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对小白菜品种进行游离小孢子培养,得到9株再生植株,其中2株为单倍体,7株为二倍体,二倍体自然加倍率为77.7%。为避免材料间差异,取单倍体和二倍体再生植株各2株,提取单株DNA,分别构建单倍体和二倍体DNA混合池。利用甲基化敏感性限制性内切酶-PCR法,结合SRAP分子标记技术,对2个DNA混合池进行筛选,共筛选768对引物,只有在未酶切的单倍体和二倍体DNA池中SRAP引物扩增无差异,而酶切后有差异的条带,才认为是单倍体和二倍体甲基化差异。试验共获得8对含有多态性的引物,试验结果证明小白菜小孢子培养获得的再生植株的倍性与基因组DNA甲基化有关。
Nine Chinese cabbage cultivars were isolated from microspore culture. Two regenerated plants were obtained, of which two were haploid, seven were diploid and the diploid natural doubling rate was 77.7%. In order to avoid the difference between the materials, taking 2 plants of haploid and diploid regenerated plants, single plant DNA was extracted to construct haploid and diploid DNA hybrid pools respectively. Two DNA hybrid pools were screened by methylation-sensitive restriction endonuclease-PCR method and SRAP molecular marker technique. A total of 768 pairs of primers were screened, and only the un-digested haploid and diploid DNA There was no difference in the amplification of SRAP primers in the pool, but there were different bands after digestion, which were considered to be haploid and diploid methylation differences. A total of eight pairs of primers were obtained. The results showed that the ploidy of regenerated plants obtained from microspore culture was related to genomic DNA methylation.