Studies on artificial gynogenesis in Haliotis diversicolor

人工雌核发育技术在育种实践和遗传学基础研究中有着广泛的应用前景，然而贝类人工雌核发育诱导至今还没有真正成功，原因之一是理化方法诱导的贝类雌核后代成活率极低。针对贝类人工雌核发育体成活率低的问题，本研究一方面细致优化了理化方法诱导杂色鲍（Haliotisdiversicolor，简为S）雌核发育程序的参数，研究了理化方法诱导杂色鲍雌核发育的细胞机理，为进一步改进杂色鲍雌核发育诱导方法、提高诱导后代成活率奠定了基础；另一方面查实了杂色鲍♀×盘鲍(H.discusdiscus，简为J)♂种间杂交F1成贝实质上是含有少量父本基因的雌核发育体，并在受精生物学和细胞遗传学研究基础上提出了杂色鲍♀×盘鲍♂产...Haliotis diversicolor (S for short) is one of most economically important mollusks cultured in the coast of Southern China. To support sustainable development of the culture industry of abalone, genetic improvement program have been proposed. Gynogenesis induction is an important chromosome set manipulation techniques with potential applications including of rapid establishment of inbred lines or ...学位：理学博士院系专业：海洋与环境学院海洋学系_海洋生物学学号：B20022700

Advances in genetics and breeding in abalone: a review

Abalones are important farming species with a high economic value. They have already been farmed for more than 50 years. As problems and new requirements rose continuously in culture industry of abalone, studies on genetic and breeding techniques are needed to improve characteristics and to gain new traits. This review concentrates on advances in genetics and breeding techniques in abalone.As for genetic studies, karyological analyses, allozyme, DNA markers and genetic diversity were reviewed. So far, karyological analyses in abalone have been performed in 12 species that can be divided into three groups according to the chromosome number. In some economically important species, loci of allozymes and microsatellites have been isolated and applied to investigate the genetic structure of natural and hatchery populations and to identify the result of chromosome set manipulation, but the related reports are only a few yet. The resultsof investigation with DNA markers and allozymes showed that the genetic structure of natural populations presents two characteristics: excessive homozygosity and subdivision.Advances of various breeding techniques, including introduction, selection, hybridization, polyploidy, gynogenesis and gene manipulation, were reviewed in the other part.Although Haliotis discus discus, introduced from Japan, has become one of the most important culture species in China, the economic, social and environmental effects of introduction have been rarely studied. Selection is one of the most important and basic breeding techniques, but the studies on selection are only a few and preliminary, referring to the relations between genetic characteristics and the traits of growth and resistance, genetic diversity and heritability of quantitative traits, and the effect of selection. Interspecific hybridization was the first breeding program carried out in abalone.Experimental hybridization have been carried out for about 20 crosses. Heterosis, such as faster growth and high survival rate, has been observed in some crosses. Triploids have been successfully induced in many species of abalone with physical or chemical shock, e.g. H. discus hannai, H. rufescens, H.diversicolor diversicolor and H. midae.Field experiments were conducted in some species of triploid abalone. In comparison with triploid, the research on tetraploid is still in quest stage. The progress of induction of gynogenesis in abalone is quite slow. Conditions of sperm inactivation,diploid restoration and nuclear behavior of gynogenetically activated eggs have been researched on in H. discus hannai. Notwithstanding the gene transfer technology in abalone is in the quest stage, the research have already involved preparation of exogenic DNA, means of gene transfer, identification integration and expression of target gene,etc. Three research directions in these topics were proposed: to investigate the germplasm resources of abalone deeply and widely, to make use of traditional breeding methods and modern biotechnique synthetically, and to combine the science research with production practice.国家863课题(2001AA621080

四种鲍45S rDNA在染色体上的比较定位

为了研究皱纹盘鲍、西氏鲍、绿鲍和杂色鲍等4种鲍的核型特征,实验利用荧光原位杂交(fluorescence in situ hybridization, FISH)技术比较定位了上述4种鲍的45S rDNA位点。皱纹盘鲍中约83%的中期细胞均检出2对45S rDNA位点,分别位于13号和16号染色体的长臂端部。西氏鲍中约75%的中期细胞均检出3对45S rDNA位点,分别位于6号染色体短臂端部、14号和17号染色体长臂端部。绿鲍中约85%的中期细胞均检出3对45S rDNA位点,分别位于4号、6号和8号染色体长臂的端部。杂色鲍中约65%的中期细胞均检出3对45S r DNA,位点,分别位于3号、4号和12号染色体短臂的端部。此外,4种鲍均有少数中期相的45S rDNA位点数高于众数,这提示,除了明确的45S rDNA位点外,4种鲍可能均有若干个不稳定的45S rDNA位点。实验结果丰富了鲍细胞遗传学研究资料,同时为鲍的遗传育种研究提供了基础数据。国家自然科学基金(U1605213)~

