异源四倍体鲫鲤雌核发育后代的RAPD分析

在优化RAPD(随机扩增多态性DNA)检测条件基础上,从134个随机引物中筛选出53个扩增较好且多态性强的引物,对异源四倍体鲫鲤第1代(G1)、第2代(G2)人工诱导的雌核发育二倍体后代群体的DNA多态性及分子标记进行了分析。结果显示,53个随机引物在G1群体和G2群体中检测到的位点数分别为541、511,其中多态性位点数分别为70、52,多态位点比例分别为12.94%、10.18%。两个群体的平均遗传距离分别为0.0732、0.0464。研究表明,经过连续2代人工雌核发育,G2的遗传多样性明显减少,种质进一步纯化。还从53个随机引物的扩增谱带中找到了2个引物(S50、S223)的特异扩增谱带,可以作为第1、2代雌核发育群体间的分子遗传标记。由计算机软件程序构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。       

异源四倍体鲫鲤群体遗传多样性的RAPD分析

湖南师范大学和湘阴县东湖渔场合作,经过10多年的研究,获得了两性可育的异源四倍体鲫鲤群体1—7,目前已经繁殖到了F14。异源四倍体鲫鲤具有4套染色体(4n=200),它们的遗传多样性水平怎么样?这是关系到异源四倍体鲫鲤这一新的种质资源能否生存和进化的问题,有必要从分子水平进行深入的研究。随机扩增多态DNA(Random Am-plified Polymorphic DNA,RAPD)是建立在PCR基础之上的一种DNA分子标记技术8,具有简便、快速、实验成本低等特点,因此被广泛应用于物种的遗传多样性分析、亲缘关系的探讨和系统进化等方面的研究9—15。本研究采用RAPD技术,检测了异源四倍体鲫鲤群体的遗传多样性水平,以期为异源四倍体鲫鲤的人工繁殖、种群复壮以及种质资源保护提供DNA水平上的依据。       

异源四倍体鲫鲤雌雄差异的RAPD标记

异源四倍体鲫鲤是从红鲫和湘江野鲤的杂交后代选育出来的,已经形成了一个遗传性状稳定的四倍体鱼新种群。用异源四倍体鲫鲤(雄性)和二倍体白鲫(雌性)生产的三倍体湘云鲫已经在全国推广应用。因此,如果能够了解异源四倍体鲫鲤的性别分化机制,人为地控制异源四倍体鲫鲤的性别分化,生产出大量的超雄鱼,这对于三倍体湘云鲫的产业化生产有重要的意义。刘少军等对异源四倍体鲫鲤的染色体组型进行了分析,并没有发现异源四倍体鲫鲤有明显的特化的性染色体,这说明通过细胞遗传学研究异源四倍体鲫鲤的性别遗传机制是有困难的。       

异源四倍体鲫鲤及其原始亲本遗传变异的微卫星标记分析

采用从鲤中分离出来的32对微卫星DNA标记,对异源四倍体鲫鲤、红鲫和野鲤的基因组DNA进行了研究。在筛选出的15对微卫星引物中,随引物不同,各等位基因数为1～8个,大小在100～420bp之间。从3个不同群体内部的遗传相似系数来看,异源四倍体鲫鲤个体之间的遗传相似系数最大,说明异源四倍体鲫鲤群体内部的遗传变异程度最低,已经形成了一个遗传性状稳定的群体。从3个不同群体之间的遗传相似系数来看,异源四倍体鲫鲤和红鲫遗传相似系数为0．5625,和野鲤的遗传相似系数为0．5125,说明异源四倍体鲫鲤接受原始母本的遗传物质比原始父本野鲤要多一些。微卫星标记与以前报道的RAPD标记的检测结果是相似的,然而由微卫星标记获得的种群内和种群间的遗传距离均大于RAPD,说明微卫星标记比RAPD标记显示出更高的个体多态性。     

异源四倍体鲫鲤的性腺发育研究

采用组织切片技术对异源四倍体鲫鲤的性腺进行了研究。结果表明：异源四倍体鲫鲤的性腺发育程序和普通鲤鲫鱼相似,可以分为6个时期。雌雄个体都能达到性成熟,其中个体性成熟年龄为一周年;雄性个体比雌性个体成熟早,150日龄即可达到性成熟。成熟卵巢的成熟系数为9．25％－15．60％,成熟精巢的成熟系数为1．65％－5．19％。成熟期卵径为1670．4－1780．5μm之间,成熟精子头径为2．3－2．4μm。繁殖期为每年3－5月份。该研究证明异源四倍体鲫鲤的性腺发育是正常的,雌雄个体都能达到性成熟。     

