| 453 | 18 | 217 |
| 下载次数 | 被引频次 | 阅读次数 |
以12个野生型龙须菜个体为材料,选择7个SSR位点,分别使用PAGE电泳银染检测和毛细管电泳荧光检测每个位点的多态性信息,并对这两种方法的检测结果进行了比较。PAGE与毛细管电泳分型结果相一致的4个位点包括全部的五碱基和六碱基重复:H88、S96、H97和H98。而分型结果不一致的3个位点则均为三碱基重复:H30、S25和S28。经测序检验,PAGE结果均准确,而毛细管电泳分型结果出现误差。在样本量少,SSR位点的变异小时,使用PAGE检测SSR位点更为准确。
Abstract：Twelve wild individuals of Gracilariopsis lemaneiformis were used to detect polymorphism of 7 SSR loci by polyacrylamide gel electrophoresis(PAGE)and capillary electrophoresis,for the purpose of comparing the detection efficiency of the two methods and exploring an accurate SSR detection system.Genotyping results of capillary electrophoresis in 4 SSR loci was consistent with that of PAGE,including all penta-and hexa-nucleotide repeats:H88,S96,H97 and H98,while all tri-nucleotide repeats:H30,S25 and S28 showed inconsistent results in two methods.At SSR locus H30,amplified bands of 12 individuals in PAGE analysis had no size difference,which indicated that there was no polymorphism.However,the size of target fragments obtained by capillary electrophoresis fluorescent detection system were compared among 12 samples,giving a result that size differences existed at this locus.Through the analysis of fluorescent peak,size difference of 1 base pair could be caused by inaccurate selection of target peak,while size difference of 3 base pairs was equal to one repeat unit of tri-nucleotide repeats,which were identified as size polymorphism.Therefore,2 alleles were detected by capillary electrophoresis at locus H30.At SSR loci S28 and S30,the genotyping results of capillary electrophoresis both showed that there was no size difference existed among 12 sample,however,amplified bands in PAGE gel displayed separation among individuals and were identified as 2 alleles,which was the evidence of polymorphism.PAGE results of the three SSR locus was further verified by gel extraction and sequencing.SSR sequences of 12 samples obtained by sequencing displayed the true polymorphic information,which were fully consistent with PAGE results.The errors existing in capillary electrophoresis detection were mainly caused by peak selection and fragment size reading,especially when repeat unit length of SSR locus was short,size difference of target fragments was small and the number of sample was small.In contrast with capillary electrophoresis,the genotyping result of PAGE was more accurate and credible.Therefore,PAGE detection was suggested to be suitable for identifying polymorphic SSR loci with small sample size and low polymorphism.
[1]Santelices B,Doty M S.A review of Gracilariafarming[J].Aquaculture,1989,78:95-133.
[2]Mchugh D J.Worldwide distribution of commercial resources of seaweeds including Gelidium[J].Hydrobiologia,1991,221(1):19-29.
[3]周峙苗,何清,马晓宇.东海红藻龙须菜的营养成分分析及评价[J].食品科学,2010,31(9):284-287.Zhou S M,He Q,Ma X Y.Evaluation of nutritional components in Gracilaria lemaneiformis[J].Food Science,2010,31(9):284-287.
[4]邓志峰,纪明侯.龙须菜和扁江篱多糖的组成及其抗肿瘤效果[J].海洋与湖沼,1995,26(6):575-581.Deng Z F,Ji M H.Composition of polysaccharides of Gracilaria sjeostedtii Kylin and G.textorii(Sur.)De-Toni(Rhodophyta)and their antitumor effect[J].Oceanologia et Limnologia Sinica,1995,26(6):575-581.
[5]陈美珍,余杰,钟秋玲,等.龙须菜藻胆蛋白免疫功能和抗氧化作用的研究[J].食品科学,2005,26(9):456-459.Chen M Z,Yu J,Zhong Q L,et al.Study on the immunity function and anti-oxidation activity of phycobiliproteins fromGracilaria lemaneiformis[J].Food Science,2005,26(9):456-459.
[6]Fei X.Solving the coastal eutrophication problem by large scale seaweed cultivation[J].Hydrobiologia,2004,512(1):145-151.
[7]Wu R S S.The environmental impact of marine fish culture:Towards a sustainable future[J].Marine Pollution Bulletin,1995,31(31):159-166.
