first , soybean ( glycine max ) is one of the most important crops for producing protein and oil .
second , it has the capacity to fix nitrogen which is one of the major problems of life science .
biological nitrogen fixation provides all the plants with 75% of nitrogen , which plays an important work in the practical production .
the completions of genome sequencing in legumes provide new ideas for the studying of symbiotic nitrogen fixation on the genome level .
large - scale duplication events have been considered important for the evolution of many organisms . about 70% of angiosperm evolution has experienced one or more polyploidization events .
polyploidy is a widespread and recursive phenomenon during plant evolution , and it could generate massive duplicated genes which is an important resource of genetic innovation [ 26 ] .
this process greatly increases the complexity of the plant and improves the adaptive capacity to new environments .
, the in - depth study of evolution after polyploidization is also needed . for comparative genomics analysis , this paper aims to study the evolution laws of duplicated genes in soybean after polyploidization .
our study identifies significant duplicated genes on the evolutionary of the two species , soybean and arabidopsis thaliana , and indicates that the whole genome duplication ( wgd ) event occurred more than once in the genome evolution of soybean .
soybean ( glycine max )   and arabidopsis thaliana   genome sequences were downloaded from plant genome duplication database ( http://chibba.agtec.uga.edu/duplication/ ) .
66210 and 27379 dna and the corresponding protein sequences of arabidopsis and soybean are obtained , respectively . and
the locations of each gene on the chromosome are also identified . in this paper ,
arabidopsis and soybean are selected as our research objects , which have common polyploid ancestors . using model organisms arabidopsis genome as a reference ,
the collinearity method is applied to infer homologous genes in the soybean genome ( figure 1 ) .
first , the similarity between soybean and arabidopsis thaliana is predicted by running blastp ( evalue < 1  10 ) .
the built - in scoring scheme for mcscan [ 10 , 11 ] is min(log10  evalue , 50 ) for every matching gene pair and 1 for each 10  kb distance between anchors , and blocks that had scores > 300 were kept .
the homology gene blocks are determined by searching the maximal match of the result by mcscan ; then , the algorithm of searching the really paralogs is listed as follows : process the aligns in output data of the mcscan to obtain the size of collinear genes blocks , such as the size of blocks of collinear genes pairs .
a - b is n1 , and a - c is n2;compare the size of the collinear genes blocks with other different genes;identify the true paralogs produced by wgd . if n1
> n2 , then , the a - b is a pair of paralogs gene .
process the aligns in output data of the mcscan to obtain the size of collinear genes blocks , such as the size of blocks of collinear genes pairs .
a - b is n1 , and a - c is n2 ; compare the size of the collinear genes blocks with other different genes ; identify the true paralogs produced by wgd .
if n1 > n2 , then , the a - b is a pair of paralogs gene .
dot plots can visualize the similarity between two proteins or nucleic acid sequences ; when the sequences match at the same location on the plot , a dot is drawn at the corresponding position .
the dna dot plot of soybean genome in figure 2 shows regional self - similarity : ( 1 ) x - axis aligned the 20 soybean chromosomes from left to right and y - axis aligned the sequence from top to bottom ; ( 2 ) based on homologous information by blast and the location of genes on chromosomes , each pair of homologous genes in the two - dimensional plan can be formed on a point ; ( 3 ) a series of consecutive points in the plane would constitute a different line . from the figure , we can find that the soybean genome contains extensive duplicated genome .
we find that 23,269 pairs of paralogs , including about 46,538 genes , could account for 70.3% of total genes in soybean genome .
all these genes are duplicated genes produced by the whole genome duplication . in total , 47 duplicated blocks located on all 20 chromosomes are identified , in which the largest block contains 1070 pairs of paralogs , 2140 genes , which are mainly distributed in the chromosomes 3 and 19 of soybean ; the smallest block contains 5 pairs of paralogs , 10 genes , which are mainly located on chromosome 5 and 6 .
a synonymous substitution is the evolutionary substitution of one based for another in an exon of a gene coding for a protein , such that the produced amino acid sequence is not modified . a nonsynonymous substitution results in a change in amino acid .
applying ng-86 model , the values of synonymous substitution rate ( ks ) and nonsynonymous substitution rate ( ka ) are calculated between homologous genes in soybean .
