A search for genetic markers associated with egg production in the ostrich (Struthio camelus)

The aim of the current study was to search for genetic markers, microsatellite loci associated with laying performance in ostriches. The material consisted of two groups of ostrich hens characterized by high or low laying performance (over 75 and less than 25 eggs per season, respectively). The investigation covered 30 microsatellite loci characteristic for the ostrich (the CAU group) and led to identification of significant differences in allele and genotype frequencies between the two groups of hens considered. Out of a total of 30 microsatellite loci examined, 28 showed different alleles in relation to analyzed performance groups. In hens of high laying performance (HP group, n = 12), specific alleles occurred in 23 microsatellite loci (40 alleles of 243 identified), while in those of low egg production (LP group, n = 12), they occurred in 22 (51 alleles of 243 identified). The results indicate the usefulness of the microsatellite loci as the potential genetic markers associated with laying performance that can be applied for genetic improvement of ostrich flocks.     

Newly developed polymorphic microsatellite markers in Job's tears (Coix lacryma‐jobi L.)

A microsatellite‐enriched library of Job's tears (Coix lacryma‐jobi L. var. Ma‐yuen Stapf) was constructed using a modified biotin–streptavidin capture method. After screening, 17 polymorphic microsatellites were used for diversity analysis among 30 Job's tears accessions. The number of alleles ranged from one to five alleles per locus with an average of 2.8 alleles. Expected heterozygosity (H_E) and polymorphism information content (PIC) ranged from 0 to 0.676 and from 0 to 0.666, respectively. The newly developed microsatellite markers are expected to be very valuable tools for evaluation of genetic diversity among large germplasm collection of Job's tears present in our Korean Gene Bank.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.     

Isolation and characterization of 12 polymorphic microsatellite markers from ladyfish (Elops saurus Linnaeus)

Ladyfish (Elops saurus Linnaeus) is an economically important marine fish species. 76 microsatellite loci were isolated from an enriched genomic library of Elops saurus. Twelve of these markers were polymorphic in a test population with alleles per locus ranging from three to nine. The number of observed, expected heterozygosity and polymorphism information content (PIC) per locus in 20 individuals ranged from 0.2000 to 1.0000, 0.1897–0.8846, 0.1769–0.8476, respectively. One markers significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of markers. As a result, 12 microsatellite markers probably should provide sufficient level of genetic diversity to investigate the fine-scale population structure, stock management and enhancement, genetic linkage map construction and molecular marker-assisted breeding in Elops saurus Linnaeus.     

Studies on resources of genetic diversity in conservative flocks of geese using microsatellite DNA polymorphic markers

The studies conducted aimed at evaluating the genetic diversity within and between varieties of conservative flocks of geese, using the polymorphism of 14 microsatellite sequences. The experimental material included conservative flocks of geese the following indigenous breeds and varieties kept in Poland: Kielecka (Ki), Kartuska (Ka), Lubelska (Lu), Suvalska (Su), Rypinska (Ry), Sub-Carpathian (SC), Hunched Beak (HB) and Pomeranian (Po). Among the 14 microsatellite sequences a total of 97 microsatellite alleles were identified. The number of alleles at one locus ranged from 3 to 19. In the overall pool of 97 alleles, 26 (26.8%) were specific for individual breeds and varieties of geese. The values of the expected heterozygosity (He) for individual geese ranged from 0.38 (Sub-Carpathian) to 0.51 (HB). Similarly, the mean values for the observed heterozygosity (Ho) ranged from 0.45 (Po) to 0.55 (Ki and Su). The polymorphic information content reached the highest value of 0.80 at loci CKW21 (Ki) and TTUCG5 (Po and Su). The greatest genetic distance was observed between the HB and Ry (0.44) and between the HB and Po (0.39) varieties, while the smallest–between the Lu and Po as well as Lu and Ki (0.028) varieties. The phylogenetic tree, elaborated on the basis of the genetic distances, clearly confirms the specificity of the HB goose as compared to the remaining breeds and varieties.     

