两种泥鳅不同群体遗传变异的RAPD分析

应用RAPD技术,分析了采自于我国黄河、长江和珠江三大水系中游的大鳞副泥鳅和泥鳅不同群体间的遗传变异。结果表明：种内不同群体间带纹相似度在0．730－0．938之间;遗传距离为0．089－0．245;种间不同群体间的带纹相似度为0．392－0．505,遗传距离为0．620－0．800。两种泥鳞采自武汉的群体,其不同个体间的相似度较之其它群体为低,表明其群体内遗传变异程度较高。     

PCR扩增泥鳅和大鳞副泥鳅SRY盒基因

以特异扩增人ＳＲＹ基因保守区的一对引物,研究了泥鳅和大鳞副泥鳅基因组中ＳＲＹ盒基因的扩增。结果表明,该引物可以在泥鳅中扩增出四条带,其长度分别为２００,５５０、９４０和１０００ｂｐ。在大鳞副泥鳞中扩增出三条带,大小为２００,５５０和９００ｂｐ。经Ｓｏｕｔｈｅｒｎ杂交显示出二者的阳性带为２００和５５０ｂｐ。阳性带在雌雄个体间和两个物种间无差异。     

人工诱导泥鳅与大鳞副泥鳅雄核发育单倍体子代的RAPD分析

用３０个经过筛选的随机引物对３组泥鳅雄核发育单倍体、２组大鳞副尼鳅雄核发育单倍体及其相应亲本进行了ＲＡＰＤ分析。结果表明,雄核发育倍体子代与其父本的ＲＡＰＤ谱带相似率为９７．０％－９７．８％,与母本相似率为３０．３％－５９．５％,子代中的非亲本谱眩为０－０．０２９,极少母本特异谱带。这一结果说明雄性发育鱼类单倍体的遗传信息主要来自父本,且存在着个体差异,雄核发育子代存在ＤＮＡ变异和母本ＤＮＡ非特异带,但并非其必要条件。     

南四湖泥鳅和大鳞副泥鳅随机扩增多态DNA分析初报

采用随机扩增多态性DNA(RAPD)技术对南四湖的泥鳅（Misgurnus anguillicaudatus）和大鳞副泥鳅（Paramisgurnus dabryanus）进行了初步分析。共用10个10bp的OPG组寡核苷酸片段作为引物,筛选出结果好的5个引物,从泥鳅4个体基因组DNA中扩增出142个片段,共有片段12个,特有片段5个;从大鳞副泥鳅3个体基因组DNA中扩增出101个片段,共有片段18个,特有片段5个,在总扩增出的243个片段中,共有片段仅4个。所有扩增片段大小介于0.2-3kb之间。用PoPGen32软件计算出个体间的遗传距离,依此进行UPGMA聚类得到的分子系统树与形态分类学的结果一致,这7个体被聚成2大类,正对应着形态学的2个种：泥鳅和大鳞副泥鳅。     

两种泥鳅不同核质关系下LDH同工酶基因表达的研究

采用聚丙烯酰胺不连续系统凝胶电泳方法分析了泥鳅和大鳞副泥鳅不同杂交组合不同核质关系下胚发育阶段（0－145h）中乳酸脱氢酶（LDH）同工酶的分化表达谱式,ＬＤＨ同工酶的基因表达随细胞质、细胞核不同及胚胎发育各个时期具有不同的个体发育谱式,以泥鳅卵子为细胞质的Ⅰ、Ⅱ两组,杂交组（Ⅱ）ＬＤＨ同工酶基因在受精后３min至原肠中期雄核基因参与表达与调控,部分基因位点比本交组（Ⅰ）启动与表达的时间要早,两组的管家酶主要来自细胞质。以大鳞副泥鳅为细胞质的Ⅲ、Ⅳ两组,各酶均以细胞质调控为主,其管家酶与泥鳅有很大不同。并具体分析和讨论了不同核质关系ＬＤＨ同工酶基因的表达和调控的时空顺序。     

两种泥鳅中Sox基因的检出

性别决定基因SRY都具有一个高度保守的基因序列——HMG-box,编码Sox蛋白,在胚胎发育过程中起重要作用。本文利用两种引物检测了泥鳅和大鳞副泥鳅的Sox基因。第一对引物特异扩增人类SRY基因的保守区。在扩增泥鳅的基因组DNA时可见长度分别为200、550、940和1000bp的四条扩增带。在大鳞副泥鳅中可以扩增出200、550、900bp三条带(Fig.1)。Southem杂交表明200和550bp两条带可以和SRY基因探针杂交(Fig．2)。第二对引物是兼并引物,可以特异扩增Sox基因的HMG-box区域。以基因组DNA为模板可以在大鳞副泥鳅中扩增出220、550、700和1500bp四条带(Fig．3);而在泥鳅中则为220、530、570乖1500bp四条带(Fig.4)。PCR产物的长度差异被认为是由于部分Sox基因的HMG-box区域中存在着内含子。没有发现性别特异扩增带。以上结果说明哺乳动物与性决定有关的组分在脊椎动物中是广泛存在的。但这些组分在鱼类中是否与性决定有关,或仅是哺乳动物性决定因子的进化前体尚不得而知。无论如何广泛深入研究鱼类的Sox基因对于揭示性决定系统和因子的进化方式是十分有意义的。     