四种鲍45S rDNA在染色体上的比较定位

为了研究皱纹盘鲍、西氏鲍、绿鲍和杂色鲍等4种鲍的核型特征，实验利用荧光原位杂交（fluorescence in situ hybridization,，FISH）比较定位了这4种鲍的45S rDNA位点。皱纹盘鲍中，约83%的中期细胞检出2对45S rDNA位点，分别位于13号和16号染色体的长臂端部。西氏鲍中，约75%的中期细胞检出3对45S rDNA位点，分别位于6号染色体短臂端部、14号和17号染色体长臂端部。绿鲍中，约85%的中期细胞检出3对45S rDNA位点，分别位于4号、6号和8号染色体长臂的端部。杂色鲍中，约65%的中期细胞检出3对45S rDNA位点，分别位于3号、4号和12号染色体短臂的端部。此外，4种鲍均有少数中期相的45S rDNA位点数高于众数，这提示除了明确的45S rDNA位点外，4种鲍可能均有若干个不稳定的45S rDNA位点。研究结果丰富了鲍细胞遗传学研究资料，为鲍的遗传育种研究提供了基础数据

Study on Changes in Cellular Ultrastructure and Soluble Proteins Constitution of Gomphrena Glolosa Leaves after Infected with NMV.

防治水仙病害是提高水仙品质和产量，发展水仙产品生产的重要措施。水仙花叶病是水仙的主要病害。研究水仙病害病理变化规律可为综合治理水仙病害提供理论依据。自40年代以来，国内外研究者陆续报道了水仙花叶病毒（Narcissusmosaicvirus,NMV）引起的症状，病毒形态、在细胞内的存在方式、体外抗性等方面的工作[10、11、13-24]。但这些研究工作对于染病过程中，病毒——宿主相互作用关系以及病理变化与病毒运动的关系涉及较少。针对此问题，本课题开展以下工作：研究NMV侵染千日红36hr、症状期，千日红叶片超微结构以及可溶性蛋白的变化；水仙健株、病株可溶性蛋白组分的变化。结果如下： （1） ...It is a important measure preventing narcissus from virus diseases to improve the quality and quantify of narcissus. Narcissus mosaic disease is a principal disease of narcissus. Although many investigators have reported many artclss about NMV from 1940s, little is known of relations of virus between host and relation of pathogenesis and virus movement. So changes of cellular ultrasturcture a...学位：理学硕士院系专业：生命科学学院生物学系_细胞生物学学号：19952601

Karyotype Analysis on Paphia undulata

取波纹巴非蛤担轮幼体为材料,经秋水仙素浸泡、低渗处理后,用卡诺氏固定液固定,滴片后经gIEMSA染色液染色镜检拍照,利用PHOTOSHOP软件排列核型,MICrOMEASurE 3.3软件测量染色体长度与臂比.结果表明,波纹巴非蛤的染色体数为38,核型公式是2n=38=14M+12SM+4ST+8T,nf=64,未发现随体染色体,染色体绝对长度为1.5~4.0μM.所给出的波纹巴非蛤的核型,为波纹巴非蛤的种质鉴定、资源保护与遗传育种研究提供了必要的基础资料.Trochophore of Paphia undulate was collected,and treated with colchicine,hypotonic solution and Karry′s fixative.Afterwards,the chromosome specimens were prepared by dropping and stained with Giemsa solution.The length and the arm ratio of chromosomes were measured and calculated with Micromeasure 3.3 software,and the karyotype was arranged with Photoshop software.The results show that the chromosome number of P.undulate is 38,and the karyotype formula can be summarized as 2n=38=14m+12sm+4st+8t.The number of chromosome arm was 64.No satellite chromosome has been found in the chromosome set of P.undulate.The length of chromosome was about 1.5-4.0 μm.This is the first report on the karyotype of P.undulate,which will provide necessary information for further study on identification,resource protection and genetics and breeding.福建省自然科学基金项目(2011J01229);海洋公益性行业科研专项(201205021-02);2010年度福建省大学生创新性实验计划项

Characterization of Pacific abalone( Haliotis discus hannai) karyotype by C-banding and fluorescence in situ hybridization with rDNA

