萝卜-芥蓝异源四倍体的合成及GISH分析

Development and GISH Analysis of Amphidiploid Raphanobrassica between Raphanus sativus and Brassica alboglabra

通过萝卜（Raphanus sativus L．，2n=18，RR）与白花芥蓝（Brassica alboglabra Bailey，2n=18，CC）杂交，F1经秋水仙碱加倍合成萝卜-芥蓝异源四倍体（Raphanobrassica，2n=36，RRCC）。经F4-F10代连续育性选择，F10单株种子产量达32．3g，每角粒数达14．9。基因组原位杂交显示F10减数分裂行为类似于二倍体物种，表明该异源四倍体的细胞学行为已经稳定。育性观察表明，可育花粉足够各代生产种子，但低世代杂种出现高频瘪粒种子，胚珠败孕可能是其主要原因。该萝卜-芥蓝异源四倍体可以用作向油菜（B．napus L．，2n=38，AACC）转移萝卜基因的遗传桥梁。

Artificial intergeneric amphidiploids Rapahnobrassica （2n = 36, RRCC） between Raphanus sativus L. （2n = 18, RR）and Brassica oleracea L. （2n = 18, CC） have been studied for more than eighty years as potential new fodder crop or bridge crop, for their disease resistance, cytoplasm male sterility and vegetative vigor. However, their use as one new crop was mainly limited for the low seed productivity. Amphidiploids synthesized from different crossing combinations showed different levels of seed fertility, which could be improved to some extent by repeated selections. To exploit the useful R. sativus genes for Brassica breeding, one new amphidiploid Raphanobrassica was produced by doubling the chromosome number of the F1 hybrids of R. sativus cv. Heqing × B. alboglabra Bailey with colchicine treatment. As previous reports, the developments of siliqua and seeds were very difficult in the early generations of the present amphidiploid （Plate Ⅰ-A, B）, most siliquas stopped growth and became shriveled after about 30 days of fertilization, and no seeds were visible in pods. A small part of short and thick siliquas formed and contained one or several seeds per pod, but most of seeds were small, shriveled and morphologically un-regular and only a minority was of full size. Selections were made from F4 to F10 with special attention for the improvement of seed fertility, and consequently siliqua number per plant and seed maturation level increased generation by generation, on the other hand the frequency of small and shriveled seeds decreased. F10 plants showed good seed fertility （Plate Ⅰ -E, F, G）, for the mean seed yield reached 32.3 g per plant, the number of siliqua per plant was about 700, the number of seed per siliqa was 14.9 （Table 1）, approximately 80% of the seeds were fully developed （Plate Ⅰ -F）. Genomic in situ hybridization （GISH） investigations on the meiosis of F10 pollen mother cells （PMCs） showed that these plants had the expected chromosome complements of the 18 chromosomes from R. sativus and the other 18 ones from B. alboglabra. The normal meiotic pairings and segregations occurred in their PMCs, for 18 bivalents were formed at diakinesis and chromosomes only paired with their homologs （Plate Ⅰ -H, I）, segregations at anaphase Ⅰ were also very regular with 18 chromosomes in each polar and without bridges and fragments （Plate Ⅰ -J, K）. These results indicated that diploid-like meiotic behavior was established in the amphidiploid. Fertility factors analysis showed that available fertile pollen grains in anthers were enough to produce seeds in every generation, and the shriveled seeds observed frequently in the early generations might be attributable to ovule abortion. The application potential of the Raphanobrassica was discussed as a fertile genetic bridge for gene transfer from R. sativus to Brassica crops.