转基因异源四倍体鲫鲤F1的研究

采用显微注射法将含有鲁鱼β－actin基因启动子的草鱼生长激素基因“全鱼”基因pCAgcGHc转入异源四倍体鲫鲤,然后使其自交得到转基因异源四倍体鲫鲤F1,对150日龄F1体重和体长进行检测,可明显看见转基因异源四倍体鲫鲤F1的生长优势;取F120尾,提取尾鲤基因组DNA,采用合适的引物,PCR方法检测转基因异源四倍体鲫鲤F1是否含有外源生长激素基因,结果150日龄F1阳性率达到90％,且有些雄性个体可以挤出少量精液,而普通150日龄异源四倍体鲫鲤无此现象,文章阐明了通过近交筛选转基因异源四倍体鲫鲤种系具有重要意义。     

异源四倍体鲫鲤的受精细胞学

异源四倍体鲫鲤的成熟卵子处于第二次减数分裂中期,精子通过受精孔进入卵内。精子入卵以来,受精孔立即被受精塞堵住。受精后8min,受精卵出现明显的精子星光,同时进入第二次减数分裂后期,即将排出第二极体;13min时,精子头部开始膨胀,趋向核化;18min时,雌雄原核均已形成,并向胚盘中央靠近;23min时,雌,雄原核开始接触;33min时,雌,雄原核完全融合成为一个合子核;38min时,受精卵开始第一次卵裂,53min后分裂形成两个子核。该研究证明异源四倍体鲫鲤和大多数二倍体鱼一样,具有正常的受精细胞学程序,受精方式为单精受精。     

异源四倍体鲫鲤的受精细胞学

异源四倍体鲫鲤的成熟卵子处于第二次减数分裂中期,精子通过受精孔进入卵内.精子入卵以后,受精孔立即被受精塞堵住.受精后8 min,受精卵出现明显的精子星光,同时进入第二次减数分裂后期,即将排出第二极体;13 min时,精子头部开始膨胀,趋向核化;18 min时,雌雄原核均已形成,并向胚盘中央靠近;23 min时,雌、雄原核开始接触;33 min时,雌、雄原核完全融合成为一个合子核;38 min时,受精卵开始第一次卵裂,53 min后分裂形成两个子核.该研究证明异源四倍体鲫鲤和大多数二倍体鱼一样,具有正常的受精细胞学程序,受精方式为单精受精.     

三倍体鲫鱼--异源四倍体鲫鲤(♂)×金鱼(♀)

利用雄性四倍体鲫鲤与雌性二倍体红色双尾金鱼交配 ,制备了 1种新型三倍体鲫鱼 ,并对Ⅰ龄三倍体鲫鱼染色体数目和组型、性腺指数和性腺结构、外形、生长速度等生物学特征进行了系统研究。结果表明 :这种三倍体鲫鱼染色体数目为 3n =15 0 ,核型公式为 33m 5 1sm 33st 33t;在繁殖季节 ,三倍体鲫鱼卵巢和精巢指数明显低于作为对照的红鲫卵巢和精巢指数 ;组织学切片表明三倍体鲫鱼的卵巢和精巢不能产生成熟的卵子和精子 ,证明它们是不育的。三倍体鲫鱼体色为青灰色 ,单尾 ,具有一些介于其父母本———异源四倍体鲫鲤和红色双尾金鱼之间的外形特征 ,如具有 1对肉眼不易观察到的短须 ,侧线鳞式为 31 6 7,背鳍为 18,体高 体长之比为 0 5 ,比湘云鲫的体高 体长之比 (0 4 1)有明显的增加。经过约 8个月的饲养 ,该三倍体鲫鱼平均重量为 35 0g,最大个体达 5 5 0g。在Ⅰ龄鱼中 ,雌性三倍体鲫鱼的个体生长速度快于雄性个体。与三倍体湘云鲫相比 ,该三倍体鲫鱼保留了生长速度快、不育等优点 ,同时新添了体高 体长比值更高、肉质更甜美的特点 ,为 1种有推广前途的新型三倍体鲫鱼     

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     

DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrid/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that at the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis.     