为了提高对皱纹盘鲍染色体的辨识水平,实验利用bA(OH)2处理显示了皱纹盘鲍染色体的C带,并用荧光原位杂交分析(fluOrESCEnCE In SITu HybrIdIzATIOn,fISH)研究了核糖体大亚基rdnA在皱纹盘鲍中期染色体上的数目与位置。核型结果显示,皱纹盘鲍染色体组包含7对中部着丝粒染色体和8对亚中部着丝粒染色体,另有3对染色体介于中部着丝粒染色体与亚中着丝粒染色体之间(M/SM)。C显带结果显示,8对染色体有稳定的着丝粒C带,5~7对染色体上有中期相间多态的端部C带,3对染色体上有同源染色体异态的臂间C带。fISH分析显示,皱纹盘鲍中期染色体上分布着4个大亚基rdnA位点,分别位于2号短臂(2S)、7号短臂(7S)、12号短臂(12S)和18号长臂(18l)的端部。研究结果为皱纹盘鲍染色体辨识提供了新的特征与标记,为进一步研究皱纹盘鲍种群的染色体多态和鲍属染色体进化提供了基础资料。The chromosomes of Pacific abalone,Haliotis discus hannai,were examined by C-banding and fluorescence in situ hybridization(FISH)with human major rDNA(18S-5.8S-28S)as probes.The chromosome set of Pacific abalone was composed of 7 pairs of metacentric chromosomes,8 pairs of submetacentric chromosomes,and 3 pairs of metacentric/submetacentric chromosomes.Stable centromeric C-bands were present on 8 pairs of chromosome.Inter-metaphase polymorphic telomeric C-bands and heteromorphic pericentric C-bands were found on 5-7 pairs and 3 pairs of chromosomes,respectively.In addition,4 major rDNA loci were mapped onto the telomere of four pairs of chromosomes(2S,7S,12S and 18L).These results provided new markers and characteristics to distinguish chromosomes in the genome of Pacific abalone,and would serve as basic data for further researches on chromosome evolution in abalone.国家“八六三”高技术研究发展计划(2012AA10A412); 海洋渔业科学与技术浙江省重中之重学科开放课题(20110216); 闽发改投资(2010)983号; 广东省教育部产学研结合项目(2012B091100085); 集美大学国家级大学生创新创业训练计划项目(Z81228

Influence factors on fertilization rate in laboratory hybridization between Haliotis diversicolor and H.discus discus

研究卵子排放后的时间、精子排放后的时间、授精精子浓度、胰蛋白酶、钙离子、pH和水温等因子对杂色鲍(Haiotisdiversicolor,以下简称S)与盘鲍(H.discus discu,以下简称J)种间杂交受精率的影响。结果表明,卵子排放后的时间和授精精子浓度是影响杂交受精率两个最重要的因素。与自繁组合相比,正反杂交受精率均较低且下降速度快得多。水温21.5℃时,两亲本自繁对照组中,卵子排放1 h对受精率均无显著影响。而SJ杂交,卵子排放时间2 min时,受精率为55.9%±8.1%,4.7 min时受精率已下降了50%;JS杂交,卵子排放时间为4 min时,受精率为31.5%±9.7%,10 min时受精率下降至4 min时的29.1%。相比之下,精子排放时间对杂交受精率的影响小得多,3 h内对正反交杂交受精率没有显著影响。杂交适宜精子浓度约为母本自繁的100倍,其中SJ杂交适宜授精精子浓度为6.0×106~2.4×107mL-1,JS杂交适宜授精精子浓度为3.8×107~7.5×107mL-1。水温对于杂交受精率也有影响,短时间内将水温控制在26~29℃可以得到较高受精率。其他实验因素,如添加胰蛋白酶、Ca2+或改变授精体系pH对杂交受精率均无正向作用。To overcome the gametic incompatibility of interspecific hybridization between Haliotis diversicolor(S) and H.discus discus(J),the effects of gametic age,insemination sperm concentrations,Trypsin,Ca~(2+),pH and temperatures on the fertilization rate were investigated.The results showed that the age of egg and insemination sperm concentration are two important factors influencing the fertilization rate.In comparinson with the pure cross,the fertilization rate was low and declined much sharply for the heterospecific cross.At 21.5℃,the fertilization rate was 100% and did not declined significantly in 1 h post egg spawned for the pure cross,while it was 55.9%±8.1% at 2 min and declined by 50% at 4.7 min post spawned for the SJ cross,and it was 31.5%±9.7% at 4 min and declined by 70.9% at 10 min post spawn for the JS cross.The concentrations of sperms yielded maximum fertilization with the least abnormality in subsequent development was 6.0×10~(6)-2.4×10~(7)mL~(-1) for the SJ cross,and 3.8×10~(7)-7.5×10~(7)mL~(-1) for the reciprocal cross,which were about 100-fold higher than that in homologous cross.Higher temperature accelerated the egg losing the ability of being activated by the heterospecific sperm.For the eggs spawned for less than 10 min,the optimal temperature for hybridization is 26-(29℃).The other tested factors,including the addition of Trypsin or Ca~(2+) into the fertilization system and alteration of pH,led to decline of fertilization rate.国家“863”计划课题资助(2003AA603240

