【作者】 李锁平；

【导师】 刘大钧；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 利用幼胚培养和秋水仙素处理产生了节节麦(Aegilops tauschii,2n=2x=14, DD)和黑麦(Secale cereale,2n=2x=14, RR)间的杂种和双二倍体。节节麦和黑麦正反交结实率和种子发育程度有较大差异,以节节麦作父本,杂交结实率为8.5%,成胚率为3.8%,培养授粉14天后的3个幼胚不能萌发,未能产生杂种植株；以节节麦作母本,杂交结实率为70.1%,成胚率为48.0%,选择授粉后14天、16天的幼胚进行胚培养,成苗率分别为36.0%(9/25)和72.2%(26/36)。利用细胞学和SSR分子标记对杂种植株进行鉴定,表明节节麦-黑麦杂种含有双亲的全套染色体。节节麦-黑麦杂种F1的花粉母细胞(PMCs)在减数分裂MI染色体平均构型为10.84Ⅰ+1.57Ⅱ+0.01Ⅲ。利用原位杂交技术,对杂种花粉母细胞中期Ⅰ染色体进行分析,三价体中R-D-R和D-R-D联会分别占66.7%、33.3%。二价体中D-D、D-R和R-R类型联会的频率分别是8.6%,83.3%和8.2%。杂种中出现D-D、R-R、D-R-D和R-D-R联会方式表明存在染色体组内非同源染色体之间的配对或不同染色体组的非同源染色体间的配对,这可能与节节麦、黑麦基因组内或基因组间非同源染色体存在重复基因和重复序列有关或进化易位有关。利用杂种幼胚愈伤组织诱导和再生技术产生了节节麦-黑麦杂种幼胚无性系,继代培养420天的再生杂种植株表现形态学变异和PMCs减数分裂MI染色体配对频率的提高,其PMCs减数分裂MI染色体构型为7.59Ⅰ+3.10Ⅱ+0.05Ⅲ+0.01Ⅳ。利用原位杂交技术分析表明其染色体结构和数量没有改变。提示体细胞无性系变异可能是增加部分同源染色体配对,增加属间物种间基因交流的一个新途径。节节麦-黑麦杂种秋水仙素加倍的研究结果表明越冬前分孽期进行处理比越冬后的返清期处理有较好的成活率、加倍率和植株结实率。越冬前分孽期对杂种幼苗利用秋水仙素处理16小时(15℃)效果最好。对节节麦-黑麦双二倍体C1到C4代的细胞学和育性研究表明,其体细胞染色体数2n＝28的植株的比例从C1的57.1%提高到C4的92.5%,PMC减数分裂MI二价体数目从11.70提高到12.25,平均结实率从24.5%提高到51.3%,表明该双二倍体逐代趋向稳定。来自不同结实率的C0双二倍体各代的结实率有一定的差异。不同结实率的C0单株,其后代的结实率和最初C0的结实率有关,C0的结实率越高,后代的平均结实率和单株最高结实率也高。利用染色体C-带和基因组原位杂交鉴定了节节麦-黑麦双二倍体的真实性。节节麦-黑麦双二倍体有较好的育性,是结合D和R基因组一体的小麦族中的新物种,开展小麦遗传进化研究和育种利用的四倍体新种质。利用两套分别扩增重复序列和单拷贝序列的酶切组合EcoRⅠ-MseⅠ、PstⅠ-MseⅠ引物对杂种F1和双二倍体的扩增结果发现,基因组的序列变异主要发生于杂种F1,序列消除是主要的。上述两套酶切组合引物扩增带中,节节麦基因组在杂种F1消失带数占其总消失带数的比例分别为70.00%和52.95%;该比例在黑麦中分别为96.90%和81.64%。染色体加倍后,亲本序列变异以很少的频率发生在双二倍体各世代中。MSAP分析表明节节麦和黑麦杂交和加倍能够导致节节麦和黑麦基因组序列甲基化状态改变,主要发生在杂种F1,以甲基化为主；加倍后主要发生在双二倍体的早期世代,在C2代以后,没有检测到甲基化的改变。本研究结果推测节节麦-黑麦双二倍体在细胞学稳定过程中序列遗传改变比表观遗传改变有更密切的相关性。更多还原

【Abstract】 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.更多还原