the changes in the proportion of polymorphic locus in different mutant population were M3>M2>M1,the offspring variation degree with EMS induced was enhanced from generation to generation,so the M3 generation of Mengnong sainfoin mutant population could also be used as the screening population of breeding parents for different breeding targets.(3)The light color flower by EMS treated and the control(pink and amaranth)were three different color systems,and the light color flower was defined as yellow-white flower according to the b*and the h°.10 kinds of flavonoids and 5 kinds of anthocyanins were detected in 3 colors of sainfoin,but 6 varieties of kaempferol derivatives.2 types of petunia derivatives,2 varieties of delphinidin derivatives and 1 types of mallowin derivative were first reported in Mengnong sainfoin.(4)A total of 81319631904 nucleotides(about 81 Gb),over 547 million reads and 53009 unigenes were obtained from the petal transcriptome of Mengnong sainfoin in different colors and different periods sequencing,and 6,202 Unigenes had SSR locus,so the distribution density was 5.86 kb;the number of repeat motifs were mainly tri-nucleotide(49.13%),followed by di-nucleotide(22.07%);AG/CT type was the highest(16.16%)among 284 repeat motifs and the next was AAG/CTT(14.30%);tri-nucleotide type of motify repeats was highest,di-nucleotide was next.And with the number of motif repeats increases,the number of nucleotides decreases.The length of SSR of Mengnong sainfoin was 12～44 bp,and that was mainly concentrated in 12^24 bp.There were 3130 SSRs which length were greater than or equal to 20 bp,that was 38.29%of SSRs had high polymorphism potential.A total of 6396 genes were annotated with KEGG metabolic pathways,and a total of 3 metabolic pathways related to flower color were found,which was the flavonoid metabolic pathway,the flavonoid and flavonol metabolic pathway and the anthocyanin metabolic pathway.There were 38 differential genes related to flavonoid metabolic pathway,10 differential genes related to flavonoid and flavonol metabolism pathway and 1 differential gene related to anthocyanin metabolism pathway.(5)Through the transcriptome sequencing and qRT-PCR verification of different flower petals of Mengnong sainfoin,7 key genes which was 4CL3,ANR,LAR,FLS,CHS,DFE and ANS respectively were found.And the increase of CHS,DFR,ANS expression led to the deepening of flower color.