Construction Of Molecular Karyotype And Strcutural Aberrance Library Of Haynaldia Villosa Chromosomes

Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum (L.) Candargy) is a wild relative of common wheat(Triticum aestivum L.). It possesses many important agronomic traits, such as resistance to powdery mildew, leaf and stem rusts, take-all, eyespot, and wheat streak mosaic virus, as well as vigorous tillering ability, more spikelets, high grain protein content, and drought and frost tolerance. Therefore, it is a potential gene resource for wheat genetic improvement. To localize, transfer and utilize interested genes of H. villosa, Firstly, we screened common wheat lines carrying structurally changed chromosomes containing single segments of H. villosa by genomic in situ hybridization. Secondly, the specific molecular markers of individual chromosomes of H. villosa were used to identify these structurally changed chromosomes and construct "Triticum aestivum-Haynaldia villosa structural aberrance library"Section I Development and identification of T. aestivum-H. villosa disomic addition linesNow two sets of T. aestivum-H. villosa disomic addition lines were developed in the world, where from is different with H. villosa of our institute, and difference in agronomic or unique useful traits. Genomic in situ hybridization combined with molecular marker analysis were applied to detect chromosome of H. villosa in backcrossed and selfcrossed generations derived from (Chinese Spring/T. durum-H. villosa amphiploid (irradiated by60Co-γ rays)//Chinese Spring) in this study. A set of disomic addition lines involved in seven H. villosa chromosomes have been identified in BC3F2and BC4F2. The chromosome pairing behavior of H. villosa chromosomes at metaphase I (MI) of pollen mother cells (PMCs) were analyzed through using genomic in situ hybridization, one pair of H. villosa chromosomes paired ring or rod bivalent. Development and identification a set of T. aestivum-H. villosa disomic addition lines were used to localized useful genes of H, villosa.Section II Construction of molecular karyotyping of H, villosa chromosomesIdentification the chromosomes or chromosome segments of H. villosa in common wheat background is first step to use the interested genes. The aim of the present work was to use two repetitive DNA sequences pSc119.2and pAs1, and two multigene families45S rDNA and5S rDNA as probes to construct molecular karyotyping of diploid H. villosa chromosomes (VV,2n=14), and identified the chromosomes or chromosome segments of H. villosa in wheat background. The results of fluorescence in situ hybridization showed the hybridization sites of the45S rDNA and5S rDNA were localized on1VS and5VS, respectively, and were cytogenetic marker for1VS and5VS. The pSc119.2as probe had a simpler distribution, whose signals were hybridized mainly on terminal or subterminal positions of both arms with the exception of the hybridization point on the short arm of two pair chromosomes. The signals generated by the pScl19.2probe were different on position and signals intensity. Meanwhile, they were uniquely identified. Furthermore, we used the pScl19.2in combination with pAsl as probes labeled with different fluorochromes to hybridize for H. villosa chromosomes. As a result, the pAsl as probe was more widely dispersed than pSc119.2over the H. villosa genome. We found pAs1hybridization sites on all of the chromosomes of H. villosa and practically on all of the chromosome arms. These hybridization points appear in terminal, subterminal, interstitial, and occasionally centromeric positions. The individual chromosomes which exhibited the fluorescence in situ hybridization signals were identified when using the pSc119.2and45S rDNA as probes to a set of T. aestivum-H. villosa disomic addition lines. Combining the genomic in situ hybridization and fluorescence in situ hybridization techniques allowed for all seven chromosomes of H. villosa for identification and constructed molecular karyotyping of H. villosa chromosomes.Section Ⅲ Construction of aberrance library of H. villosa chromosomesTo induce as many as T. aestivum-H. villosa intergeneric translocation chromosomes involved in different chromosomes and chromosome segments of H. villosa, T. durum-H. villosa amphiploid was irradiated with60Co-γ rays, and pollens collected from the spikes after irradiation was pollinated to emasculated common wheat cv. Chinese Spring by Bie Tongde and Cao Yaping. Mass of H. villosa chromosomal aberrances were detected using genomic in situ hybridization technique in the M1and many of them could transmit to offspring. Firstly, we screened common wheat lines carrying structurally changed chromosomes containing single segment of H. villosa by genomic in situ hybridization. Up to date,140chromosomal structural changes were screened,41of small alien segment translocation,32of large alien segment translocation,44of whole arm translocation,18of chromosomal deletion and5of intercalary translocation of H. villosa, respectively.30homozygous plants were screened in these chromosomal structural changes,7of small alien segment translocation,9of large alien segment translocation,10of whole arm translocation,2of chromosomal deletion and2of intercalary of H, villosa, respectively.In order to better identify these structural chromosomal changes, we screened1576primer pairs and89of them could be used for tracing individual chromosomes of H. villosa,2for1V,14for2V,8for3V,8for4V,41for5V,2for6V and14for7V, respectively. These90specific molecular markers add to the existing markers earlier screened and developed in H. villosa. The telosomic chromosome or whole arm translocation of chromosome3V and7V were absent in our laboratory. Two telosomic chromosomes and two whole arm translocations were hybridized with pSc119.2and pAsl as probes. The results showed that the plant of TV60-1-8-27and TV60-1-8-18was telosomic chromosome of3VS and3VL, respectively. Meanwhile, the plant of TV54-5-5-22and TV54-5-5-21was7VS·W and W-7VL, respectively. Furthermore, the specific molecular markers of chromosome3V and7V were localized on corresponding chromosomal arm by using these aneuploid,8on3VS,1on3VL,11on7VS and7on7VL, respectively.Finally, these structural changes involving different chromosome and region of H. villosa were identified with124specific markers to individual chromosome. Now,67chromosomal structural changes were identified,8involving1V,11involving2V,5involving3V,10involving4V,9involving5V,13involving6V and11involving7V, respectively, and these aberrances involved different chromosomal region of H, villosa. We constructed "T. aestivum-H. villosa chromosomal aberrance library" based on above studies and the specific markers of H. villosa were localized on different chromosomal regions. The gene controlling bristles on the glume ridges was further located on2VS (FL:0.00-0.33) through observed the phenotype of involving chromosomal structural changes of2VS.