RAPD法对罗曼蛋鸡基因组DNA多态性分析初报

利用RAPD（随机扩增多态DNA）方法,选用37种10bp的随机引物,对罗曼蛋鸡基因组DNA进行多态性分析,发现其中两种引物（OPS－08,OPY－06）在三代7个品系中都能扩增出多条带并且反映其基因组DNA的多态性,可用于检测出不同品系间的差异,并且这两种引物的碱基序列有80％是对应互补的。     

用RAPD标记检测六种蛇基因组DNA多态性

用 ＲＡＰＤ技术检测了乌梢蛇 Ｚａｏｃｙｓ ｄｈｕｍｎａｄｅｓ、王锦蛇 Ｅｌａｐｈｅ　ｃａｒｉｎａｔａ、黑眉锦蛇 Ｅ．ｔａｅｎｉｕｒａ、双斑锦蛇 Ｅ． ｂｉｍａｃｕｌａｔａ、赤链蛇 Ｄｉｎｏｄｏｎ ｒｕｆｏｚｏｎａｔｕｍ和日本蝮短尾亚种 Ａｇｋｉｓｔｒｏｄｏｎ ｂｌｏｍｈｏｆｆｉｉｂｒｅｖｉｃａｕｄｕｓ等６种蛇的基因组ＤＮＡ的多态性。经２０个随机引物扩增得到２５３个多态片段，其中锦蛇属３个种共有片段８条，游蛇科５个种共有片段５条。在这些共享片段中未检测出种内多态现象。片段分布和片段共享度聚类分析所得的游蛇科５种蛇之间的亲缘关系与染色体、颅骨和半阴茎研究的结果基本一致。     

条纹斑竹鲨基因组的RAPD分析初报

采用11种随机引物对4 条条纹斑竹鲨基因组进行了RAPD检测.结果表明,11种引物在每条个体上扩增的 DNA片段总数在77～84之间,单个随机引物扩增的DNA片段数目由1至11条不等,平均为7.5条 DNA 片段,片段的大小在 300～2 800bp之间.个体之间的相似率在90%以上.     

杜鹃属基因组DNA提取及RAPD的检定

以杜鹃花属常绿杜鹃亚属井冈山杜鹃树叶为材料,分别采用修改的CTAB法、碱裂解法、SDS法提取基因组DNA。实验表明,三种方法所提的DNA纯度及产率有差别,从综合结果看,以CTAB法最好,其所提到的DNA大小与λDNA（21kb）相似,经检验该法适合杜鹃花属植物总基因组DNA的提取,所得DNA不需纯化就可直接用于RAPD分析。     

用RAPD技术检测不同生态类型蜘蛛代表品种的基因组DNA多态性

蜘蛛有3种生态类型,即洞穴型、结网型和游猎型。利用RAPD技术检测3种生态类型蜘蛛的代表品种,即白额巨蟹蛛(游猎型)、漏斗蛛(结网型)、虎纹捕鸟蛛和七纺器蛛(洞穴型)的基因组DNA的多态性。用11个随机引物对3种生态类型的代表蜘蛛的基因组DNA进行扩增,平均每个品种观察到约22.5个标记,单个引物获得的标记在4～13个之间。实验结果经统计学分析表明,L纺器蛛和虎纹捕鸟蛛的随机扩增多态DNA共享度(F)高,在分子水平上进一步证实了同生态类型蜘蛛的亲缘天系最近,聚类结果表明,3种类型蜘蛛的总体演化趋势为:洞穴型 结网型—游猎型,与以往的形态学研究相符。     

RAPD条件优化及天麻基因组DNA多态性分析

建立了RAPD扩增条件快速优化程序与方法．并应用于天麻基因组DNA扩增条件的优化及多态性的测定：获得了天麻基因组DNA的RAPD扩增优化条件和DNA指纹图谱;分析了模板DNA、引物、dNTP、Taq DNA聚合酶等的浓度和退火温度对RAPD扩增的影响．结果表明：天麻基因组DNA用引物S1扩增的片段具有更明显的多态性,这种指纹图谱更适合于天麻遗传分化研究;而用引物S12扩增的DNA指纹图谱具有更大的相似性,这种指纹图谱更适合于天麻真伪鉴别．该方法使RAPD扩增条件优化过程实现了程序化和数量化,是获得RAPD优化条件的简便快速、经济实用方法．应用该方法进行RAPD扩增,可获得图谱清晰、稳定可靠的实验结果．     

