EST-derived SSR marker has been developed in many species,but few methods of high efficiency have been reported for the exploitation of EST-SSR markers.Thus,a high efficiency method for mining millions of redundant EST data is needed.A modified method for the EST-SSR development with high efficiency was established based on the redundant EST data of soybean in this study.The method achieved its function through classifying ESTs according to the same SSR motif and detected candidate loci with redundant sequences.In this study,a total of 80 polymorphic EST-SSR markers of soybean were developed,50 of them were exploited by this modified method which proved the higher speed and efficiency of this method.All the 80 polymorphic EST-SSRs were mapped on soybean physical map through in silico mapping and 15 markers were integrated on a genetic map constructed in previous study.A software named hpSSR（high polymorphic SSR） was programmed based on the concept of the up-built method for EST-SSR development.This method is not only pragmatic for EST-SSR exploitation in soybean,but also effective for the development of the marker in other species if the redundancy EST data is available.
MicroRNAs （miRNAs） are derived from distinct loci in the genome and play crucial roles in RNA-mediated gene silencing mechanisms that regulate cellular processes during development and stress responses of plants. The miRNAs are approximately 21 nucleotides long and code for the complementary strand to a larger genic mRNA. They are often found within the complementary primary transcript （pri-miRNAs）. In the past few years, a growing number of soybean miRNAs have been discovered, however, little is known about the transcriptional regulation of these miRNAs. In this study, promoters and cis-acting elements of soybean miRNAs were analyzed using the genomic data for the first time. A total of 82 miRNAs were located among 122 loci in genome, some were present as double or multiple copies. Five clusters that included ten miRNAs were found in genome, and only one cluster share the same promoter. A total of 191 promoters from 122 loci of the soybean miRNA sequences were found and further analyzed. The results indicated that the conserved soybean miRNA genes had a greater proportion of promoters than that of non-conserved ones, and the distribution of the transcript start sites （TSSs） and TATA-boxes found had different motif styles between conserved and non-conserved miRNA genes. Furthermore, the cis-acting elements 5＇ of the TSSs were analyzed to obtain potential function and spatiotemporal expression pattern of miRNAs. The data obtained here may lead to the identification of specific sequences upstream of pre-miRNAs and the functional annotation of miRNAs in soybean.
LIU Yong-xinHAN Ying-pengCHANG WeiZOU QuanGUO Mao-zuLI Wen-bin
The importance of microRNA （miRNA） at the post-transcriptional regulation level has recently been recognized in both animals and plants. In recent years, many studies focused on miRNA target identification and functional analysis. However, little is known about the transcription and regulation of miRNAs themselves. In this study, the transcription start sites （TSSs） for 11 miRNA primary transcripts of soybean from 11 miRNA loci （of 50 loci tested） were cloned by a 5＂ rapid amplification of cDNA ends （5＂ RACE） procedure using total RNA from 30-d-old seedlings. The features consistent with a RNA polymerase II mechanism of transcription were found among these miRNA loci. A position weight matrix algorithm was used to identify conserved motifs in miRNA core promoter regions. A canonical TATA box motif was identified upstream of the major start site at 8 （76%） of the mapped miRNA loci. Several cis-acting elements were predicted in the 2 kb 5＂ to the TSSs. Potential spatial and temporal expression patterns of the miRNAs were found. The target genes for these miRNAs were also predicted and further elucidated for the potential function of the miRNAs. This research provides a molecular basis to explore regulatory mechanisms of miRNA expression, and a way to understand miRNA-mediated regulatory pathways and networks in soybean.
LI JingLIU Yong-xinHAN Ying-pengLI Yong-guangGUO Mao-zuLI Wen-bin
The importance of microRNAs （miRNAs） at the post-transcriptional regulation level has recently been recognized in both animals and plants. In this study, the simple and most effective method of comparative genomic approach was used. First known plants miRNAs BLAST against the soybean genome, and then the located candidates were searched for novel miRNAs by RNA folding method in the vicinity （±400 nt） of the candidates. The results showed that a total of 521 novel soybean miRNA genes, including 236 mature miRNAs, were identified. All these mature miRNAs were grouped into 58 families, of which 21 of them were novel family in soybean. The upstream 2 000 nt of potential pre-miRNAs was used for promoter prediction, in order to investigate prediction of miRNAs and detect transcript unit and clustering. In this study, rniRNA genes less tend to be present as clusters in soybean. Only 9 clusters, containing 2l miRNA genes （accounted for 4.0% of the total）, were observed as part of polycistronic transcripts. Detailed analysis of sequence characteristics of novel miRNAs in soybean and all previous known plants miRNAs, were carried out. These results of this study provide a reference point for further study on miRNAs identification in plants, and improve the understanding of genome in soybean.
LIU Yong-xinCHANG WeiHAN Ying-pengZOU QuanGUO Mao-zuLI Wen-bin
