The Molecular And Cytogenetic Analysis Of Aegilops Tauschii-Secale Cereale Intergeneric Hybrids And Amphidiploids

Intergeneric hybrids and amphidiloid between Aegilops tauschii (2n=2x=14, DD) as female parent and Secale cereale (2n=2x=14, RR) were produced through embryo culture and colchicine treatment. The apparent differences of the seed set and embryo recovery in the reciprocal cross between Ae. tauschii and S. cereale in were observed. The seed set rate and embryo recovery frequency using rye as the female parent was 8.5% and 3.8%, no hybrid plant was obtained by culturing 3 hybrid embryos 14 days after pollination。While in the reciprocal cross, the seed set and embryo recovery was 70.1% and 48.0%, respectively. Of the 36 hybrid embryos rescued from 16 days after pollination,26 (72.2%)embryos germinated, the survival rate was higher than 36.0%(9/25)of the hybrid embryos rescued from 14 days after pollination.The F1 hybrids were characterized by both cytological and SSR molecular marker analysis, and the results proved that the hybrids contained complete set of choromosomes from both parents. The average chromosome configuration of pollen mother cells (PMCs) of the F1 haploid hybrids at metap.hase I was 10.84Ⅰ+1.57Ⅲ+0.01Ⅲ. By genomic in situ hybridization (GISH), three types of bivalents were observed in the haploid F1 hybrids, including D-D pairing, R-R pairing, and D-R pairing with frequencies of 8.6%, 8.2% and 83.3%, respectively. Two types of trivalents, D-R-D and R-D-R pairing, were also observed and their frequencies were 33.3% and 66.7%. These indicated that some bivalents or trivalents were involved in chromosomes belonging different homoeologous groups. The presence of D-R-D and R-D-R associations in the F1 hybrids (DR) were possibly related to evolutionary translocations of the rye chromosomes. The occurrence of chromosome pairing between different homoeology groups in the Ae. tauschii×S. cereale hybrid may be associated with the existence of both duplicate genes and families of repetitive DNA sequence located in nonhomologous chromosomes.The somaclones from hybrid embryo were produced by the embryo-callus-regenerated plants. The average chromosome configuration of PMC of the some plants from subculture 420 days was 7.59Ⅰ+3.10Ⅱ+0.05Ⅲ+0.01Ⅳ, the frequencies of the chromosomes pairing was much higher than that of the hybrid embryo-rescued F1 plants. No variation in chromosomes number or structures in these regenerated plants was revealed by GISH. The results showed that somaclone variation was possibly a new way of increasing chromosome pairing and genetic transfer beween the related species.The survival rates, doubling rates and seed set of the colchicine-treated hybrid plants at tiller stage before winter were higher than those at return green stage after winter. The best results with 100% survival rate and 100% doubling rate was obtained from the F1 hybrid colchicine-treated 16 hours at 15℃at tiller stage before winter. The fertility and cytology studies of the Ae.tauschii-S. cereale amphidiploids from C1 to C4 generations showed that the percentage of its progenies with chromosome number of 2n=28 increased from 57.1% to 92.5%, the mean numbers of bivilants in the PMCs from11.70 to 12.25, and the mean seeds set from 24.5% to 51.3%, with the maximum seed set of one C4 plant reaching 70.5%. These results indicated that the amphidiploid had a tendence of stability. The amphidiploid was further confirmed by chromosome C-banding and GISH analisis. This amphidploids with good seed set chould be a new tetraploid species with both R and D genome and useful genetic resources for wheat and Triticale improvement as well as evolution study.The AFLP analysies were conducted using two primer comibinations of EcoRⅠ-MseⅠ(E/M) and PstⅠ-MseⅠ(P/M), which primarily amplify repetitiv and low copy sequences, repectively. The results showed that the main type of genetic changes was sequence elimination with about 70.00% and 52.95% of Ae.tauschii parental bands losts, and 96.90% and 81.64% of the rye parental bands losts occurred in the F1 plants for E/M and P/M primers, respectively. The MSAP analyses showed that the cytosine methylation alterations, with its main types of methylation modification were only observed in the F1 hybrids and amphidiploids C1 progenies, but not in amphidiploids C2, C3 and C4 progenies. These results indicated that the increasement of fertility of amphidiploid from C1 to C4 progenies was possibly related to the DNA sequence elimilation of F1 hybrids and amphidiploids.