Development And Application Of Annotation And Marking Of Common Bean Mutator Transposable Elements

Phaseolus vulgaris is an important vegetable crop,which is widely planted all over the world.It has high nutritional value and economic value.The identification of common bean germplasm resources can delay and solve a series of problems such as variety degradation and variety confusion.The Mutator transposable elements is one of the DNA transposons with the most copy number.It has the characteristics of wide distribution,high copy number,high transposition frequency,high polymorphism,ease of use,and close functional gene association,which is in line with the characteristics of molecular marker development.Therefore,based on the method of structure search and homology comparison,this paper systematically identified Mutator transposable elements in two different common bean genomes,including elements quantity analysis,elements insertion bias,chromosomes distribution characteristics and phylogenetic analysis.Then a new method was developed to identify the common bean germplasm resources based on molecular markers of common bean Mutator elements,which can delay and solve common bean varieties a series of problems such as degradation and chaotic varieties.This study provides a feasible method for the identification of common bean germplasm resources,and provides a theoretical basis for the cultivation of new varieties with good quality and considerable yield.The main results are as follows:1.Identification and quantitative analysis of Mutator transposable elements in common bean genomeBased on the method of structural feature search and homology comparison,we identified 2040 Mutator transposable elements with clear boundaries,clear insertion positions,and relatively complete structures from the G19833 genome of common bean.The same method was used to identify the complete Mutator transposable elements in the BAT93 genome.There are 2130 Mutator elements.The number of Mutator transposable elements in the two genomes is not much different,but both contain their own unique Mutator transposable elements.There are 158 unique elements in the G19833 genome and124 unique elements in BAT93 element.Analysis of the Mutator elements in the G19833 genome found that the total length of these elements was about 2.07 Mb,84 % of the elements were within the range of less than 1 Kb,the smallest element was only 179 bp,and the longest element was as high as 10872 bp.2.Analysis of insertion bias of common bean Mutator transposable elementsPerformed a statistical analysis of the 20 bp and 9 bp TSD(Target Site Duplication,TSD)of the flanking sequences on both sides of all Mutator elements in the G19833 genome.The average GC content at the 9bp TSD position was about 24 %,which was much lower than the 49 bases average GC content of about 31 %.The GC content of the entire common bean genome is 36 %.The insertion site of the Mutator transposable elements has a strong bias,and it is obviously biased to insert into the region with less GC.At the same time,there is a significant positive correlation between the density of Mutator transposable elements and the density of functional genes.3.Phylogenetic analysis of Mutator transposable elements in common beanPhylogenetic analysis of all Mutator elements containing Mutator transposase in the G19833 genome of common bean showed that it can be roughly divided into five branches.Among them,clade Ⅰ,clade Ⅲ and clade Ⅴ are very rich,and the number of enzyme-containing elements is relatively large.The clade II clade contains 2enzyme-containing elements and two families.Compared with the first three branches,this branch is relatively stable in evolution.However,clade IV has only one enzymatic element and the family of this elements is the family with the most abundant copy numbers,Family1.4.Development of molecular markers based on common bean Mutator transposable elements for germplasm identificationMolecular markers were developed based on the characteristics of the broad distribution of Phaseolus vulgaris Mutator transposable elements,high copy number,high transposition frequency,high polymorphism,and relatively lack of insertion specificity.Based on the unique Mutator elements in the two common bean genomes,25 pairs of primers were randomly designed,and 32 common bean varieties from different provinces and regions were selected as supply varieties for germplasm resource identification.After PCR amplification,we screened out 13 pairs of core primers to distinguish all common bean varieties,and the discrimination rate was as high as 100 %.We also carried out genetic diversity analysis on these 32 common bean varieties.The genetic similarity coefficient is between 0.24 and 0.88,and the average genetic similarity coefficient is 0.56.At the genetic similarity coefficient of 0.24,the 32 common bean varieties were divided into 2 categories,and at the genetic similarity coefficient of 0.45,all the common bean varieties were divided into four subcategories.Finally,a unique molecular ID card code is constructed according to the presence or absence of bands.Different common bean germplasms can be distinguished more intuitively,conveniently and simply.The above results indicate that the Mutator transposable elements in the common bean genome are consistent with the characteristics of molecular marker development.This marker technology can be applied to the identification of common bean germplasm resources,and can provide a selection basis for the selection and breeding of common bean varieties,and provide molecular support for the cultivation of high-quality,high-yield varieties.