两种泥鳅中PdSox8和PdSox9基因的染色体定位

应用染色体原位杂交技术,以地高辛标记的大鳞副泥鳅ＰｄＳｏｘ８和ＰｄＳｏｘ９基因片段为探针,研究了二者在泥鳅和大鳞副泥鳅染色体组中的定位。结果表明,在大鳞副泥鳅中ＰｄＳｏｘ８、ＰｄＳｏｘ９分别于端部着丝粒染色体第４和第２号即１４和１２染色体上,信号位点距着丝粒的相对距离分别为４０．２％和６７．５％,在泥鳅中,则分别位于ｔ９、ｔ６上,信息位点距着丝粒的相对蹁分别为５８．３％和３０．８％。     

鄱阳湖区泥鳅遗传多样性研究

采用同工酶电泳、RAPD引物PCR扩增和线粒体DNA细胞色素b基因全序列3种方法,对鄱阳湖3种斑纹泥鳅的遗传结构进行分析。同工酶电泳显示3种斑纹泥鳅在LDH、MDH和EST这3种同工酶之间存在群体和组织差异;RAPD试验结果显示6条RAPD引物在18尾鄱阳湖泥鳅个体中共扩增出了169条不同分子量的RAPD标记,其中多态性带101条,多态性带的比例为59.56%,平均每个引物产生9.39条带和5.61条多态性带,不同斑纹泥鳅的遗传多样性指数显示3种斑纹泥鳅的遗传水平处于较高水平。以特异性引物进行PCR扩增,获得了1140bp的细胞色素b基因全序列。结果显示,3种不同斑纹泥鳅中检测出了1个共有单倍型和3个特有单倍型。与共有单倍型相比,大花斑泥鳅特有单倍型有91个变异位点,而小花斑泥鳅与无花斑泥鳅特有单倍型的变异位点为20和24个。基于单倍型构建的NJ树和MP树,显示大花斑泥鳅的特有单倍型EU145023处于独立的分支上,这反映出鄱阳湖地区不同斑纹的泥鳅之间在线粒体DNACytb基因中出现了一定距离的分化。       

中国啤酒大麦品种RAPD标记的遗传多样性分析

采用RAPD技术对中国38个啤酒大麦品种的遗传资源进行了聚类分析。结果表明：从筛选出的28个有多态性的随机引物中,共扩增出153条谱带,其中91条谱带具有多态性,占59．4％。每个引物可扩增出1～8条多态性谱带,平均3．3条。聚类分析表明,在遗传距离GD值0．27水平上38个啤酒大麦品种可聚成两大类,下分5个亚类。品种间遗传距离GD变幅为0．00952～0．37846。RAPD标记揭示出这38个啤酒大麦品种遗传变异较小,遗传基础比较狭窄。     

中国泥鳅属和副泥鳅属鱼类的分类整理

根据形态学特征及线粒体Cyt b和COⅠ基因对我国泥鳅属(Misgurnus)和副泥鳅属(Paramisgurnus)鱼类进行了分类整理。结果表明: 泥鳅属和副泥鳅属鱼类在鳞焦大小, 须长及基枕骨末端愈合程度等特征上存在差异。基于联合基因分析, 泥鳅属和副泥鳅属属间遗传距离(15.2%—17.5%)接近于泥鳅属种间遗传距离(10.7%—18.4%); 分子系统树显示我国泥鳅属鱼类未构成单系, 副泥鳅属嵌套分布于泥鳅属中; 北方泥鳅(Misgurnus bipartitus)和泥鳅(Misgurnus anguillicaudatus)亲缘关系近, 其次是大鳞副泥鳅(Paramisgurnus dabryanus), 黑龙江泥鳅(Misgurnus mohoity)位于系统树的基部。结合形态特征与分子数据, 副泥鳅属应作为泥鳅属同属异名, 大鳞副泥鳅应归入泥鳅属; 北方泥鳅曾为黑龙江泥鳅的同物异名, 与黑龙江泥鳅存在明显的形态差异, 且两者亲缘关系较泥鳅远, 北方泥鳅为有效种。     