Analysis of DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrids/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that of the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 195–205. The text was submitted by the authors in English.     

Rapid genomic DNA changes in allotetraploid fish hybrids

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F₂₀) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine–cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F₁ hybrids (2nF₁) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF₁ and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF₁. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F₂₀ hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.     

A more accurate method of comparing first-generation maize hybrids with their parents

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A more accurate method of comparing first-generation maize hybrids with their parents

     

Differential response of antioxidant enzymes in leaves of necrotic wheat hybrids and their parents

The leaves of necrotic hybrid of wheat ( Triticum aestivum L.) exhibited high superoxide content associated with increased lipid peroxidation and membrane damage in earlier studies (Khanna-Chopra et al. 1998, Biochem Biophys Res Commun 248: 712–715; Dalal and Khanna-Chopra 1999, Biochem Biophys Res Commun 262: 109–112). In the present study, we investigated the activities of the antioxidant enzymes in the leaves of necrotic wheat hybrids, Kalyansona×C306 (K×C) and WL711×C306 (WL×C) and their parents at different developmental stages. The K×C hybrid exhibited more severe necrosis than WL×C. In K×C, superoxide dismutase (SOD) activity showed no increase over the parents, while WL×C showed an early increase, but it was possibly insufficient to scavenge increased superoxide. Activities of guaiacol peroxidase, ascorbate peroxidase and glutathione reductase were enhanced, while catalase exhibited a decrease in activity, with the appearance of visible necrosis in both the hybrids. The isozyme profile of the antioxidant enzymes was similar in the hybrids and their parents. One existing isoform of guaiacol peroxidase showed an early appearance in the hybrid and increased in intensity with the progression of necrosis. The results reveal a differential response of antioxidant enzymes in necrotic wheat hybrids as compared to their parents. The response differed in magnitude at developmental stages of the leaves, which might be related to the intensity of necrosis expressed by the hybrids.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

Photosynthetic characteristics in Brassica carinata hybrids and their parents as influenced by moisture stress

The response of Brassica carinata hybrids and their parents to moisture stress at different growth stages was studied. B. carinata 226 was found to be susceptible to stress at pre-flowering and post-flowering stages while B. carinata 241 at flowering stage. Neither the changes in stomatal conductance nor in chlorophyll content could fully explain the reduction in net photosynthetic rate (P_N) induced by stress. B. carinata 241 had higher leaf water potential (ψ_w) although, it had lower P_N compared to B. carinata 226. Both the parents had lower P_N as well as leaf ψ_w. The stress response of PN in hybrids followed that in their respective female parents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis

Electrofusion of mesophyll protoplasts from two male sterile dihaploid Solanum tuberosum genotypes. DHAK-11 and DHAK-33, was performed. Selection of putative fusion products was based on vigorous callus growth. Regeneration of rooted putative hybrid plants was scored 14 weeks after fusion. Characterization of hybrids was performed by use of morphological assessment, random amplified polymorphic DNA (RAPD), and cytological and isozyme analysis. The rate of regenerated hybrids from callus was ca 6%. Of the putative hybrids, 45% were confirmed as true hybrids. Morphological assessment of the putative hybrids revealed that tetraploid and neartetraploid hybrids were vigorous plants with intermediate characteristics between the two parental phenotypes in respect to internode length, leaf size and shape, and purple pigmentation on the abaxial side of the leaves. Near-hexaploid hybrids were slender plants with small leaves and short petioles. Selected RAPD primers showed unique marker bands for the two parental genotypes. Hybrid plants revealed the unique marker bands from both parents. A total of 53 randomly chosen decamer primers were tested and 26 primers (49%) detected polymorphism between the two dihaploid parentals. Two primers revealed that one parental marker band was missing in two aneuploid hybrids. However, of 51 putative hybrids, a double test with two independently chosen primers showed unequivocally the hybrid character of 23 plants. The ploidy level of the hybrids was analysed by chromosome numbers in root tip cells and by number of guard cell chloroplasts. A strong correlation between the chromosome number and the number of chloroplasts was obtained. The hybrid nature of all RAPD-verified hybrids was confirmed by isozyme analysis with malate dehydrogenase.