杂交三倍体泥鳅GISH技术反应体系的建立及应用

Establishment and application of GISH reaction system in triploid loach hybrid

为研究杂交三倍体泥鳅Misgurnus anguillicaudatus的形成机制,探寻其产业化生产的有效途径,通过对GISH的多项试验参数进行优化,建立杂交三倍体泥鳅GISH技术反应体系,并对天然四倍体泥鳅(4n=100)与大鳞副泥鳅Paramisgurnus dabryanus(2n=48)正、反杂交后代进行GISH分析。结果表明:杂交三倍体泥鳅GISH技术反应体系中,探针DNA在温度为105℃、持续时间为2 min时,片段化后探针DNA片段长度为500~1000 bp;封阻DNA在温度为110℃、持续时间为10 min时,片段长度为100~500bp;标记后的探针DNA与片段化后的封阻DNA混合比(P/B)在1∶25和1∶50时均可以得到较好的杂交信号;以大鳞副泥鳅全基因组DNA为探针DNA、天然四倍体泥鳅全基因组DNA为封阻DNA,与二者正、反交后代进行基因组原位杂交试验,结果显示,来源于亲本大鳞副泥鳅24条染色体在着丝点处均有杂交信号,无杂交信号染色体50条,来源于亲本泥鳅。该研究结果不仅为杂交三倍体泥鳅后代的鉴定提供了最直观的证据,也为中国天然四倍体泥鳅是含有四套染色体组的遗传四倍体并能产生2n的配子提供了分子细胞遗传学基础数据,对泥鳅三倍体育种研究具有一定的指导意义。

In this study,reaction system of genomic in situ hybridization（ GISH） as a powerful means to identify hybrids was optimized under several experimental parameters and was used to evaluate hybridization between Paramisgurnus dabryanus（ 2 n = 48） and natural tetraploid loach Misgurnus anguilicaudatus（ 4 n = 100） to study mechanism of natural tetraploid loach and to realize industrial production of tetraploid loach. The results showed that the probe DNA had 500 bp to 1000 bp in length at 105 ℃ for 2 min; the blocking DNA had 100 bp to 500 bp in length at 110 ℃ for 10 min in the GISH reaction system of triploid hybrid loach.The screening of probe/blocking DNA ratio showed better hybridization signal at both 1 ∶ 25 and 1 ∶ 50. GISH analysis of reciprocal cross offspring by whole genome DNA of loach as probe DNA and by the whole genome DNA of natural tetraploid loach as blocking DNA revealed that twenty-four chromosomes from parental loach showed hybridization signal at the centromeres. No hybridization signal points was observed in fifty chromosomes from parental tetraploid loach. The findings offered most intuitive evidence for identification of triploid hybrid loach offsprings and provided molecular genetics basis for further provement of the tetraploid loach in China as the heritable tetraploid with four genomes,which could produce gamete of 2 n,guiding breeding of triploid loach.