七种蟋蟀基因组DNA的RAPD多态性研究（直翅目：蟋蟀总科）

应用10种随机引物,对西北地区常见的3属7种蟋蟀进行RAPD多态性检测,共筛选出2种引物S142,S8可以对7个种扩增出清晰稳定的多态性片段,多态性片段共计58条,相对分子质量在320bp-2400bp之间。应用UPGMA（非加权配对算术平均法）对多态性片段进行聚类分析,构建树状图,推测系统发生关系。每一种蟋蟀均先各自聚为一类,棺头蟋属与油葫芦属间的遗传距离最小,亲缘关系最近,斗蟋属4个种间的亲缘关系较为复杂,明显分为2个支系,与传统分类并不一致。     

真核生物基因组DNA多态性

对真核生物基因组中小卫星ＤＮＡ多态性,微卫星ＤＮＡ多态性,限制性酶切片段长度多态性,随机扩增多态ＤＮＡ以及单链构象多态性的形式机理和表现形式进行了概述。     

野生稻基因组随机扩增多态性DNA(RAPD)分析

用18个随机引物对2份栽培稻、12份包含有六个基因组型的野生稻DNA进行了扩增,共获得147个多态性DNA片断,把这些多态性DNA片断作为遗传位点用UPGMA法计算出各材料间的遗传相似性系数,并作了聚类分析．主要结果如下：1普通野生稻同栽培稻的亲缘关系很近,其中江永普通野生稻更接近于粳稻．2．CCDD组的Oryzalatifolia和EE组的O．australiensis遗传多态性相似。3．B、C、D、E组的遗传多态性相似,组成一个复合体,此复合体与A组的遗传多态性也相似,而F组则相距较远．4．O．mcyeriana和Rhynchofyzasabulata尚未确定组型,RAPD测定结果表明,前者与其它组型的种亲缘关系较远,后者则与AC复合体的种较近．     

洋葱细胞质雄性不育系70及其保持系71基因组DNA的RAPD分析

采用RAPD技术,对洋葱细胞质雄性不育系70及其保持系71的基因组DNA的多态性进行分析。结果表明,在被检测的100条随机引物中,46条引物具有扩增产物,5条引物扩增出多态性条带。对这5条引物进行重复筛选和单株验证,只有G02扩增出的多态性片段,且表现稳定。推测该标记与育性有关。     

A non‐invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout

A procedure previously used for sex steroids was adapted to extract free cortisol and cortisone from water samples taken from rainbow trout Oncorhynchus mykiss tanks. Both corticosteroids could be readily detected by radioimmunoassay (RIA), with cortisol being predominant. All stages of the sampling, extraction and RIA procedure were validated for cortisol. An intermittent problem with poor replication was traced to the use of diethyl ether during the extraction procedure, and was overcome by the use of ethyl acetate. Other modifications were also introduced to speed up the procedure. The concentration and time course of release of both corticosteroids were shown to be related to the degree of stress that the fish had been subjected to. It was confirmed that cortisol concentrations in water and estimated cortisol release rates increased in response to handling stress, and that both were correlated with plasma cortisol concentrations. The potential for using water cortisol concentration and release rates to assess the primary stress response of fishes as a non‐invasive alternative to blood sampling is discussed.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

Identification of sex chromosome molecular markers using RAPDs and fluorescent in situ hybridization in rainbow trout

The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphic bands amplifying preferentially in males of the Mount Lassen and Scottish strains of rainbow trout. Chromosomal localization using fluorescent in situ hybridization of a 900 bp probe developed from one of these markers revealed a brightly defined signal on a chromosome that could morphologically be classified as the Y chromosome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of growth rate on white muscle dynamics in rainbow trout and brook trout

In trout, fast growth stimulated white muscle fibre hypertrophy ( P 0·001) and hyperplasia ( P <0·01) in outer fibres but not in deep fibres. Glycogen was most prevalent in outer fibres ( P <0·01) and in brook trout ( P <0·01) that on average had three to four times larger fibres than rainbow trout.     

Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

The effect of acidification on oogenesis of rainbow trout during sex differentiation

Rainbow trout larvae were kept in acid water from 16 to 55 days after hatching. Initially, the acid environment stimulated a significant increase of gametes in the gonads of females at all developmental stages, but after longer exposure the stimulation was replaced by inhibition of gonadal development in the treated fish. In some fish, gamete reserves increased, while in others there was increased growth and maturation of late-generation oocytes.     

Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

Branchial release of free cortisol and melatonin by rainbow trout

Individual rainbow trout Oncorhynchus mykiss were held in a specially constructed tank that enabled water to be collected separately from the anterior and posterior ends of the fish. Measurement by radioimmunoassay showed that >95% of the cortisol and melatonin released into the water originated from the anterior end (dominated by the gills). High performance liquid chromatography confirmed the identity of both hormones.