Cost‐effective development of highly polymorphic microsatellite in Japanese quail facilitated by next‐generation sequencing

Next‐generation sequencing technologies permit rapid and cost‐effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild‐derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3–4.1 and 0.11–0.53 respectively. Compared with the wild‐derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (F_ST) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.     

跨种扩增筛选大鳄龟微卫星标记及遗传多样性分析

跨种扩增是一种能够快速、有效地获得物种微卫星标记的方法。本研究利用在近缘种中已发表的微卫星DNA引物,对大鳄龟（Macroclemys temminckii）进行跨种PCR扩增,在合成的69对引物中获得8对具有多态性的微卫星位点。对PCR扩增产物进行统计,得出观测杂合度（H_o）的范围是0.041 7~0.954 5,平均为0.384 8;期望杂合度（H_E）的范围为0.041 7~0.811 8,平均为0.479 1;多态信息含量范围为0.040 0~0.759 2,平均为0.423 2;经过卡方检验后,部分微卫星位点符合哈迪-温伯格平衡。总体来说,这些位点是研究大鳄龟遗传结构的良好分子标记。     

Microsatellite markers of genetic diversity and population structure of Carica papaya

We assessed the genetic diversity of 96 papaya accessions by molecular characterisation using microsatellite markers. Fifteen polymorphic primers were selected. Accessions, which were classified as Common, Formosa and Solo according to fruit types, were evaluated for allele frequency, heterozygosity, polymorphism information content (PIC), inbreeding coefficient (f) and the genetic diversity structure. Fifteen primers amplified 68 alleles with an average of 4.53 per locus. PIC values ranged from 0.19 to 0.69. The observed heterozygosity (H_O) was low for all selected microsatellites. High f estimates (0.58) and excess of homozygotes indicated inbreeding, mainly caused by the tendency to select hermaphrodite plants for succeeding generations. Analysis of molecular variance showed that most of the variation (98%) resides within subpopulation. The genetic analysis based on Bayesian statistics proved to be sensitive enough to detect relationships among the papaya accessions, grouping them into six clusters, irrespective of their classification types.     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

Development and polymorphisms of microsatellite markers for hinoki (Chamaecyparis obtusa)

Fifteen microsatellite markers were developed from a microsatellite‐enriched library and characterized using 32 Chamaecyparis obtusa individuals. The number of alleles ranged from two to 27 per locus, with observed heterozygosity ranging from 0.281 to 0.906. The polymorphism information content (PIC) was also calculated for each marker and the average was 0.796 ± 0.024. These microsatellite markers will be useful for investigating population genetics, reproductive ecology, tree improvement and constructing linkage maps of the species.     

Characterization of Indian cattle breeds,Ongole and Deoni (<Emphasis Type="Italic">Bos indicus</Emphasis>) using microsatellite markers

Molecular characterization of cattle breeds is important for the prevention of germplasm erosion by cross breeding. The present study was carried out to characterize two Indian cattle breeds, Ongole and Deoni using microsatellite markers. Using 5 di-and 5 tri- nucleotide repeat loci, 17 Ongole and 13 Deoni unrelated individuals were studied. Of the ten loci, eight revealed polymorphism in both the breeds. The di-nucleotide repeats loci were found to be more polymorphic (100%) than tri-nucleotide repeat loci (60%). A total of 39 polymorphic alleles were obtained at 4.5 alleles per locus in Ongole and 4.1 in Deoni. The average expected heterozygosity was 0.46 (+0.1) and 0.50 (+0.1) in Ongole and Deoni breeds, respectively. The PIC values of the polymorphic loci ranged from 0.15 to 0.79 in Ongole and 0.13 to 0.80 in Deoni breeds. Six Ongole specific and three Deoni specific alleles were identified. The two breeds showed a moderate genetic relationship between themselves with a F _ST value of 0.10.     

