基因组原位杂交鉴定牛筋草AA基因组在穇子染色体上的分布及探针长度优化方法

Distribution of Eleusine indica AA Genome on E. coracana (Poaceae ) Chromosomes Identified by Genomic in situ Hybridization and Optimization Method of Probe Length

摘要:

采用基因组原位杂交(Genomic in situ hybridization, GISH)方法研究了牛筋草(Eleusine indica)AA基因组在穇子(E. coracana)染色体上的分布,并探讨了AA、BB基因组的同源关系。用超声波破碎法进行预剪切,以缺口平移法标记的牛筋草总DNA为探针,BB基因组的E. floccifolia (Forssk.) Spreng.总DNA为封阻,与AABB基因组穇子的中期染色体进行杂交。结果表明,牛筋草AA基因组分布在穇子的18条染色体上。不加封阻或加过量封阻均不能鉴别AA基因组,说明AA和BB基因组间的分化程度不大,双方共享的重复序列较多。牛筋草与E. floccifolia总DNA分别用超声波破碎2 min和3 min后,可得到峰值为300~750 bp的DNA片段,这说明不同物种的超声波破碎时间需要调整,以获得合适长度的探针。

Abstract:

Genomic in situ hybridization (GISH) was used to detect the AA genome distribution of Eleusine indica (L.) Gaertn. on chromosomes of E. coracana (L.) Gaertn. (AABB), and the homoeologous relationship between AA and BB genomes was studied. The ultrasonic processor pre-sheared total DNA of E. indica further labeled by nick translation mix as probe and the sheared total DNA of E. floccifolia (Forssk.) Spreng. (BB) as blocking DNA, and then they were hybridized to the metaphase chromosomes of E. coracana. The results showed that the AA genome of E. indica was distributed in 18 chromosomes of E. coracana. When not adding or adding excess blocking DNA, the AA genome was not detected on the chromosomes of E. coracana, suggesting that the AA and BB genomes may have not yet been well differentiated from each other, and that E. indica and E. floccifolia may share abundant repeated sequences. The 300-750 bp fragment peaks were presented on the 1% agar gel after shearing total DNA of E. indica and E. floccifolia 2 min and 3 min, respectively. The difference in the time of DNA shearing of the two species suggests that the ultrasonic processor method should be adjusted depending on species in order to obtain DNA probes with appropriate lengths.