利用SLAF-seq技术开发带鱼(Trichiurus japonicus)微卫星标记以及跨物种扩增检测

带鱼(Trichiurus japonicus)是广泛分布于东亚大陆架海域的暖温性近底层鱼类,长期位居我国单鱼种渔获量第一位。自上世纪70年代以来,持续过度捕捞和海洋环境变化导致中国近海带鱼资源基础和遗传多样性受到严重影响,但有关带鱼种群的微卫星DNA标记研究却较为缺乏,不利于该物种遗传资源评估和保护。为此,本研究采用基于高通量测序平台的SLAF-seq技术,从带鱼的195 308个SLAF标签中识别出25 704个二至六碱基重复微卫星位点。经过引物扩增验证,最终筛选出36个具有多态性的微卫星标记。各位点等位基因数4～35,均值14.47。观测杂合度0.214～1.000,期望杂合度0.456～0.979,均值分别为0.620和0.803。所有位点的多态信息含量值均大于0.25。经Bonferroni校正,21个位点符合哈迪-温伯格平衡,且各位点间不存在连锁不平衡。这21个多态性微卫星标记可为带鱼的种群遗传资源研究提供新的有效分子标记和技术支撑。Bottleneck分析结果表明,宁波近海带鱼群体未检测到近期的遗传瓶颈效应,这可能与目前东海区带鱼野生群体数量还比较庞大、遗传变异仍然较为丰富有关。跨物种扩增结果显示,分别有12、16、4和3个带鱼微卫星标记在带鱼属(Trichiurus)、沙带鱼(Lepturacanthus savala)、窄颅带鱼(Tentoriceps cristatus)及小带鱼(Eupleurogrammus muticus)中具有较好的通用性,这些微卫星标记为今后带鱼科相应属、种的系统进化关系研究提供新的分析手段和契机。     

东海竹筴鱼的食性

以2008年5月、8月、11月和2009年2月东海灯光围网采集到的453条东海竹筴鱼为研究对象,对其胃含物进行分析,应用K-W非参数检验、卡方检验、聚类分析等方法,对不同季节和发育阶段条件下东海竹筴鱼的食性进行研究.结果表明:东海竹筴鱼的饵料生物有124种(包括未鉴定种),浮游甲壳类和小型鱼类为其主要饵料类群.优势饵料生物依次是麦氏犀鳕(IRI％=39.2％)、长尾类糠虾幼体(IRI％=18.4％)、短尾类大眼幼体(IRI％ =7.6％)和太平洋磷虾(IRI％=6.6％)等.季节和叉长对东海竹筴鱼的摄食强度均有显著影响(P＜0.01),东海竹筴鱼春季摄食强度最高,而冬季最低;叉长140 ～ 159 mm的竹筴鱼摄食强度最高,叉长45～99 mm的幼鱼的摄食强度较高,其余叉长的鱼摄食强度相对较低.聚类分析结果表明,叉长100 mm是东海竹筴鱼摄食取向的拐点.东海竹筴鱼四季的平均营养级为3.51,属于低级肉食性鱼类.     

Isolation and characterization of 22 polymorphic microsatellite DNA markers of Japanese sea bass (Laterolabrax japonicus)

Japanese sea bass (Laterolabrax japonicus) inhabits the coast of East Asia and is cage‐cultured currently in China as well. Twenty‐two microsatellite DNA markers were developed and used to type 35 individuals collected along the Chinese coast. The number of alleles per locus ranged from three to 23. The expected heterozygosity ranged from 0.111 to 0.938, while the observed heterozygosity ranged from 0.114 to 0.914. All 22 loci are neutral and independent of each other; two deviated significantly from Hardy–Weinberg equilibrium. These microsatellite DNA markers are moderately informative, which will certainly facilitate the management and exploitation of the genetic resource of L. japonicus.     

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.     

