Isolation Of P-genome Specific Sequences From Agropyron Cristatum(L.) Gaertn And Application In Wheat–A. Cristatum Derivatives

Genome-specific sequences are very important for the development of genome-specific molecular markers and the study of structure and evolution of P genome chromosomes. Grain number per spike(GNPS) is one of the three factors which determine wheat yield. It is of great significance to produce novel germplasm with high GNPS through wide hybridization for broadening the genetic basis and improving the wheat breeding. In this study, P-genome specific sequences were isolated by chromosome microdissection and DOP-PCR, and P-genome specific molecular markers were developed on basis of these sequences. Then we used them as tools to determine the distribution patterns of P-genome specific sequences on P genome chromosomes, to identify the alien A. cristatum chromosome, and to uncover molecular markers available for detecting wheat–A. cristatum germplasm with multi-kernel. Meanwhile, we evaluated the agronomic traits of novel wheat–A. cristatum germplasms. The aim of this study was to provide basic data and marterials for wheat high yield breeding and provide molecular evidences for illuminating the gene/QTL of high GNPS derived from A. cristatum P genome.1. Chromosome microdissection and DOP-PCR amplification of Chromosomes 6PS and 6PL. Chromosomes 6PS and 6PL were microdissected from wheat–A. cristatum 6PS and 6PL addition lines, and DOP-PCR amplification products from microdissected 6PS and 6PL chromosomes were used as probes to hybridize with mitotic metaphase chromosomes of diploid A. cristatum, wheat–A. cristatum addition lines 6PS, 6PL and 6P. The results showed that the distribution patterns on A. cristatum chromosomes using DOP-PCR products from microdissected 6PS as the probe were similar to those using DOP-PCR products from microdissected 6PL as the probe. They both distributed along both arms of all chromosomes, and there was no difference between chromosome 6P and other chromosomes according to the hybridization pattern. There were no signals on wheat chromosomes indicating that the DOP-PCR products contains P-genome specific sequences, and they could be used to identify P genome chromosomes and chromosome fragments in wheat–A. cristatum introgression lines.2. Isolation of A. cristatum specific sequences. The DOP-PCR products from chromosome 6PS were cloned into p MD19-T cloning vector, and dot-blot hybridization was used to screen polymorphic DNA clones between A. cristatum accession Z559 and common wheat Fukuho. Then the polymorphic DNA clones were sequenced and analyzed by BLAST. Seventy-six A. cristatum specific sequences were obtained. Among them, eighteen sequences had the characterization of retrotransposon, and six sequences were DNA transposon; a sequence belonged to tandem repeat sequence; three sequences contained simple repeat sequences; twelve sequences had no similarity with wheat genome sequences.3. Development of molecular markers on the basis of A. cristatum specific sequences. 96 primer pairs were designed based on 76 specific sequences. The marker analysis showed that 48 markers were polymorphic between Z559 and Fukuho, and 41 markers were specific to Z559 and 6P addition line 4844-12. The analysis of specificity using 16 Triticeae genomes showed that only 18 primer pairs were specific to P genome. But there were 30 molecular markers producing A. cristatum specific bands in all the tested addion lines; there were 6 markers specific to 6PS: two of them located in 6PS-0.00-0.15 and the other four located in 6PS-0.15-1.00.4. Distribution patterns of specific sequences on P genome chromosomes. Eighteen out of Seventy-six A. cristatum sequences could hybridize to A. cristatum chromosomes by FISH analysis. They could be classified into five types: the first type consisted of ten sequences distributing all over the A. cristatum chromosomes; the second type comprised two sequences distributing in the pericentromere region; there were four sequences distributing in the distal region included in the third type; the fourth type is a centromere sequence, and the fifth type was a sequence distributing in the terminal of chromosomes. Seventeen wheat–A. cristatum addition lines could be distinguished into four groups using p Ac TRT1, p Acp CR2, and p Ac TR1 as probes which distributing in the end, pericentromere region and distal region of chromosomes respectively. All chromosomes of diploid A. cristatum could be identified using p Ac TRT1 and p Acp CR2 as probes and its model map was constructed based on the two probes. Meanwhile we demonstrated that there were structural polymorphisms in A. cristatum chromosomes.5. Application of P-genome specific sequences and their molecular markers in wheat–A. cristatum derivative lines with high GNPS. Application in wheat–A. cristatum 6P addition lines: FISH using the p Ac TRT1, p Acp CR2, p Ac TR1 probes and six molecular markers specific to chromosome 6PS confirmed that there were differences among different 6P chromosomes. Application in translocation lines:(1) a wheat–A. cristatum translocation line was identified as T1AS-6PS-1AS·1AL using GISH-FISH and specific markers, and the 6P chromatin with pleiotropic effects originated from 6PS-0.00-0.15;(2) the GISH results of translocation line WAT-31 using P genomic DNA as a probe were consistent with that of FISH using p Ac PR1 as a probe, indicating that p Ac PR1(the first type of sequence) could instead of P genomic DNA in identification of translocation line. Meanwhile WAT-31 contained 42 wheat centromeres and two A. cristatum centromeres using the CRW(centromeric retrotransposon of wheat) and p Ac CR1(the centrmeric sequence we obtained in this study) probes. Application in introgression lines: 74 wheat–A. cristatum derivative lines were detected by 353 molecular markers including 41 PCR markers developed based on A. cristatum specific sequences and 312 STS markers designed by A. cristatum ESTs. We obtained 15 molecular markers which could totally detect 65 wheat–A. cristatum derivative lines by molecular markers analysis. The fifteen markers included six markers specific to P genome, and nine STS markers: seven markers from the long arm of 6P, and two markers from the short arm of 6P. It would provide important foundation for high yield breeding and marker-assisted selection.6. Evaluation of comprehensive traits in wheat–A. cristatum derivative lines. The investigation of agronomic traits of 128 wheat–A. cristatum derivative lines was carried out in Xinxiang, Henan province, through two years planting. Meanwhile the powdery mildew resistance and high molecular weight glutenin subunit(HMW-GS) composition were evaluated. The results showed that 80 of 128 wheat–A. cristatum derivative lines conferred GNPS exceeding 80, and 85 lines had powdery mildew resistance and there were 41 lines conferring high quality subunit composition. Moreover, 12 wheat–A. cristatum novel germplasms conferred not only GNPS exceeding 80, but also powdery mildew resistance and high quality subunit composition, which indicating these germplasms had excellent comprehensive properties. It would be an important material basis for breeding new wheat varity with high-yield, high-quality, and powdery mildew resistance.