form the figure , we can see two obvious peaks ; the ks is about 0.15 and 0.42 , respectively .
in addition , the ks which is about 520 pairs of paralogs is almost 0 , and about more than 1200 pairs of paralogs are greater than 1.5 .
the data showed that the multiple of massive gene duplication event may occur in the genome of soybean .
the values of synonymous substitution rate ( ks ) between duplicated genes are very small .
the results showed that the duplicated genes were produced by recent duplication event , which also showed the existence of the ongoing duplication event in the soybean genome .
the distribution of duplicated genes on the chromosome produced by the whole genome duplication may be related to its physical location . to infer the potential rule here ,
we divide all the soybean genes into two categories : ( 1 ) duplicated genes produced by wgd and ( 2 ) nonduplicated genes .
firstly , computing the distance of each duplicated genes and nonduplicated genes to chromosome ends and , secondly , comparing the average distance of duplicated genes and nonduplication genes to the chromosome ends , we applied the paired two - sample analysis .
for these two sets of distance dn ( to the ends of chromosomes ) , we can state that the null hypothesis is that the means between the two distance sets are equal , respectively .
the two - sample test is a hypothesis test for answering questions about the mean where the data are collected from two random samples of independent observations . for a duplicated gene in soybean , if the existence of distance dn1 of n1 paired duplicated genes and distance dn2 of n2 nonrepetitive genes which calculates the mean of n1 and n2 is x1 and x2 , test statistic of z - test is analyzed to test whether the difference between two means is equal to a number ; statistics are 
 ( 1)z=(x1x2)d012/n1+22/n2  .
assume that d0 is the mean difference of dn1 ( the mean of duplicated genes ) and dn2 ( the mean of nonduplicated genes ) , where the mean difference is assumed to be 0 ; 1 , 2 are the variance of the two samples .
our analysis found that duplicated genes are often located near the chromosome ends , and whole genome duplication and gene physical location are shown in table 1 .
the average distance of duplicated genes to chromosome ends in soybean is 6.6  mbp ( p_value = 0  0.05 ) , while the average distance of all genes is 11  mbp ( p_value = 0  0.05 ) . in soybean , about 40% of duplicated gene pairs are distributed close to the ends of chromosomes where the distance is 4  mb , as shown in table 1 . as shown above ,
the physical location of genes on the chromosome is closely related to whole gene duplication , while duplicated genes produced by wgd are distributed close to the ends of chromosomes .
first , there is a large number of redundancies of more conservative sequence far from centromere ; second , gene collinearity is often found in areas of high density ; third , the original copies are often near the ends of chromosomes . dna rearrangement and gene codon mutation reduced the similarity between sequences , resulting in the emergence of new gene sequences , which may have new functions to support the organism to adapt to the new environment .
our studies suggest that the whole genome duplication event occurs more than once in the genome evolution of soybean .
based on the collinearity method , the duplicated genes which cover 70.3% of the soybean genome identified are produced by the whole genome duplication .
one of the reasons is that the original copies are often near the ends of chromosomes .
our findings also raise the possibility that the ongoing duplication event exists in the soybean genome .
identifying the scale of gene multiplication in genome can provide important information for the study of genetics .
dna rearrangement and gene codon mutation reduced the similarity between sequences , resulting in the emergence of new gene sequences . although an increasing number of reports have revealed the phenomenon of gene duplication , the origins and mechanisms of duplicated gene and the evolutionary fate of duplicated gene still lack sufficient research . revealing the contribution of gene and genome duplications to the evolution and domestication of these plants
is a significant research ; we hope that our study will promote the research in the field .
 soybean ( glycine max ) is one of the most important crop plants for providing protein and oil . 
 it is important to investigate soybean genome for its economic and scientific value . 
 polyploidy is a widespread and recursive phenomenon during plant evolution , and it could generate massive duplicated genes which is an important resource for genetic innovation . improved sequence alignment criteria and statistical analysis are used to identify and characterize duplicated genes produced by polyploidization in soybean . 
 based on the collinearity method , duplicated genes by whole genome duplication account for 70.3% in soybean . from the statistical analysis of the molecular distances between duplicated genes , our study indicates that the whole genome duplication event occurred more than once in the genome evolution of soybean , which is often distributed near the ends of chromosomes . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion