Cloing Rye 1RS Specific Conserved Sequence And Expression Of 1BL.1RS Translocation Lines LTR Retrotransposon

Wheat (Triticum aestivum L., AABBDD) is our second largest food crop after rice, also one of the world’s major food crops. Due to the characteristics of high-yielding and disease-resistant wheat varieties (lines) carrying 1BL.1RS translocation chromosome has planted a large number of the world. However, due to the IRS single source, most people consider the question is how to broaden the genetic diversity of 1BL.1RS translocation system. The current development of IRS specific molecular markers has provide a convenience for 1BL.1RS application in wheat breeding, but for many of the reported IRS specific molecular markers, its versatility and signature of the amplified sequences are lack of detailed study. Furthermore, wheat genome contains a large number retrotransposon sequence and has a certain biological function. Currently, the research about the expression of the retrotransposon sequences in 1BL.1RS translocation varieties (lines) is very little. Therefore, in this experiment we selected nine representative 1BL.1RS translocation or 1RS.7DL,7DS.1BL translocation varieties (lines) as well as 9 sister 1BL.1RS translocation lines fostered by our group for materials, studied the versatility of 142 pairs of the published IRS-specific primers, some products were cloned and sequenced Simultaneously, we also studied the expression of retrotransposon LTR (long terminal repeat) in the sister 1BL.1RS translocation lines.In this study, first, we used in situ hybridization of cytological analysis to identify wheat varieties; second,we screened the IRS specific markers through PCR-based molecular markers, and then analysed these conserved sequences amplified from IRS-specific markers through clone and sequence, finally, we conducted a preliminary analysis about the expression of retrotransposons LTR in sister 1BL.1RS translocation lines. The innvovative results are as follows:1. Cytology identification of the experimental materials, used to determine 7 wheat varieties and hybrids of 9 the lab created sister were 1BL.1RS translocation lines (In addition.there are two wheat varieties of LM11 and JN16 for lRS.7DLand 7DS.1BL translocation lines), and the rest of the seven control materials are not contain rye chromatin.2.Through PCR amplification, among the 142 markers that were reported to be located on IRS, only 27 markers amplified IRS-specific bands from all the nine 1BL.1RS and 1RS.7DL,7DS.1BL cultivars/lines and these bands did not exist in CS, MY11, CN27, CM107, J1025, R345 and R297. The 27 markers are TSM39, TSM81, TSM86, TSM92, TSM103, TSM109, TSM111, TSM123, TSM264, TSM294, TSM303, TSM306, TSM391, TSM460, TSM634, TSM642, TSM716, Top1017, Sec-1, BmacO213, STSWE126, P6M12-P, ora003, ora004, ora007, ora011 and ora012.3. From the 27 specific primers, the products,which amplified by the 15 markers, selected randomly,were cloned and sequenced, the results showed that for each of the markers ora004, ora007, ora011, ora012, P6M12-P, Top1017, TSM103, TSM109 and TSM111, the sequences amplified from the nine cultivars/lines were identical; Four different sequences were amplified by marker Bmac0213; Among the 27 sequences amplified by marker TSM294, three kinds of sequences were observed; Two kinds of sequences amplified by markers ora003, TSM39 and TSM123; marker TSM264 produced 13 different sequences from the nine cultivars/lines; the length of the sequence amplified by TSM39 and Bmac0213 are not same,and even the length of the sequence amplified by primers TSM294, ora003, TSM123, TSM264 is same, but different nucleotide. The results show that genetic diversity analysis with molecular markers based on PCR only through the size of the bands to determine is not accurate.4. According to the conserved region of LTR sequence of 19 family Tyl-copia like retrotransposons, we designed 21 primers to analysis the 9 sister 1BL.1RStranslocation linesand the parent MY 11 with cDNA as template, the results show that the size of the products amplified by 6 pairs of primers are consistent with the expected fragment size. Among them,5 Primersin 10 materials amplified products. But there is no product in 14T-80-1 translocation lines amplified by the primer TREP228. The products amplified by primers Angela-3 and TREP228 were cloned and sequenced. Sequence alignment analysis showed that the similarity of these sequences and sequences in the databases of LTR conserved region is all above 90%, indicating that these sequences amplified by the primer Angela-3 and TREP228belong to the sequence of retrotransposons LTR, and these retrotransposon LTR had transcriptional activity in 1BL.1RS translocation lines, but the expression of retrotransposons LTR, there are differences existed in different sister translocation lines.5.The results of this study provide 9 pairs of universal and conserved IRS specific markers, and provide a basis for the investigation of the polymorphism of IRS.In addition, it will provide a theoretical basis for the further study about the expression and function of the retrotransposons in 1BL.1RS translocationins lines.