大鳞副泥鳅、泥鳅和北方泥鳅肉质比较分析

文章采用组织切片、生化组分分析以及实时荧光定量PCR等方法, 研究了大鳞副泥鳅(Paramisgurnus dabryanus)、泥鳅(Misgurnus anguillicaudatus)和北方泥鳅(Misgurnus bipartitus)肉质差异。结果显示: 大鳞副泥鳅、泥鳅和北方泥鳅的肌纤维横截面积分别为 (3589.17±2326.01)、(2809.7±1818.69) 和(2511.93±1949.03) μm2。粗蛋白和粗脂肪含量均是大鳞副泥鳅最高[分别为(17.07±0.31)%和(2.57±0.38)%], 依次为泥鳅[分别为(14.57±0.59)%和(1.37±0.12)%]和北方泥鳅[分别为(12.33±0.15%和0.57±0.06%]。必需氨基酸指数(EAAI)依次为泥鳅(74.38)、大鳞副泥鳅(65.11)和北方泥鳅(60.14); 呈味氨基酸含量依次为泥鳅(32.60±1.64)%、大鳞副泥鳅(27.75±2.13)%和北方泥鳅(24.86±1.00)%; 除亚油酸以外的总多不饱和脂肪酸含量依次为北方泥鳅(24.43±0.26)%、泥鳅(24.18±1.99)%和大鳞副泥鳅(7.86±0.24)%。大鳞副泥鳅的肌肉生长相关基因myod的表达量高, myog和mrf4的表达量低; 泥鳅和北方泥鳅的myog和mrf4的表达量高, myod的表达量低。elovl5等8个脂肪代谢相关基因的表达特征: 大鳞副泥鳅整体表达水平最高, 北方泥鳅次之, 泥鳅最低。结果表明, 大鳞副泥鳅肉质油润, 但是质地相对粗糙; 泥鳅营养价值高且鲜味程度高; 北方泥鳅肉质细嫩, 但是氨基酸营养价值不高, 鲜味程度较差。三种鳅的上述肉质差异可能与肌肉生长和脂肪代谢活动不同有关。     

水稻抗褐飞虱基因SCAR标记的获得

采用282个随机引物对药用野生稻1665和栽培稻桂99远缘杂交的抗褐飞虱近等基因系B3F4分离群体的不抗池DNA和抗池DNA进行了特异性RAPD标记筛选,从中筛选到一个具有明显的特异性扩增带谱的RAPD标记S1159,序列分析表明,S1159序列长度为1408bp,与基因库中已报道的水稻第四号染色体的BAC克隆(编号OSJNBa0070O11)序列(67114-69100)有51.86%的同源性。为了提高所找到的RAPD标记S1159在应用上的稳定性,将RAPD标记转化为SCAR标记检测近等基因系群体,结果表明与RAPD标记结果一致,说明该研究得到的RAPD标记具有较好的稳定性和重复性,为进一步的研究打下了良好的基础。     

利用RAPD标记鉴定甜菜无融合生殖的同一性

目的：对具有无融合生殖特性的带有白花甜菜染色体的栽培甜菜单体附加系M14三代材料进行同一性鉴定。方法：通过RAPD随机扩增技术对M14三代材料的基因组DNA的扩增多态性进行分析。结果表明,M141998年、1999年、2000年三代材料的RAPD位点高度一致,遗传相似度S＝1,遗传距离D＝0,从而表明M14三代个体完全一样。结论：初步确定M14材料无融合生殖基因在其后代中是稳定传递的。     

Identification,cloning and sequence analysis of a dwarf genome-specific RAPD marker in rubber tree [<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> (Muell.) Arg.]

High-yielding dwarf clones of Hevea brasiliensis are tolerant to wind damage and therefore useful for high-density planting. The identification of molecular markers for the dwarf character is very important for isolating true-to-type high-yielding dwarf hybrid lines in the early stage of plant breeding programs. We have identified a dwarf genome-specific random amplified polymorphic DNA (RAPD) marker in rubber tree. A total of 115 random oligonucleotide 10-mer primers were used to amplify genomic DNA by PCR, of which 19 primers produced clear and detectable bands. The primer OPB-12 generated a 1.4-kb DNA marker from both natural and controlled F₁ hybrid progenies (dwarf stature) derived from a cross between a dwarf parent and a normal cultivated clone as well as from the dwarf parent; it was absent in other parent (RRII 118). To validate this DNA marker, we analyzed 22 F₁ hybrids (13 with a dwarf stature and nine with a normal stature); the dwarf genome-specific 1.4-kb RAPD marker was present in all dwarf-stature hybrids and absent in all normal-stature hybrids. This DNA marker was cloned and characterized. DNA marker locus specificity was further confirmed by Southern blot hybridization. Our results indicate that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern based on the presence/absence of specific DNA markers than dye-stained gels or Southern blot analysis of RAPD blots using probes made from purified PCR products. Detection of RAPD markers in the hybrid progenies indicates that RAPD is a powerful tool for identifying inherited genome segments following different hybridization methods in perennial tree crops.     