Development of thirty new polymorphic microsatellite primers for <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Four new microsatellite primer pairs were developed in tree peony (Paeonia suffruticosa) based on the database mining and other twenty-six primer pairs by fast isolation by AFLP of sequences containing repeats (FIASCO) method. The polymorphism of each locus was further evaluated in 40 individuals of one population plus 5 tree peony related species. The number of alleles per locus ranged from 3 to 7 and the expected (H_e) and observed (H_o) heterozygosity at each locus ranged from 0.42 to 0.78 and 0.28 to 0.59, respectively. These microsatellite markers will be useful for investigating genetic diversity and studies of population genetic structure of tree peony.     

猪F2设计资源家系中4条染色体微卫星标记分析

利用中国地方猪种蓝塘猪(16头母猪)与外来品种长白猪(8头公猪)按F2设计建立资源家系, 根据美国肉畜中心(USDA-MARC 2.0)公布的猪连锁图谱, 在1、4、7和8号染色体上间隔10~20 cM选择一个微卫星标记, 共31个标记, 采用WAVEÒ核苷酸片段分析系统和ABI 377 DNA序列分析仪检测资源群体的P、F1和F2代个体微卫星的基因型, 对其基因频率、杂合度和多态信息含量等进行统计分析。结果发现: 利用ABI 377检测的猪1、4和8号染色体上的有效微卫星标记21个, 其中13个标记的18个等位基因片段大小超过了网上已报道的结果, 发现新等位基因的标记占62%; 在31个微卫星标记中, 杂合度(h)在0.043~0.7855之间, 总平均杂合度为0.6460, 其中70%座位的h>0.60; 总平均多态信息含量(PIC)为0.5949, 有77.4%位点的PIC>0.5。统计分析结果表明, 选用的微卫星标记能够较好地提供标记信息, 为进一步在该家系中进行猪重要性状的QTL定位打下了良好的基础。     

Development of microsatellite markers in black locust (Robinia pseudoacacia) using a dual‐supression‐PCR technique

Black locust (Robinia pseudoacacia) is an economically and ecologically important tree species in the world. We isolated seven polymorphic microsatellite loci from R. pseudoacacia using a dual‐suppression‐PCR technique. These loci provided microsatellite markers with high polymorphism ranging from three to 12 alleles per locus and expected heterozygosity between 0.538 and 0.944. The markers are now available for more detailed investigation of population genetic structure and pollen and seed dispersal.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

我国地方鸡品种保种群微卫星多态性分析与分子标记档案的建立

利用20个微卫星标记对国家家禽品种资源基因库中保存的11个地方鸡品种保种群进行了遗传检测,计算各群体的等位基因频率、平均基因杂合度、平均多态信息含量及各群体间的遗传距离,并用类平均法进行聚类分析。研究结果表明：20个微卫星标记在11个地方鸡品种保种群共检测到176个等位基因,平均为8.8个,基因频率分布在0.013～0.838之间。检测到等位基因中,有45个等位基因为11个地方鸡品种所共有;11个地方鸡品种平均杂合度在0.6800～0.7432之间。其中藏鸡最高,为0.7432;狼山鸡最低,为0.6800;平均多态信息含量在0.6329～0.7023之间,均大于0.5,表现为高度多态性;11 个鸡品种聚为4类。丝羽乌骨鸡、茶花鸡、仙居鸡、藏鸡、萧山鸡聚为一类,鹿苑鸡、狼山鸡聚为一类,固始鸡、北京油鸡、大骨鸡聚为一类,河南斗鸡单独聚为一类;通过利用20个微卫星基因座检测不同世代群体中等位基因及其频率、群体基因平均杂合度和多态信息含量,建立地方鸡品种保种群微卫星标记档案,并分析世代间的差异,预期可以达到监测保种效果的目的。     