Quantitative assessment of behavioural response to the netting panel in the jack mackerel Trachurus japonicus

The behavioural response of the jack mackerel Trachurus japonicus when encountering a netting panel was shown to be influenced by swimming speed and the diagonal mesh-size ratio (vertical:horizontal).     

Hatch date‐dependent differences in early growth and development recorded in the otolith microstructure of Trachurus japonicus

Otolith microstructure of 308 juvenile jack mackerel Trachurus japonicus sampled in the southern Sea of Japan between June and September 2002 were examined by light microscopy. Hatch‐date distribution indicated a protracted spawning season from 16 January to 30 May 2002. The first secondary primordium (SP) was formed at ages 19–54 days, with a mean ±  s . d . of 30·4 ± 6·1. The number of SP in an otolith ranged from two to 15, with a mean of 6·1 ± 1·7. Age of the first SP formation, number of SP and increment width varied with hatch date. In general, late‐hatched fish were younger when the first SP formed, and had a higher number of SP and wider increments, than early‐hatched fish. As increment width is a linear function of somatic growth and formation of the first SP is associated with metamorphosis in jack mackerel, these variations in otolith microstructure indicated that rates of growth (as inferred from wider increments) and development (as inferred by younger age of first SP formation) were higher in late‐hatched fish relative to early‐hatched fish. Higher temperature experienced by late‐hatched fish during early life stages may contribute critically to their higher growth and development rates compared to early‐hatched fish.     

Development and characterization of polymorphic microsatellite markers in Linum usitatissimum

Thirty-five microsatellite loci were isolated and characterized in Linum usitatissimum using enriched genomic libraries. These loci were screened in eight cultivars from different countries and regions and were found to be polymorphic, with the number of alleles per locus ranging from two to six, and observed and expected heterozygosities ranging from 0.125 to 0.375 (mean 0.013) and from 0.233 to 0.842 (mean 0.601), respectively. These polymorphic new microsatellite loci will be useful for genetic linkage map construction, germplasm classification and identification, gene identification and quantitative trait loci mapping, and marker-assisted selection in breeding in L. usitatissimum.     

Isolation and characterization of ten polymorphic microsatellite markers for the blue mussel (Mytilus edulis)

In this study, we isolated 10 novel polymorphic microsatellite markers in Mytilus edulis by using the magnetic beads enrichment procedure. The characteristics of these loci were estimated by using a sample of 32 individuals of M. edulis. The number of alleles at 10 polymorphic microsatellite loci ranged from 2 to 15 with an average of 5.667. HO and HE ranged from 0.2667 to 1.0000 (0.6800 in average) and from 0.4723 to 0.9226 (0.6190 in average), The PIC value of 6 loci was more than 0.5, and that of the other 4 was between 0.25 and 0.50, Significant deviation from Hardy–Weinberg Equilibrium was observed at ME8, ME115 and ME153 after Bonferroni correction (P < 0.004, adjusted value), which possibly was due to the presence of null alleles. This study will be useful for the analysis of population genetic diversity, and the management of this important M. edulis resource.     

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

Eleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.     

花鲈微卫星标记分离及其多态性分析

该文利用FIASCO法(fast isolation by AFLP of sequences containing repeats)和GenBank数据库搜索法开发花鲈微卫星标记,并对筛选的标记进行多态性检测.两种方法共获得54条能够设计引物的序列,扩增结果显示15对引物具有多态性,多态性微卫星位点的等位基因数为2～10个.15个多态性位点中,4个位点偏离了Hardy-Weinberg平衡;各位点间没有连锁不平衡现象;仅位点SP52可能存在无效等位基因;除SP17和SP468外,其余引物的P1C值均在0.5以上,可用于花鲈群体遗传分析等研究.     

