基于转录组测序的大熊猫多态性微卫星标记筛选

结合已公布的大熊猫Ailuropoda melanoleuca基因组和本实验室所测6只大熊猫的转录组数据,筛选多态性微卫星位点并分析其组成及特征。结果显示:共获得326个多态性微卫星位点,其中二碱基多态性微卫星最多,共228个,占69.93%;三、四、五、六碱基所占比例分别为9.51%、14.11%、5.21%、1.22%。根据分析结果中缺失率与标准差2项指标以及位点序列长度,选取20个多态性二碱基微卫星位点,用于25只大熊猫个体血液DNA进行PCR验证并做后续分析。结果表明:不同位点的等位基因数为2~8,平均等位基因数为3.70,观测杂合度、期望杂合度分别为0~1.000、0.280~0.784,平均值分别为0.472和0.532。在Bonferroni校正后,证实4个位点显著偏离哈迪-温伯格平衡,所有位点未观察到显著连锁不平衡(P>0.01)。20个位点多态信息含量(PIC)在0.246~0.734,其中具有高度多态性的位点9个(PIC>0.50),11个位点呈中度多态性(0.25相似文献   

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

微卫星DNA与生化标记分析对长爪沙鼠群体遗传分析的比较

目的比较生化标记和微卫星DNA标记方法对长爪沙鼠群体遗传分析的可靠性。方法应用27个生化位点和13个微卫星DNA位点,采用已建立的生化标记和微卫星DNA标记分析方法对国内2个长爪沙鼠群体进行遗传分析,计算并比较两种方法测得的各群体遗传参数。结果生化基因位点中有13个位点在整体中呈现遗传多态性,多态率为48.1%;微卫星位点中有11个位点在整体中表现出多态性,多态率均为84.6%。两种方法测得的平均有效等位基因数趋于一致,微卫星DNA的多态位点百分率和平均杂合度均明显高于生化标记方法。但生化标记和微卫星DNA检测对两个长爪沙鼠群体的遗传多样性差异反映一致,所反映的群体平衡状况也基本一致。结论生化标记分析和微卫星DNA方法均可较好地反映长爪沙鼠群体遗传结构。     

Twelve polymorphic microsatellite loci from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius)

In the study, 34 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese Spanish mackerel (Scomberomorus niphonius). And 12 microsatellite loci were found to be polymorphic between 3 and 8 alleles. The number of observed and expected heterozygosity per locus in 23 individuals ranged from 0.6087 to 1.0000 and 0.8908 to 0.9773, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 12 microsatellite loci probably located on different chromosome pairs and these polymorphic microsatellite loci should provide sufficient level of genetic diversity to investigate the fine-scale population structure and evaluate the breeding strategy in S. niphonius. The authors Shi-Chao Xing and Gen-Bo Xu contributed equally.     

利用双重PCR技术研究长爪沙鼠的微卫星结构

目的为了建立快速检测长爪沙鼠群体遗传多样性的方法及获得Z：ZCLA长爪沙鼠封闭群现用微卫星位点的结构。方法利用17个微卫星位点（9个来自长爪沙鼠,8个来自大小鼠）进行了PCR反应体系及反应条件的优化,组合了6组双重PCR及两个复合式点样,用上述8个组合对普通级Z：ZCLA长爪沙鼠封闭群43、444、5三个世代核心群各100只种鼠进行遗传检测。结果三个世代的300只种鼠的检测结果表明,9个长爪沙鼠位点均为微卫星,其中7个位点为完全型的微卫星,1个为复合型,1个为不完全型,多态性主要表现在核心序列的重复;来自大小鼠的8个微卫星位点,有7个在Z：ZCLA长爪沙鼠核心群中得到有效扩增,只有3个位点在三个世代中均有出现,对测序结果分析后发现,其核心序列均为小卫星。结论来自长爪沙鼠的位点,无论结构还是遗传方式均符合微卫星遗传标记的特点,可用作检测长爪沙鼠的群体遗传多样性。     

微卫星标记应用于凡那滨对虾家系鉴别的研究

以人工选育建立的凡那滨对虾（Litopenaeus vannamei）全同胞家系为实验材料,探讨了微卫星标记对混养家系进行亲权鉴定的可能性。Cervus模拟分析表明从10个微卫星基因座组合中选取的多态性信息含量最高的6个进行组合与原组合的累积排除概率均在0.99,多态性信息含量最高的6个基因座的组合判定正确率为0.97,置信度为95%。在家系混养的模拟实验中使用这6个高多态性的微卫星基因座从20个候选雌虾中找到真正母亲的概率为88%,从30个候选雄虾中找到真正父亲的概率为78%,低于理论预测值,分析可能与微卫星基因座中的无效等位基因,等位基因的突变以及PCR过程中Taq酶发生链滑移等因素有关。      