[8]Troell M,Halling C,Nilsson A,et al.Integrated marine cultivation of Gracilaria chilensis(Gracilariales,Rhodophyta)and salmon cages for reduced environmental impact and increased economic output[J].Aquaculture,1997,156(1-2):45-61.
[9]Zhou Y,Yang H,Hu H,et al.Bioremediation potential of the macroalgaGracilaria lemaneiformis(Rhodophyta)integrated into fed fish culture in coastal waters of north China[J].Aquaculture,2006,252:264-276.
[10]Messier W,Li S H,Stewart C B.The birth of microsatellites[J].Nature,1996,381(6582):483.
[11]Schl9tterer C,Tautz D.Slippage synthesis of simple sequence DNA[J].Nucleic Acids Research,1992,20(2):211-215.
[12]Jiao Y,Jia H,Li X,et al.Development of simple sequence repeat(SSR)markers from a genome survey of Chinese bayberry(Myrica rubra)[J].BMC Genomics,2012,13:201.
[13]Cheng X,Xu J,Xia S,et al.Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus[J].Theoretical and Applied Genetics,2009,118(6):1121-1131.
[14]Luo H,M9rchen M,Engel C R,et al.Characterization of microsatellite markers in the red algaGracilaria gracilis[J].Molecular Ecology,1999,8(4):700-702.
[15]Billot C,Rousvoal S,Estoup A,et al.Isolation and characterization of microsatellite markers in the nuclear genome of the brown algaLaminaria digitata(Phaeophyceae)[J].Molecular Ecology,1998,7(12):1778-1780.
[16]Zhou W,Hu Y,Sui Z,et al.Genome Survey Sequencing and Genetic Background Characterization of Gracilariopsis lemaneiformis(Rhodophyta)Based on Next-Generation Sequencing[J].PLoS One,2013,8(7):e69909.
[17]曲鲁江,李显耀,杜志强,等.微卫星PCR产物变性与非变性PAGE-银染检测方法的比较[J].遗传,2004,26(4):522-524.Qu L J,Li X Y,Du Z Q,et al.The comparison of denatured and non-denatured PAGE-silver-staining detection of PCR product of microsatellites[J].Hereditas,2004,26(4):522-524.
[18]陈雅琼,李凤霞,李锡坤,等.烟草SSR荧光标记与毛细管电泳检测技术研究[J].中国烟草科学,2011,32(2):66-70.Chen Y Q,Li F X,Li X K,et al.Fluorescent detection and capillary electrophoresis in tobacco SSR[J].Chinese Tobacco Science,2011,32(2):66-70.
[19]易红梅,王凤格,赵久然,等.玉米品种SSR标记毛细管电泳荧光检测法与变性PAGE银染检测法的比较研究[J].华北农学报,2006,21(5):64-67.Yi H M,Wang F G,Zhao J R,et al.Comparison of two maize SSR detection methods:Capillary electrophoresis with fluorescence detection method and denaturing PAGE silver_staining detection method[J].Acta Agriculturae Boreali-Sinica,2006,21(5):64-67.
[20]郝晨阳,王兰芬,贾继增,等.SSR荧光标记和银染技术的比较分析[J].作物学报,2005,31(2):144-149.Hao C Y,Wang L F,Jia J Z,et al.Comparison of fluorescence and silver-staining detection systems of microsatellite markers[J].Acta Agronomica Sinica,2005,31(2):144-149.
[21]Lehmann T,Hawley W A,Collins F H.An evaluation of evolutionary constraints on microsatellite loci using null alleles[J].Genetics,1996,144(3):1155-1163.
[22]Angers B,Bernatchez L.Complex evolution of a salmonid microsatellite locus and its consequences in inferring allelic divergence from size information[J].Molecular Biology&Evolution,1997,14(3):230-238.
[23]Grimaldi M C,Crouau-Roy B.Microsatellite allelic homoplasy due to variable flanking sequences[J].Journal of Molecular Evolution,1997,44(3):336-340.
基本信息:
DOI：10.16441/j.cnki.hdxb.20170031
中图分类号:S644.6
引用信息:
[1]胡依依,隋正红,彭冲,等.聚丙烯酰胺凝胶与毛细管电泳检测龙须菜SSR位点的比较研究[J],2018,48(03):65-72.DOI:10.16441/j.cnki.hdxb.20170031.
基金信息:
“十二五”农村领域国家科技计划项目(2012AA10A411);; 国家自然科学基金项目(31372529);; 中国博士后科学基因项目(2016M590662)~~