Development And Application Of CpSSR Molecular Markers In Sophora Flavescens And Its Response To Temperature Stress

Sophora flavescens Ait.is a perennial plant of Sophora genus in the legume family.Using root as medicine,it has the effect of clearing away heat and dampness,killing insects and diuresis.Its main functional components have good anti-inflammatory,analgesic and anti-tumor effects.With the wide application of Sophora flavescens in medicine and agriculture,the demand of Sophora flavescens in Chinese medicine market is increasing day by day.In recent years,the artificial cultivation area of Sophora flavescens is increasing year by year.It is particularly important to carry out research on the germplasm resources of Sophora flavescens and select and breed excellent varieties.This article analyzes the chloroplast genome of Sophora flavescens,developed cp SSR molecular markers based on it,distinguished and evaluated different germplasm resources of Sophora flavescens,so as to promote germplasm innovation and breeding of new varieties,and provide a certain theoretical basis for improving the yield and quality of Sophora flavescens.At the same time,combined with the characteristics of wide distribution and rich ecological types in Sophora flavescens,the research on temperature stress response of Sophora flavescens was carried out to explore its resistance and defense ability to high and low temperature stress,which laid a foundation for molecular level research on temperature stress response of Sophora flavescens.The results are as follows:1.Illumina sequencing and functional annotation were performed on the chloroplast genome of Sophora flavescens.The total length of the chloroplast genome in Sophora flavescens was 154165 bp,showing a typical four-segment structure.The total number of annotated genes was 123,among which the number of protein coding genes,t RNA genes and r RNA genes were 77,38 and 8,respectively.A total of104 SSR loci in chloroplasts were identified by MISA software,and the main repeating type was A/T.Codon bias analysis showed that the codon bias in the chloroplast genome of Sophora flaveschinensis was weak and preferred to use A/T.The amino acid conversion at RNA editing sites was mainly hydrophilic to hydrophobic.Sequence alignment of the chloroplast genomes of published Sophora flavescens was conducted,and there were differences in 4 genes.Phylogenetic analysis of Sophora flavescens and 16 other legumes using Mega X indicated that Sophora flavescens was the most closely related genus of Sophora alopecuroides.2.Based on 104 cpSSR loci,18 cpSSR loci were selected to design the primers.The amplified sequence bands of 18 pairs of primers were clear and the PIC average value was 0.886,indicating that 18 pairs of primers had high polymorphism.Genetic diversity analysis showed that 18 pairs of primers had high genetic diversity and had certain generality.3.Genetic diversity analysis of 150 Sophora flavescens germplasm from 15 producing areas showed that the average values of Na,Ne,I and h were 5.415,4.395,1.535 and 0.748,respectively,indicating that Sophora flavescens germplasm had high genetic diversity.AMOVA analysis showed that the intra-species genetic variation rate was 92%,and the inter-species variation rate was 8%,indicating that the genetic variation within the germplasm of Sophora flavescens was greater than that between germplasm.Through the analysis of genetic structure,the germplasm of Sophora flavescens from 15 producing areas were grouped into two groups,which showed a certain regional correlation.Meanwhile,the division of population structure showed that there was a certain genetic differentiation among the germplasm of Sophora flavescens.4.The high temperature(38℃)and low temperature(4℃)stress tests were conducted on Sophora flavescens seedlings at different times.The plants changed significantly during the stress process,the color of the leaves gradually turned yellow,the leaves wilted,and the phenomenon of lodging occurred.With the prolongation of the stress time,the chloroplast fluorescence parameters Fv/Fm and Fv/Fo showed a downward trend as a whole.Under low temperature stress,Fo first increases and then decreased,while under high temperature stress Fo showed a rising trend;under high and low temperature stress,the relative conductivity and proline content both showed an increasing trend.Under low temperature stress,SOD increased,POD and CAT first decreased and then increased,MDA first increased and then decreased.However,under high temperature stress,the activities of these three enzymes,MDA showed a completely opposite trend.Temperature stress caused some damage to Sophora flavescens seedlings,but the plants showed some resistance during the stress process.5.In order to screen the internal reference genes suitable for temperature stress study of Sophora flavescens,using RT-q PCR technology,3 kinds of software and comprehensive evaluation by Excel,the optimal combination of internal reference genes was determined to be β-actin and EF1α.