Development Of E-chromosome Specific PCR Markers For Thinopyrum Elongatum

Repeat sequences constitute major fraction of the higher plant genomes. It was reported that 75% of wheat genome sequence was occupied by them. Unhomologous recombination, illegitimate recombination and self-mutations, as genomic evolution power, cause many kinds of mutations in DNA level, which provide abundant diversity in plant genomes and form genome-specific or species-specific repeat sequences. Some of them are not only used as useful molecular tool to detect genetic introgression between genomes or species, but also useful for developing molecular markers to map genes and enrich genetic linkage map. Diploid species of Thinopyrum elongatum is one of the valuable relatives for wheat quality and yielding improvement. For better utilization of the genes or superior characters deposited in Th. elongatum species, wheat-Th. elongatum disomic additional and substitution lines have been obtained through cross between wheat and Th. elongatum. However, the markers for detection and tracking of alien genetic material of Th. elongatum were very limited and thus impeded us better to use this plant resources in wheat breeding. In this study, seventy four E-chromosome specific molecular markers were acquired and three of them were successfully converted into the chromosome specific PCR markers. The results present there would provide new ways for rapid detection of alien genetic material transferred through Th. elongatum into common wheat. The main results were as follows:1. Screening of E-chromosome specific fragments of Th. elongatum11 primers combined into 35 primer combinations were used to screen Triticum aestivum cv. Chinese Spring, Th. elongatum and CS-Th. elongatum disomic additional lines for development of E-chromosome specific molecular markers of Th. elongatum. It was showed that 74 fragments which were specific for 1E to 7E except 2E chromosome were obtained. The specific fragments distributed in 1E, 3E, 4E, 5E, 6E and 7E chromosomes were 7, 7, 15, l9, 9 and17, respectively. Abundance of specificity in different chromosomes was different, such as specific fragment of 2E chromosome was not found. We also found that specificity of E genome in the context of wheat revealed by different primer combinations had a certain degree of difference. In three types of primer combinations, IRAP and ISSR amplified specific fragments fewer, were 8 and 7 respectively, and REMAP amplified specific fragments most, were 59. 2. Sequencing and blasting of specific fragmentsOne 5E and five 7E chromosome specific fragments were cloned and sequenced. After sequence alignmented in NCBI GenBank, it was indicted that one specific fragment of 5E and two specific fragments of 7E whose vectors were removed were 445, 757 and 325bp, respectively. They were so numbered Z2-445, Z21-757 and Z12-325. Apart from about 100bp of barley retrotransposon BARE-1 LTR DNA sequence, these three sequences had no similar sequence. Because the primers were designed based on LTR sequence, they could amplify about 100bp of LTR sequence actually. Non-homologous sequences were likely to be partial specific sequence of Th. elongatum. We surmised that the primers designed based on such sequences could amplify the specific fragment, which could be observed through the agarose gel electrophoresis directly, easily and quickly. And thus they were converted into specific PCR markers.3. Development of E-chromosome specific PCR markers for Th. elongatumWe designed three pairs of primers and numbered 5E-1,P4,ZCP1 according to sequences of Z2-445,Z21-757,Z12-325, which could amplify fragments 304,329 and 105bp, respectively. After screening CS, Th. elongatum and CS-Th. elongatum disomic additional and substitutional lines, 5E-1,P4 and ZCP1 successfully amplified the anticipated fagments in Th. elongatum and CS-Th. elongatum disomic additional and substitutional lines. Among them, 5E-1 only existed in Th. elongatum, CS 5E additional and substitutional lines, while P4 and ZCP1 only presented in Th. elongatum, CS 7E additional and substitutional lines. Then we used P4 and ZCP1 to screen 7ES and 7EL ditelesomic additional lines, they only existed in 7EL ditelesomic additional line. They were thought to Th. elongatum 7EL chromosome specific PCR markers. The results showed that Z2-445,Z21-757 and Z12-325 could be converted into chromoseme specific PCR markers. Successful transformation 5E and 7E chromosome specific PCR markers were repeatable, stable, easy, quick and low cost after several tests. They were particularly suitable for development of molecular markers when there was no related genomic sequence information. They could be used for identifying the translocation lines or the alien genetic materials of Th. elongatum in the introgression in wheat chromosome engineering, as well as providing more molecular markers for gene cloning.