Isolate New Repetitive DNA Sequence From Daypyrum Breviaristatum Genome And Its Application In Identifying New Resistant Wheat Germplasm

The genus Dasypyrum consists of two species, D. villosum (L.) Candargy (syn. Haynaldia villosa (L.) Schur) and D. breviaristatum (Lindb. F.) Frederiksen (syn. H. hordeaceae Coss. et Dur.). D. villosum is an annual allogamous diploid species (2n = 2x = 14, VV) mainly distributed from the Mediterranean to the Caspian sea. D. breviaristatum consist of diploid (2n = 2x = 14, VbVb) and tetraploid (2n = 4x = 14, VbVbVbVb) cytotypes, they are perennial allogamous species restricted to two mountainous regions in northwest Africa and in Greece and Morocco. The genus Dasypyrum belongs to Triticinae of Triticeae, and these species contain a large number of useful genes for resistance to powdery mildew, leaf rust, stem rust, take-all, and they have strong ability of tiller, more spicules, bear drought, cold, slat and have high quantity of protein, which will be useful for quality and yield improvement in wheat breeding program.So far, there have been four shortcomes on Dasypyrum researches:(1) the phylogenetic relationship of the tetraploid D. breviaristatum in genus Dasypyrum is still discussed controversial, particularly on the evolution relationships between D. breviaristatum and D. villosum, lacking of molecular and cytogenetic evidences. (2)The molecular markers specific to genus Dasypyrum whole genome or single chromosome still need to be exploited. (3)We have already started a project to transfer powdery mildew and stripe rust resistance from tetraploid D. breviaristatum to wheat, and we have already bred a large quantity of wheat-D. breviaristatum introgression lines, however, the homologous relationships of the D. breviaristatum chromatin in these introgeressions still unknown which need further identification. (4)The resistant or high yielding wheat-D.villosum translocations refer to chromosome 1V, 4V and 6V have already been established, but there is no any report on compensating Robertsonian translocations involving D. villosum chromosme 2V, 3V, 5V and 7V.In this research, we will solve the four shortcomes mentioned above. We will further analyze the evolutionary relationships of the Dasypyrum species using molecular and cytogenetic methods. We developed and identified wheat-tetraploid D. breviaristatum addition lines using EST-STS (Expressed sequences Tags-Sequence Tag Site) markers and PLUG (PCR-based Landmark Unique Gene) markers. We induced and identified wheat- D.villosum 2V, 3V, 5V and 7V robertsonian translocations. The reseach results reported as follows:1.(1)D. villosum, tetraploid D. breviaristatum, Th. intermedium, S. africanum, common wheat of Chinese Spring and some other species in Triticeae were analyzed using random amplified polymorphic DNA(RAPD)technique. The cluster result showed that D. villosum and tetraploid D. breviaristatum were not clustered into one group, indicating that tetraploid D. breviaristatum did not originated from D. villosum. (2)Giemsa C-banding showed that diploid and tetraploid D. breviaristatum had mainly weak interstitial C-bands, while D. villosum had mainly large telomeric and distal C-bands. (3)We cloned the chloroplast infA genes, mitochondrial genes trnfM and rrn18 from Dasypyrum three species. Two evolutionary trees were built based on the chloroplast and mitochondrial genes independently using bootstrap analysis. Both evolutionary trees support the hypothesis that tetraploid D. breviaristatum was originated from diploid D. breviaristatum but not from D. villosum. (4)Genomic in situ hybridization (GISH) and Fluorescence in situ hybridization (FISH) using the ribosomal gene probe pTa71 showed that tetraploid D. breviaristatum was dirived from diploid D. breviaristatum. (5) FISH using the rye genome repetitive DNA probe pSc74 showed that not only chromosome rearrangement but also repetitive DNA amplification happened in the course of diploid D. breviaristatum genome doubled to form tetraploid D. breviaristatum2. (1)We isolated a new Long Terminal Repeat (LTR) Sabrina-like retrotransposon sequence pDb12H (or called OPH12) from the D. breviaristatum genome. Based on the sequence of pDb12H, a pair of specific primer was designed. A sequence characterized amplified region (SCAR) marker was