Isolation and characterization of 15 polymorphic microsatellite DNA loci from Argyrosomus japonicus (mulloway), a new aquaculture species in Australia

Fifteen microsatellite loci were isolated for population genetic studies of mulloway (Argyrosomus japonicus), a commercial/recreational fish species in Southern Australia. A genomic library was screened randomly for di- tri- and tetranucleotide repeats. Fifteen microsatellite marker loci were developed that were highly polymorphic (allele number ranged from four to 18). Observed and expected heterozygosities ranged from 0.17 to 1 and from 0.27 to 0.91, respectively. These markers have proven useful for estimating genetic variation and for evaluating population structure across the species' natural distribution. They also provide powerful tools for optimizing hatchery practices to conserve genetic diversity.     

Isolation and characterization of polymorphic microsatellite markers in the red snow crab (Chionoecetes japonicus)

A total of 12 polymorphic microsatellite DNA loci were isolated from the red snow crab, Chionoecetes japonicus (Brachyura: Majidae), one of important fisheries resources in the Far East. The number of alleles observed at each locus ranged from two to 19, with the observed and expected heterozygosities of 0.125-0.875 and 0.156-0.949, respectively, suggesting these loci to be a useful molecular marker for population analysis in this species. Of the 12 loci, seven also were available for genotyping of the snow crab, Chionoecetes opilio, implying these loci as a useful molecular marker in the genus Chionoecetes.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

Development and characterization of polymorphic microsatellite markers in Rhododendron simsii (Ericaceae)

A set of 14 polymorphic microsatellite markers has been developed and characterized using FIASCO (fast isolation by AFLP of sequences containing repeats) methods for the garden plant, Rhododendron simsii Planch. Forty‐one R. simsii individuals showing large morphological differences were used to identify these markers. The total number of alleles for each locus ranged from 2 to 6, with an average value of 3.643. The expected heterozygosities and observed heterozygosities ranged from 0.137 to 0.778 and 0.000 to 0.769, respectively. Eight loci exhibited significant deviations from Hardy‐Weinberg equilibrium. Among these microsatellites, seven could successfully be transferred to four related species (R. pulchrum, R. hybrida, R. alutaceum and R. molle (Blume) G. Don). These novel microsatellite markers could be further used for assessment of genetic diversity and population structure, phylogeographic analysis and marker‐assisted selection for R. simsii and other Rhododendron species.     

Development and characterization of polymorphic microsatellite markers in Momordica charantia (Cucurbitaceae)

? Premise of the study: Polymorphic microsatellite markers were developed for Momordica charantia L. to investigate the genetic diversity and population structure within and between M. charantia and its four related species (Cucurbita pepo L., Luffa cylindrical L., Lagenaria siceraria L., and Cucumis sativus L.). ? Methods and Results: Using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) method, 16 polymorphic microsatellite loci were identified in 36 individuals of M. charantia. Across all the M. charantia samples, the number of alleles per locus ranged from three to eight. Seven primers successfully amplified in the four related species. ? Conclusions: These markers will be useful to study population ecology and population differentiation among M. charantia species and its related species.     

Development and characterization of polymorphic microsatellite markers in Cyclobalanopsis glauca (Fagaceae)

? Premise of the study: We isolated and characterized polymorphic microsatellite loci in Cyclobalanopsis glauca (Fagaceae), an evergreen broadleaved monoecious tree, to provide tools for analyzing parentage and mating system. ? Methods and Results: Thirteen polymorphic microsatellite markers were developed and tested in three C. glauca populations. The number of alleles per locus varied from two to 22. The observed and expected heterozygosities within populations were 0.000-0.967 and 0.033-0.949, respectively. ? Conclusions: These polymorphic primers showed high levels of polymorphism within tested populations, and can be used in parentage analysis and mating system estimation of C. glauca.     

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta).

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.     

Development of twenty-five polymorphic microsatellite markers for the endangered red-cockaded woodpecker (Picoides borealis)

Twenty-five polymorphic microsatellite markers were developed for the endangered red-cockaded woodpecker (Picoides borealis). The number of alleles ranged from two to five and observed heterozygosities ranged from 0.036 to 0.750. These loci should be useful tools for conducting research towards the management and conservation of this species.