Study On The Phenotypic Traits And Transcriptomic Analysis Of Red Clover With Different Isoflavone Contents

Red clover（Trifolium pratense L.）is a multifunctional herb,which not only has high nutritional value,medicinal value and ornamental value,but also has good nitrogen fixation ability and characteristics of preventing soil erosion.Red clover contains a large amount of isoflavones,these isoflavones can remove free radicals and active oxygen which may cause diseases in the human body,it has a good antioxidant effect.It is of great significance to explore an effective way to cultivate red clover that can take into account both high medicinal value and high feed value.In this study,the content of red clover isoflavones in the field was measured,and then the morphological characteristics,agronomic traits,yield and transcriptomics of red clover populations with different isoflavone contents were compared,then,identification of key genes in the biosynthetic pathway of red clover isoflavones.It provides a theoretical basis for the research on the biosynthesis of red clover isoflavones and molecular assisted breeding.Below are key research findings:（1）The content of isoflavones in red clover at the early flowering stage ranged from15.65 to 36.33 mg·g^-1,and the average isoflavone content was 23.68 mg·g^-1.It can be clustered into 7 red clover populations（H1～H7）by cluster analysis.Observation of the phenotypic characteristics of seven red clover populations showed that most of the red clover populations with high isoflavone content（H1）had prostrate（56.00%）,purple flowers（66.90%）and smaller leaf area（63.50%）features.（2）The agronomic traits of seven red clover populations with different isoflavone contents were measured,and it was found that two red clover populations with higher isoflavone content（H1 and H2）had significantly higher absolute plant heights（35.20cm and 33.65cm）,larger crown width（55.95cm and 53.55cm）,significantly faster growth rate（0.93cm/d and 0.90cm/d）and significantly higher leaf rate（0.71 and 0.70）specialty.The yields of 7 red clover populations with different isoflavone contents were measured,and it was found that the fresh weight（385.75g and 382.26g）and dry weight（116.08g and118.21g）of the red clover H1 and H2 populations were significantly higher than those of other group,while the ratio of fresh to dry（3.33 and 3.25）was also significantly lower（P<0.05）.（3）Screening the DEGs of red clover in different phenological stages found that 4665DEGs were unique to red clover with high isoflavone content.The main enriched pathways of these high-isoflavone-specific DEGs are:biosynthesis of secondary metabolites,phenylpropane biosynthesis,sesquiterpene and triterpene biosynthesis,and monoterpene biosynthesis;GO terms include:responses to other microorganisms,responses to external stimuli,defense responses,and responses to biological stimuli.（4）The transcription factors of the unique DEGs gene set of red clover with high isoflavone content were analyzed,and 96 related differential transcription factors were found,belonging to 21 families.Among them,19 MYB＿superfamily,14 AP2/ERF family,8 NAC family and 8 GRAS family were the most,and these families were all involved in plant stress response and secondary metabolism.（5）The weighted Gene co-expression network was constructed for the DEGs of red clover with different Isoflavone content at the same phenological stage,and 18 gene modules were copolymerized.According to the annotation of main reference module（turquoise）genes in phenylpropane synthesis pathway,flavonoid synthesis pathway and Isoflavone synthesis pathway,key genes for Isoflavone synthesis such as PAL,C4H,4CL,CHS,CHR,CHI,HIDH,IFS and IOMT were screened.The expression time of CHS,CHI,IFS and HIDH in red clover with high Isoflavone content is earlier than that in red clover with low Isoflavone content.