| The genetic basis of Brassica napus L. (2n=38, AACC), one of the most important oilseed crops worldwide, is quite narrow. The species originated in Europe and was introduced into China as an oil crop in the 1950s. In view of its important role in economic manufacture, how to exploit valuable characters like yellow-seed and disease resistant genes from related species into B. napus using modern biotechnology is in face of breeders. Because of better nutrition and processing quality, yellow-seeded trait is becoming an increasingly important objective of rapeseed breeding. None yellow-seed exists in natural B. napus, and even the existed yellow rapeseed is bred via interspecific hybridization of Brassia species, novel yellow-seeds introduced from the related genus has not been reported.Sinapis alba L., a member of the Brassicaceae, possesses desirable agronomic characteristics such as yellow seed color, tolerance to drought stress, reduced pod shattering, resistance to virus diseases, blackleg disease, black spot and beet cyst nematodes. Somatic hybrids of B. napus and S. alba obtained by electrofusion have been described previously. For creation of high yielding rapeseed lines with improved disease resistance and seed quality, these hybrids were subsequently backcrossed with B. napus and self-pollinated to obtain a BC3F1 generation with valuable agronomic characteristics derived from S. alba. This material contains various interesting phenotypes that may be useful for rapeseed breeding.The objective of this study was to carry out character observation and trace the chromosome behaviors through genomic in situ hybridization (GISH). On the other hand, we obtained yellow-seed variants in the BC3F1 progenies, which were characterized by both molecular and cytological analysis. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques were applied to demonstrate the presence of S. alba chromatin and putative S. alba genome introgressions in B. napus and S. alba interspecific progenies. In addition, we used minisatellite core sequence 33.6 as primer also obtained a 300bp band in D244 which was specific to S. alba. FISH using the PCR-derived fragments of DNA hybridized to mitotic metaphase chromosomes of D244, enabled dependable discrimination of S. alba chromatin introgression, showing a significant hybridization signal on one of the 38 chromosomes.According to flavonoid biosynthetic genes of Arabidopsis thaliana, 27 primers were designed for PCR identification of S. alba, B. napus and intergeneric hybrid progenies, of which 16 primers derived from 13 genes amplified polymorphism between both parents, suggesting that significant deviation of flavonoid biosynthetic genes exists between B. napus and S. alba. Further identification of yellow seed progenies with above 16 primers indicated that TT2-2 yielded a S. alba-specific band (1800bp) in progeny lines, whereas amplified bands of progenies by other primers were consistent with'Yangyou 6'. This result further confirmed that part of S. alba genes was transferred to B. napus through introgressions. A gene fragment of 1672bp in length results from the 1800bp band of D244-18, which was amplified by primer TT2-2 was sequenced. BLAST analysis indicated segment similarity of two base region 61-468 and 664-1709 to B. napus BAC clone (LOCUS: AC189336, GI: 199580018) was 97% and 99%, respectively. No matched data in genebank was found resemble to the 195 bases of region 469-663. Another primer Sa1, designed according to the sequencing result, obtained a prospective 1408bp fragment in S. alba and yellow seed lines, which was differ from the 1200bp band of'Yangyou 6', proving S. alba gene exists in yellow seed progenies. In addition, above S. alba specific gene fragment was absent in other three yellow seed varieties produced through interspecific hybridization, illuminating our yellow seed germplasm was different from some of other yellow seed resources tested. |
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