developed using the primer pair designed, this marker can be used to screen the population of resistant wheat-D. breviaristatum introgression and wheat-D.villosum translocations. FISH analysis showed that pDbH12 is present on Dasypyrum (V genome), Hordeum (H genome) and Thinopyrum intermedium Js genome with the FISH sites covering the entire chromosomes. However, pDbH12 did not hybridize to the genomes of Secale, Triticum, Lophopyrum, Pseduoroengeria, Aegilops, Agropyron desertorum and Elymus. Results from SCAR PCR and dot blot both support the FISH results, and the combined results suggest that amplification of Sabrina-like LTR retrotransposons pDb12H is an important factor, which was involved in the speciation process. This retrotransposon lost its activity in early generations when transpose to Triticinae species genomes, and what is more, Dasypyrum V genome and barley H genome may involved the formation of Th. intermedium Js genome. Kishii et al. reported that the rye R genome and Thinopyrum bessarabicum J genome are involved the formation of the Th. intermedium Js genome using GISH and molecular markers. Combining these results, we suggest the genomic formula of Th. intermedium can be tentatively re-designated as StJs(V-J-R-H)s. pDbH12 can serve as a cytogenetic marker for tracing chromatin from V or Vb, H and JS genomes in wheat-alien introgression lines. (2)943 pair of EST-STS primers, which belong to wheat homolous group one to seven, were screened using tetraploid D. breviaristatum and Chinese Spring as material, all PCR product were digested by four different DNA cutter enzyme HaeIII, MspI, RsaI and AluI, totally 474 polymorphism marker were obtained. Meanwhile, we also screened 56 pair of PLUG primers (four for each wheat chromosome arm), the PCR products were digested by DNA cutter enzyme TaqI and HaeIII, totally 6 PLUG markers were obtained.3. Resistant wheat- D. breviaristatum introgressions were primarily screened by marker pDb12H. The introgeression which can amplify pDb12H namely had D. breviaristatum chromatin in wheat background, were secondarily investigated by Geimsa C-banding. The result showed that there are only two different wheat- D. breviaristatum additions Y93-1-6-1 and Y93-1-A6-4 in the set of introgression lines. The former mentioned EST-STS and PLUG marekers were used to identify these two additions. The results showed that the D. breviaristatum chromosomes in additions Y93-1-6-1 and Y93-1-A6-4 rearranged a lot. In addition Y93-1-6-1, the D. breviaristatum chromosome long arm was 6VbL, while for the short arm, there was chromosome segment exchange between 6VbS and 7VbS; in addition Y93-1-A6-4, the D. breviaristatum chromosome rearranged much more complicated, the short arm was 6VbS and 7VbS rearrangemnt,and the long arem was 1VbL, 2VbL and 7VbL rearrangment. This was the first report about D. breviaristatum chromosome rearrangement. 4. The monosomic stock was crossed with corresponding wheat-D.villosum disomic addition stock to make the double monosomic plants which were self pollinated. The self pollinated seed were used to screen wheat- D.villosum translocations. (1)Totally 660 progenies derived from double monosomic 40+2V+2D were screened. Four heterozygous T2VL.2DS translocations and three heterozygous T2VS.2DL translocations were obtained. (2)A total of 496 progenies derived from double monosomic 40+3V+3D were screened. Five heterozygous T3DS.3VL translocations and three heterozygous T3DL.3VS translocations were obtained. (3)A total of 342 progenies derived from double monosomic 40+5V+5D were screened. Telwe heterozygous T5DL.5VS translocations were obtained but no T5DS.5VL was found. (4)A total of 384 progenies derived from double monosomic 40+7V+7D were screened. Six heterozygous T7DS.7VL translocations and two heterozygous T7DL.7VS translocations were obtained. The former mentioned heterozygous translocations selfing seeds were screened, and till now, all the homologous translocations were obtained.In conclusion, based on the cluster analysis, FISH and GISH, we systemically researched the phylogenetic relationship of genus Dasypyrum species. Dasypyrum genome specific markers were also exploited and these markers developed in the present study were all used to identify the new germplasm which were developed by the current author.