长江中上游两个鲢群体遗传变异的微卫星分析

对长江中上游2个鲢群体使用39个微卫星标记进行了遗传多样性分析, 计算并统计了平均观测等位基因数、平均有效等位基因数、多态信息含量、遗传杂合度、Hardy-Weinberg平衡偏离指数、遗传相似系数、遗传距离等遗传参数。结果表明: 万州鲢和监利鲢群体所检测微卫星位点的平均观测等位基因数分别为6.128和4.974; 平均有效等位基因数分别为4.107和3.395; 多态位点百分率分别为100和94.87; 39个微卫星标记共有等位基因259个, 173个等位基因为两群体所共有; 多态微卫星位点的PIC在0.077~0.865之间变动,平均为0.617; 两群体所检测位点平均观测杂合度为0.834和0.775, 平均期望杂合度为0.713和0.623; 两个群体间的遗传相似系数为0.618, 群体间的遗传距离为0.482。结果显示长江中上游两个鲢群体间存在显著遗传分化, 应隶属于不同的种群。     

Isolation of microsatellite DNA and analysis on genetic diversity of endangered fish,Hucho taimen (Pallas)

Hucho taimen (Pallas) is an endangered species in China. To evaluate the species’ genetic diversity and population genetic structure, we isolated six polymorphic microsatellite markers from its genomic libraries by (ACA)₁₆ enriched, and characterized using 61 wild individuals. The number of alleles per locus ranged from two to seven, expected heterozygosity ranged from 0.3200 to 0.7410, polymorphism information content ranged from 0.3047 to 0.6896. These markers will be useful for the genetic variation assessment of taimen.     

Assessment of genetic diversity and chemical composition among seven black locust populations from Northern China

Black locust (Robinia pseudoacacia L.) is one of the important biomass sources used to produce bioenergy and bioethanol. In this study, we examined variations in chemical composition as well as genetic diversity and differentiation among 165 black locust plants from seven populations in five provinces (Beijing, Hebei, Gansu, Shanxi, Shandong) of Northern China using microsatellite markers(SSR). The contents of cellulose, hemicellulose, lignin varied widely among seven populations. Of the microsatellite markers analysed, 14 showed polymorphisms, and 45 alleles were identified. The polymorphism information content (PIC) ranged from 0.2885 (Rops4) to 0.6837 (Rp200), and most of the microsatellite loci had PIC values > 0.5. Expected heterozygosity (He), observed heterozygosity (Ho), and Shannon's information index (I) detected relatively high genetic variation among populations. The percentage of polymorphic loci in three populations was 100%, and the average among all populations were 95.92%. Analysis of molecular variance (AMOVA) indicated that the degree of genetic differentiation among the seven populations was low (G_ST = 0.058; N_m = 4.05), and chemical compositions had no relationship with genetic or geographic distance. This study demonstrates that microsatellite markers efficiently assess of genetic diversity and genetic differentiation in black locust populations, and all seven populations exhibit high genetic diversity. The population from Feixian has the potential to be lingo-cellulosic biomass for bioenergy and bioethanol.     

Characterization of new microsatellite loci in Pieris amamioshimensis (Ericaceae), a species nearly extinct in the wild

A set of 11 polymorphic microsatellite markers has been developed and characterized for the critically endangered species Pieris amamioshimensis. Fifty‐nine individuals of an ex‐situ population were used to identify these markers. The total number of alleles for each locus ranged from 3 to 9, with an average of 5.4. The expected heterozygosities (H_S) and observed heterozygosities (H_O) ranged from 0.47 to 0.77 and 0.22 to 0.88, respectively. In total, four loci exhibited significant deviations from Hardy–Weinberg equilibrium: two loci showed significant heterozygosity excess and the other two loci showed significant heterozygosity deficit. The polymorphism information content (0.43 ≤ PIC ≤ 0.73), the probability of exclusions (PE₁ = 0.9565, PE₂ = 0.9969 and PE₃ = 0.9999) and probabilities for identity (PI = 3.78 × 10^?9 and PI‐Sib = 2.35 × 10^?4) suggest that these markers are useful for estimating not only genetic diversity but also parentage, for the ex‐situ conservation management of populations.