Screening Of Linkage Group -Specific BAC Clones In Chinese Cabbage And FISH Analysis In Cabbage-Chinese Cabbage Monosomic Alien Addition Lines 1# And 4#

Chinese cabbage (Brassica campestris L. ssp. pekinensis syn. rapa) and cabbage (Brassica oleracea var. capitata L.), belonging to the same genus Brassica, are both vegetable crops grown extensively in the world and important materials for studying the origination, evolution and relationship between A and C genomes in Brassica. Because both are small chromosomes species and their adjacent chromosomes are very similar, it is difficult to identify them. The building of bacterial artificial chromosome library (BAC) provides a good "platform" to identify the Chinese cabbage chromosomes upon cabbage background with BAC-FISH technology. In this study, several linkage group–specific SSR makers used to screen BAC clones, some of which were then used as probes to perform the FISH system. At the same time, the hybridization signals distribution of 25SrDNA probe and its behaviors in meiosis were analyzed in Cabbage-Chinese Cabbage Monosomic Alien Addition Lines 1# and 4# by 25SrDNA FISH technology. The study results are as follows:1. Eighteen positive single clones corresponding to linkage group A05, A06, A08, A10 were obtained through PCR screening from 50 primary mixed pools with linkage group-specific SSR makers in Chinese cabbage. . One single clone was identified by one marker ENA17 located on A05, one single clone was identified by one maker ENA10, three single clones were identified by markers ENA19 located on A06, six single clones were identified by BRMS-033 located on a08, one single clone was identified by one marker ENA18 located on A10, six single clones were identified by one maker B036L21-2.2. In this study, the effect of Formamide deionized concentration, Salmon sperm DNA concentration on the BAC-FISH hybridization were discussed.The primary results indicated that the appropriate hybridization solution with less unspecific singles is 30%FAD, 10%DS, 2×SSC, probe 1μg, 1μg/μL ssDNA. Hybridization signals were detected by single clone probes 91M11, and signals were detected by mixed probes 97D24 and 91M11, and signals were detected by mixed probes 91 pool M row.3. Diploid cabbage served as the control, reproductive traits of Cabbage-Chinese cabbage monosomic alien addition lines 1# and 4# were surveyed. The results showed that the growth vigor and branching capabilities of CA1 and CA4 were very strong, with ovalleaves and fertility, as with the diploid parents. However, there were large differences in plant height, branching angle, size and shape of bud and petal. CA4 plant was significantly lower than that of CA1 and parents, with blunt rounded flower buds, pale yellow flowers and flat petals, and big lateral branch angle. CA1 had slender buds, yellow flowers, slightly wrinkled petals similar to parent, but the lateral branch angle was small and plant was compact.4. Five 25S rDNA hybridization signals were detected at mitotic metaphase in cabbage-Chinese cabbage Monosomic Alien Addition Lines CA1, four of which were detected on chromosome 5 and 9 in cabbage, one of which was detected on the introduced chromosome 1 of Chinese cabbage. The chromosome configuration forms at diakinesis were mainly 9Ⅱ+Ⅰ, chromosomes were distributed on equatorial plate at metaphaseⅠ, the separation form of chromosomes was mainly 10-9 at anaphaseⅠ, the form 10-9-10-9 was mainly observed at anaphaseⅡ. Furthermore chromatid dissociation in advance at anaphaseⅠand chromosome segregation imbalance were observed in some cells.5. Five 25S rDNA hybridization signals were detected at mitotic metaphase incabbage-Chinese cabbage Monosomic Alien Addition Lines CA4, four of which were detected on chromosome 5 and 9 in cabbage, one of which was detected on the introduced chromosome 4 of Chinese cabbage. The chromosome configurations at diakinesis were diversity, with bivalents, trivalents, univalents, and multivalents. And single univalent and bivalents was observed on equatorial plate at metaphaseⅠ, the separation form of chromosomes was 10-9 at anaphaseⅠ, the form 10-9-10-9 was mainly observed at anaphaseⅡwith a few exceptions of 9-10-8-11, the hybridization signals were separated by 3-2-3-2 at anaphaseⅡ.Chromosome segregations were not simultaneous or were imbalance in some cells.