Thirty-three microsatellite loci for noninvasive genetic studies of the giant panda (Ailuropoda melanoleuca)

Limited microsatellite markers useable in noninvasive genetic methods have hampered the studies of dispersal patterns and mating systems of giant pandas. Therefore, we describe in this paper the characterization of 15 novel microsatellite loci from genomic DNA-enriched libraries and 18 redesigned microsatellite loci from published papers on the giant panda. The number of alleles per locus in 60 individuals ranged from 2 to 13, the average observed heterozygosity per locus from 0.168 to 0.800, and the average expected heterozygosity per locus from 0.152 to 0.882. All loci followed Hardy-Weinberg expectations. Four pairs of significant linkage association were found among all these loci. Moreover, the 33 microsatellite loci showed high amplification successes rate in noninvasive samples, which indicated that these loci will be of use in studying dispersal patterns and mating systems of giant pandas using noninvasive genetic methods.     

秦川母牛群体遗传特性的微卫星标记研究

为了从DNA分子水平揭示秦川牛群体遗传多态性和群体遗传结构,寻找可用于秦川牛的微卫星标记,本研究选择了12个普通牛（Bos taurus）微卫星标记检测了90头秦川母牛各微卫星位点的遗传变异及多态性。结果表明,在秦川母牛群体中,12个微卫星位点共检测到了247个等位基因,各位点的等位基因数在13（INRA005）～33个（HEL13）之间,平均每个微卫星位点的等位基因数为21个;总有效等位基因数和平均每个位点平均有效等位基因数（Ne）分别分为142．6229和11．8852。各位点平均基因频率取样方差（V（pij））为2．6036×10^-4。12个微卫星位点平均观察杂合度（Ho）和平均期望杂合度（He）在0．7842（INRA005）～0．9775（BM315）和0．7952（BM315）～0．9446（HEL13）之间。12个位点平均多态信息含量（PIC）在0．7653（INRA005）～0．9420（HEL13）之间,平均为0．8965．12个微卫星位点均属于高度多态位点,这表明秦川母牛群体中所检测各微卫星位点具有丰富的遗传多态性,具备较大的选择潜力。12个微卫星位点的平均固定指数（F）为-0．0076,即各位点杂合子的缺陷度不高,即偏离Hardy—Weinberg平衡的程度不大。     

Mitochondrial microsatellite variability in common wheat and its ancestral species

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.     

Isolation and characterization of microsatellite loci in Acer opalus (Aceraceae), a sexually-polymorphic tree, through an enriched genomic library

An enrichment protocol was used to isolate and characterize microsatellite loci in Acer opalus, a Mediterranean tree species. Highly polymorphic microsatellite loci were required for paternity analyses in a population of this species. Eight microsatellite loci were amplified and a total of 87 alleles were detected in a sample of 142 individuals from one population, allowing the identification of each individual with a unique multilocus genotype. The paternity exclusion probabilities varied from 0.261 for locus Aop820 to 0.806 for locus Aop450, and the parent-pair exclusion probabilities varied from 0.433 for Aop820 to 0.940 for Aop450. The cumulative probabilities of exclusion for paternity and parentage of the eight loci were both higher than 0.999, supporting the usefulness of these microsatellite loci for future paternity and parentage analyses in A. opalus. Cross-species transferability was also assayed, supporting their potential use in other eight Acer species.     

Isolation and characterization of microsatellite loci in Hexagrammos otakii based on 2b-RAD method

In this study, we isolated 20 novel microsatellites loci associated with the growth of Hexagrammos otakii (Fat greenling) by using 2b-RAD sequencing method. The characteristics of 20 microsatellite loci were amplified in 105 H. otakii individuals which came from four different groups and tested by Capillary electrophoresis. The number of allele of the 20 microsatellite loci ranged from 8 to 26 with an average of 19.95. PIC value ranged from 0.1659 to 0.9227 with an average of 0.7555. Observed and expected heterozygosity varied from 0.0667 to 0.8571 and from 0.1710 to 0.9314, respectively. Highly polymorphic characteristics were observed in each microsatellite loci of the study except HO6. Further research showed that 10 microsatellite loci had transferability in Hexagrammos agrammus. In this study, high polymorphism and genetic diversity using the 15 polymorphic microsatellite loci suggest that they are suitable for investigating the fine-scale population structure, genetic relationship and further evaluating the artificial reproduction strategy of H. otakii.     