Genetic variation and relationship of alfalfa populations and their progenies based on RAPD markers

The aim of investigation was to evaluate genetic variation and relationship among alfalfa populations and their offspring, with minimal cost, by using DNA marker analysis. RAPD analysis was performed on bulked DNA samples of five alfalfa parental populations and their progenies: 20 F1 populations from reciprocal diallel crosses and five S1 populations from self-pollination. Twenty primers generated 217 bands, ranging in size from 300 to 6000 bp, with the average number of bands per primer of 10.85 and polymorphism information content of 0.246. Percentage of polymorphic loci, effective number of alleles, expected heterozygosity and Shannon’s information index were used to estimate genetic variation. Higher diversity was observed in F1 progeny populations, while genetic variation in parental populations and S1 progenies remained similar. The genetic relatedness of alfalfa populations was analysed by UPGMA and Bayesian model-based clustering approach. In both types of analysis selfpollinated progenies were grouped. Furthermore, the hybrid offspring where Zuzana, and RSI 20 were maternal parents were placed in separate groups. The results indicate that use of RAPD markers on bulked DNA samples can be fast and cost-effective way for differentiation of alfalfa parental populations and their offspring, as well as for evaluation of their genetic relationships.     

BALB/c小鼠遗传质量检测的新方法

目的比较随即扩增多态性方法（RAPD）、微卫星方法（STR）与生化标记方法对近交系小鼠遗传质量检测的差异,为近交系动物遗传质量控制提供一种分子生物学方法。方法提取近交系小鼠BALB/c基因组DNA,用6条RAPD引物和20对STR引物对其进行PCR扩增,用生化标记法检测13个位点。结果在6条RAPD引物中,引物2（p2）、引物3（p3）、引物5（p5）和引物6（p6）这四条引物扩增的条带出现差异,表现为不同的RAPD图谱;在20对STR引物中,引物2、4、10和11,这四对引物扩增的条带出现差异,表现为不同的STR图谱;13个生化标记位点中,过氧化氢酶-2（Ce-2）等6个生化位点发现杂合基因。结论RAPD和STR可用于验证生化标记方法的实验结果,并用于保证近交系动物的遗传质量。     

Genetic fingerprinting for determining the mode of reproduction in Paspalum notatum, a subtropical apomictic forage grass

 Paspalum is an important genus of the family Gramineae that includes several valuable forage grasses. Many of the species are polyploid and either obligate or facultative apomicts. Cyto-embryological observations of several tetraploid genotypes of P. notatum were performed to determine their mode of reproduction. Afterwards, selfed progenies of the genotypes F131, Q3664 and Q4117 were analysed using RFLP and RAPD genetic fingerprints to identify maternal and non-maternal (aberrant) plants, and to establish the degree of apomictic reproduction. Five maize clones and six primers were used for detecting genetic deviations from the maternal profile. Maize clones umc379, umc384 and umc318 and primers OPG10 and OPI4 were the most informative for discriminating between maternal and aberrant individuals within the progenies of F131 and Q3664. The combined results of three RFLP clones or 4–6 RAPD primers were necessary to ascertain the mode of reproduction in plants F131 and Q3664. The results obtained with the RFLP and RAPD markers were in agreement with the cyto-embryological studies in ascertaining the mode and degree of apomictic reproduction. Plant F131 showed a completely sexual reproductive behaviour, Q3664 an elevated expression of sexuality, while Q4117 was highly apomictic. A fingerprint analysis of an outcrossing population, aimed at the identification of hybrid plants, was also performed. Maize clones um318 and umc379 and primers OPC2 and OPC9 were used. The presence of specific bands belonging to the male parent permitted a rapid and easy detection of hybrids. The methodology described here can be applied both for the characterisation of P. notatum populations and to identify hybrid progenies in Paspalum breeding programs. Received: 5 March 1997 / Accepted: 13 May 1997     

Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice

The random amplification of polymorphic DNA (RAPD) using primers of arbitrary nucleotide sequence has been extremely valuable in identifying heritable markers in a variety of systems. The present studies examined whether the RAPD technique can identify large numbers of polymorphisms that can be used to construct genetic maps in inbred strains of mice. By screening the inbred mouse strains C57BL/6J and DBA/2J with 481 random 10-mer oligonucleotide primers, we identified 95 polymorphisms and mapped 76 of these by use of the BXD series of recombinant inbred (RI) strains. The results clearly demonstrate that the RAPD technique allows for the identification of large numbers of DNA-based polymorphisms that distinguish these two inbred strains of mice,and that such markers can readily be used to construct molecular genetic linkage maps.     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质