Preliminary Studies on Hybridization Between Japanese Stock and Taiwan Stock of Haliotis diversicolor

采用杂色鲍日本群体(RB)和台湾群体(TW)进行群体间杂交及群体内自繁,得到RB♀×TW♂、TW♀×RB♂、TW♀×TW♂和RB♀×RB♂4个组合的F1代.对这些交配组合的卵径、受精率、胚胎发育速度、幼体附着率、幼体变态率、幼体存活率以及稚贝早期生长进行了比较.结果表明,杂交组与自繁组在卵径、受精率以及胚胎发育速度方面并无显著的差别,但杂交组在幼体附着率、幼体变态率、幼体存活率以及稚贝早期生长方面,与自繁组相比表现出不同程度的杂种优势.TW♀×RB♂组8 d的存活率杂种优势达到40.6%,RB♀×TW♂组25 d壳长的杂种优势达到48.6%.实验初步显示,不同地理群体间的杂交将可能是养殖杂色鲍遗传改良的有效途径.F_1 generation of 4 groups,Japanese(RB)♀× Taiwan(TW)♂,TW♀×RB♂,TW♀× TW♂and RB♀×RB♂were obtained from mating and crossing within and between Japanese stock and Taiwan stock of Haliotis diversicolor.Comparisons of egg size,fertilization rate,embryonic development rate,larval settlement rate,larval metamorphosis rate,larval survival rate and postlarval growth among four groups were made.The results revealed that there were no significant differences on egg size,fertilization rate,and embryonic development rate.But the hybridization groups showed heterosis of various degrees on larval settlement rate,larval metamorphosis rate,larval survival rate and postlarval growth compared to self-fertilized groups.Heterosis of survival reached 40.6% in TW♀×RB♂(Day 8) and heterosis of growth achieved 48.6% in RB♀×TW♂(Day 25).It is indicated that the cross between geographically different stocks may be an effective way for genetic improvement of small abalone Haliotis diversicolor.国家“863”计划(2003AA603240)资

Cytological studies on artificially gynogenetic Haliotis diversicolor

应用组织切片方法,比较观察了杂色鲍普通二倍体、人工诱导三倍体、人工诱导雌核发育单倍体和雌核发育二倍体在成熟分裂和第一次卵裂期间的细胞学变化,以探讨人工诱导杂色鲍雌核发育的细胞学机制。结果表明,精子经紫外线辐射进行遗传失活处理后,仍然能够进入卵子,并激动卵子发育,卵子能够正常完成两次减数分裂,与普通受精卵没有明显差别。精子入卵后形成精核,大多能够逐渐解凝、液化并膨大,最终形成形态正常的雄性原核,但在第一次卵裂早期退化成浓缩的染色质小体(DCB),并在胞质分裂时随机地分配到其中一个子细胞里的分裂沟附近。在人工诱导三倍体和雌核发育二倍体受精卵中,受精卵经细胞松弛素B处理后,染色单体在第二次成熟分裂期分离,但胞质分离受抑制,留在卵质中的两套染色体形成两个染色质团,并发育成两个雌性原核。To obtain useful information about the mechanism of the artificial induction of gynogenesis in Haliotis diversicolor,we observed the cytological images of diploid control group,triploid group,haploid gynogenetic group and diploid gynogenetic group during meiosis and first cleavage.After being incorporated into the egg cytoplasm,the UV-irradiated sperm nuclei could activate eggs to complete meiosis,and sperm nuclei discondensed gradually,liquefied and expanded.Most sperm nuclei eventually developed into the male pronuclei,just like the normal sperms,except that sperm nuclei in a few zygotes kept dense throughout the fertilization process.Dense chromosome body(DCB)was seen at the nuclear area during the prophase of first mitosis,and in the middle of spindle at metaphase of first mitosis.At completion of cytokinesis of first cleavage,the DCB was seen in the cytoplasm of one of the two blastmeres near the cleavage furrow.In the cytochalasin B-treated group,both sets of female chromosomes hold in cytoplasm for chromosomes segregated as in normal zygote but the cytoplasm division was inhibited,formed two chromatin bodies and eventually developed into two pronuclei.国家“八六三”高技术发展计划(2006AA10A407