A microsatellite-based genetic linkage map for channel catfish, Ictalurus punctatus

Microsatellite loci were identified in channel catfish gene sequences or random clones from a small insert genomic DNA library. Outbred populations of channel catfish contained an average of eight alleles per locus and an average heterozygosity of 0.70. A genetic linkage map of the channel catfish genome (N = 29) was constructed from two reference families. A total of 293 microsatellite loci were polymorphic in one or both families, with an average of 171 informative meioses per locus. Nineteen type I loci, 243 type II loci, and one EST were placed in 32 multipoint linkage groups covering 1958 cM. Nine more type II loci were contained in three two-point linkage groups covering 24.5 cM. Twenty-two type II loci remained unlinked. Multipoint linkage groups ranged in size from 11.9 to 110.5 cM with an average intermarker distance of 8.7 cM. Seven microsatellite loci were closely linked with the sex-determining locus. The microsatellite loci and genetic linkage map will increase the efficiency of selective breeding programs for channel catfish.     

Isolation and characterization of twenty-one polymorphic microsatellite loci in the Tibetan macaque (<Emphasis Type="Italic">Macaca thibetana</Emphasis>)

Twenty-one microsatellite loci were isolated from AC-enriched library of Tibetan macaque (Macaca thibetana). The number of alleles at the 21 microsatellite loci ranged from 8 to 15, with an average of 12.2 per locus. Polymorphism information content (PIC) ranged from 0.805 to 0.910 with an average of 0.873. The observed and expected heterozygosities ranged from 0.208 to 0.792 and from 0.843 to 0.938, respectively. These microsatellite loci will be useful for future studies that relate to the genetic diversity and population structure of Tibetan macaque.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.     

Microsatellite markers in peach [Prunus persica (L.) Batsch] derived from an enriched genomic and cDNA libraries

Twenty‐four and 12 microsatellite loci were developed in peach [Prunus persica (L) Batsch cv. Akatsuki] by using an enriched genomic and fruit cDNA libraries, respectively. The microsatellite loci obtained from an enriched library produced 1–9 alleles per locus, 24 in total, of which 22 showed polymorphisms. The average values of observed and expected heterozygosities among the 24 loci were 0.15 and 0.68, respectively. The microsatellite loci derived from cDNA showed 1–7 alleles per locus. Eight sequences showed significant homology to the registered genes in a database.     

Microsatellite markers for the ectomycorrhizal basidiomycete Lactarius mammosus

We report the characterization of polymorphic microsatellite markers in the ectomycorrhizal fungus Lactarius mammosus Fr. Two enrichment protocols were used to isolate microsatellite loci and polymorphism was explored within 31 sporocarps originating from a forest site in northern Sweden. We found nine variable microsatellite loci with the number of alleles per locus varying between 2 and 5, and expected heterozygosities ranging from 0 to 0.84. These loci are available for the analysis of genetic structure and gene flow in L. mammosus populations.     

Isolation and characterization of fourteen microsatellite loci for striped field mouse (<Emphasis Type="Italic">Apodemus agrarius</Emphasis>)

We describe the isolation and characterization of 14 microsatellite loci in the striped field mouse from genomic DNA-enriched libraries in this paper. The 14 new loci were tested in 24 individuals from four populations in Southwest China. These loci were highly polymorphic with numbers of alleles per locus ranging from 6 to 14 and expected heterozygosities from 0.789 to 0.925. All loci followed Hardy–Weinberg expectations except SFM4, SFM11 and SFM13 loci. No significant linkage association was found among all loci. The 14 novel polymorphic microsatellite loci will be useful in studying phylogeography and population genetics of the striped field mouse.     

Isolation and characterization of polymorphic microsatellite loci in the field vole,Microtus agrestis,and their cross‐utility in the common vole,Microtus arvalis

We developed nine polymorphic microsatellite loci for the field vole, Microtus agrestis. The number of alleles ranged from five to 15 and observed heterozygosities ranged from 0.40 to 1.00. We also tested the microsatellite loci for amplification and polymorphism in the congeneric species Microtus arvalis. Five of the nine loci were successfully analysed in this species. The microsatellite markers will be employed in studies of reproductive success and fine‐scale spatial genetic structure.