FISH技术在栉孔扇贝和虾夷扇贝杂交子代染色体识别中的运用初探

PRELIMINARY STUDY ON FISH IDENTIFICATION OF CHROMOSOMES IN HYBRIDS FROM CROSS BETWEEN PATINOPECTEN YESSOENSIS AND CHLAMYS FARRERI

核糖体间隔序列(ITS)是扇贝基因组中进化较快的基因片段,研究利用虾夷扇贝(Patinopecten yessoen-sis)与栉孔扇贝(Chlamys farreri)在核糖体间隔区ITS-1基因的序列差异,构建了虾夷扇贝的种特异性荧光原位杂交(Fluorescence In Situ Hybridization,FISH)探针,并成功地将其运用于两种扇贝杂交子代的染色体识别和遗传变异分析中。研究结果表明,在适当的杂交和洗脱条件下,两扇贝ITS-1基因20.3%的差异已足够使虾夷扇贝ITS-1探针产生明显种特异性,探针只在虾夷扇贝分裂相第3对和第5对亚端部染色体或间期细胞上产生稳定的4个杂交信号,但在栉孔扇贝上并不产生明显的杂交信号;采用该探针对两扇贝的正反交子代染色体进行分析,结果表明所有杂交子代染色体分裂相均产生半数的杂交信号,与杂交种含半数的虾夷扇贝亲本染色体相符,杂交信号在杂交种染色体上的数目和位置保持恒定,并未产生明显的变异;该结果证实了两扇贝的杂交为真正的精卵结合意义的杂交,子代对双亲遗传物质的继承仍以单倍染色体组的叠加为主;同时研究也表明ITS-1探针可有效地运用于杂交子代中特定染色体识别和跟踪过程中,对今后扇贝遗传变异系的识别和鉴定及进一步育种具有重要意义。

Intra or inter-specific hybridization may provide important tools for germplasm improvement in shellfish breeding programs.Attractive of this strategy for aquaculture is the rapid generation of novel genetic types compared to conventional breeding.Hybridization between scallop Chlamys farreri and Patinopecten yessoensis were conducted since 2003 in our laboratory either in small scale of experiment or in larger scale of breeding program.In most cases,obvious heterosis was observed in hybrids in both aspect of growth rate and disease resistance,compared to their parents,suggesting a potential resolution to current problems,principally diseases and high mortality during the farming,which has been intimidating the scallop farming industry along the coast of China in recent years.But to date,the genetic constitution and identification of possible variants in the hybrid offspring were still not intensively conducted.Since lots of studies about intra or inter-specific hybridization have disclosed great variation in hybrids,which proved to be of great importance in further genetic breeding.This situation of our program will certainly hamper the full usage of the breeding potentials of this interspecific hybridization.A intensive study on genetic make up of the hybrid and genetic variation possibly existed in the offspring,especially at the chromosome level,is badly needed.In this paper,in order to investigate the chromsome constitution of scallop hybrids,a noval Fluorescence in situ Hybridization（FISH）probe was developed based on the sequence variation between scallop C.farreri and P.yessoensis in internal transcribed spacer regions（ITS） of ribosomal RNA transcript units.We did this because ITS regions are known to evolve faster than the coding region in scallop genome and more likely to produce species-specific signals when used as a FISH probe,thus helpful to indentify specific chromsomes.Genome DNA was extracted from scallop P.yessoensis and was used to amplify the internal transcribed spacer I gene.Probe labeling was performed by using PCR protocol described by Roche company to incorporate biotin-16-dUTP and the amplied gene was sequenced.In situ hybridization protocol was also accordingly optimized.ITS-1 probe was finally hybridized to the interphase neclei and mataphase chromosome spread of both parent scallops and their hybrids.Results showed that under the optimized FISH condition,20.3% sequence variation in internal transcribed spacer region had been enough for constructing a species-specific FISH probe in scallops.When applying this ITS-1 probe to metaphse spread of parent scallops,4 signal loci were detected on both metaphase chromosomes and interpase nuclei of P.yessoensi,which was later verified to locate on short arms of sub-telocentric choromosome 3 and 5,while none was detected on either chromosomes or interpase nuclei of C.farreri.When hybridizing the probe to the metaphase spread of reciprocal hybrids,only 2 signal loci were detected on both metaphase chromosomes and interpase nuclei of each type hybrid,which conformed to the fact that half sets of chromosomes in hybrid had come from P.yessoensis.The number and location of the signals remained stable on each metaphse spread of hybrid,with each signal locating only on short arms of sub-telocentric choromosome 3 or 5 originally from P.yessoensi,and no obvious chromosome variation was detected.These results provided another evidence that the hybridization between the two scallop species might be another case of chromosome level hybridization,commonly found in shellfish hybridization.The combination of different haploid genome from the duplex parental species might construct the nuclei genome in most hybrids.This probe will provide a new way for fast identification of hybrid scallop,and would act as a useful tool in chromosome recognition and chromotin tracing in hybrids,and predict potentially wide application in intensive breeding of scallops in the future.