Mining Of SSR Marker Related To Ovule Development From Transcriptome Information And Analysis Of Differential Expressed Gene In Pinus Tabulaeformis Carr.

Pinus tabulaeformis Carr. is the primary species in greening and afforestation and shelterbelt throughout China. The research on the mechanism of ovule development of Pinus tabulaeformnis has important theoretical and practical significance for improving the seed yield and quality. The studys show that ovule development is the results of gene sequential expression in space, and the regulation of gene expression is affected by many factors in vivo and vitro. Recently increasing experimental evidences indicated that SSR inside plants is one of the important factors involved in the gene expression and regulation. In Xing Cheng Seed Orchard, Liao Ning province, female gametophte free nuclear of No.28 sterile clones stopped division in early stage, which caused the ovule abortion. To study the reason of stopping division in this clone, the research group used high-throughput sequencing to analyze Pinus tabulaeformis ovule transcriptome of fertile and sterile clones respectively in different stage of free nuclear, and got 63449 unigenes related the development. On the basis, this research investigated the characterization of SSR in transcriptome, and screened the SSR markers related to development; illustrated the expression rule of target genes in different stages of fertile and sterile clones. The main results are as follows:1. Using the program MISA to mine and analyze the transcriptome to seek the SSR locus, a total of 1412 SSRs were identified in 1274 unigenes with one SSR per 2.15 kb. Among 87 SSR repeat motifs existing in ovule transcriptome, (A/T) n was in maximum proportion, accounting for 45.89%. The highest frequency repeat types are mononucleotide and trinucleotide, accounting for 46.88% and 34.99% respectively. The majority of microsatellites were below 20bp in length. The proportion of microsatellites over 20bp were only 5.67%. The result showed a negative correlation between the frequency of occurrences and the length of microsatellites. Most of SSRs were in untranslated regions, and only 232 SSRs located in protein-coding regions, while 18 in both of them. Trinucleotide repeats occupied the largest proportion in protein-coding regions (105,45.26%). Compared with the others, the genes of the ancient plants such as Pinus tabulaeformis Carr. and Ginkgo biloba, Taxus chinensis var. mairei were more stable, so SSRs occurred with lower frequency, less species and smaller range of length.2. RPKM values of genes contained SSR were acquired by high-throughput sequencing technology. The differentially expressed genes between fertile and sterile clones were chosen. The software Primer 6.0 was used to design primer based on sequences of these genes.12 pairs primers were produced to amplify by PCR. Agarose gel electrophoresis were used to test the result. A 300 bp fragment was expressed specially in sterile clones and a 250 bp fragment was expressed specially in fertile clones. The two fragments were clear and stable. We speculated that they may involve in the development process of ovule and have impact on sterility, but further validation is needed.3. RT-qPCR was used to analyze the expression pattern of 8 primary target genes in different stages. Four target genes were amplified by primer 24、31、36、38. The amplification curves and dissociation curves were stable, which indicated reliable amplification results and the available primers. Then the expression differences of these four genes in fertile and sterile clones were quantified relatively by 2-ΔΔCT.We get the different multiple but the same rule:in prophase of stage, the expression level of target genes in sterile clones was higher than in fertile clones. It was more obvious in metaphase, and showed the opposite result was shown in anaphase. It is concluded that these four genes may take directly part in the free nuclear division of female gametophyte, and play important roles in ovule development.