Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

Background

Cynomolgus macaques (Macaca fascicularis) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.

Results

We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.

Conclusion

BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.     

An initial genetic linkage map of the rhesus macaque (Macaca mulatta) genome using human microsatellite loci

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate species in biomedical research. To create new opportunities for genetic and genomic studies using rhesus monkeys, we constructed a genetic linkage map of the rhesus genome. This map consists of 241 microsatellite loci, all previously mapped in the human genome. These polymorphisms were genotyped in five pedigrees of rhesus monkeys totaling 865 animals. The resulting linkage map covers 2048 cM including all 20 rhesus autosomes, with average spacing between markers of 9.3 cM. Average heterozygosity among those markers is 0.73. This linkage map provides new comparative information concerning locus order and interlocus distances in humans and rhesus monkeys. The map will facilitate whole-genome linkage screens to locate quantitative trait loci (QTLs) that influence individual variation in phenotypic traits related to basic primate anatomy, physiology, and behavior, as well as QTLs relevant to risk factors for human disease.     

Sixteen novel microsatellite loci developed for the giant panda (Ailuropoda melanoleuca)

Sixteen novel microsatellite DNA loci were developed from the giant panda (Ailuropoda melanoleuca) using a magnetic-bead capture method. A total of 115 alleles were obtained for these markers, ranging from 4 to 12 alleles per locus (average 7.188). These loci exhibited high levels of polymorphic information content and expected heterozygosity, 0.558–0.855 (average 0.729) and 0.628–0.885 (average 0.778), respectively. Therefore, the allelic polymorphism and heterozygosity show that the giant pandas raised in China Research and Conservation Center possess abundant genetic variation. In addition, if the three markers showing null alleles were excluded, the remaining 13 microsatellite loci still presented extremely low non-exclusion probabilities of parentage (0.002), paternity (0.000) and identity (0.000). As a result, this new suit of microsatellite markers would be a very informative tool for the genetic and conservation studies of giant pandas.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

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。     

Simple sequence repeat (SSR) polymorphisms for colony management and population genetics in rhesus macaques (Macaca mulatta)

Cross-species amplification of 72 SSR (predominantly tetranucleotide) loci from the DNA of six rhesus macaques of diverse regional origins was conducted using human primers for the polymerase chain reaction (PCR). Thirteen of these primer pairs, which consistently and unambiguously amplified polymorphic fragments from these six samples and which exhibited Mendelian properties, were also used to amplify SSR loci for 176 male rhesus macaques that are founders of six different captive breeding colonies. These include four groups of macaques originating in India and one group of macaques each that originated in China and Thailand. Gene diversity based on the SSR loci provided a reliable estimate of average heterozygosity but was between two and four times higher than that for 9 protein coding loci. Based on the SSR loci, Chinese rhesus were more genetically diverse and unique than were rhesus from India or Thailand, a conclusion not consistent with data based on protein coding loci. The six most informative SSR loci are unlinked and provide probabilities of single parent exclusion and genetic identity exceeding 0.99 and one in one million, respectively, both of which are reasonable standards for colony management purposes. Am. J. Primatol. 42:199–213, 1997. © 1997 Wiley-Liss, Inc.     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Isolation and strategies of novel tetranucleotide microsatellites with polymorphisms from different chromosomes of the rhesus monkey (Macaca mulatta)

A total of 45 tetranucleotide chromosome-specific microsatellite markers with polymorphism were developed successfully based on three reference rhesus monkey genomes and on In-silico PCR prescreening. The polymorphic information content (PIC) values of 45 polymorphic microsatellite loci ranged from 0.487 to 0.879, with an average of 0.715, which were proven to be moderate to highly polymorphic. We detected 315 alleles on 45 microsatellite loci in 24 Rhesus monkeys. The number of alleles ranged from 3 to 15 and the mean number of alleles was 7 for each locus. Accordingly, the observed and expected heterozygosities obtained were between 0.417 and 1.0 and between 0.550 and 0.908, with an average value of 0.736 and 0.767, respectively. Genetic information demonstrated that 10 loci significantly deviated from Hardy–Weinberg equilibrium (P?<?0.05). All 45 primers were not significant with regard to linkage disequilibrium (P?>?0.001). Pearson correlation indicated that the PIC value exhibited a significant negative correlation with the loci number (r?=?? 0.741, P?=?0.022), whereas the positive correlation with the number of the samples (r?=?0.847, P?=?0.070) was not significant. This may be attributed to the presence of random particularities within the loci. The T test of the sample groups indicated that the PIC difference was not significant when the number of samples was set at 10 and/or?≥?15 (P?=?0.7472?~?0.8564). These polymorphic and valuable microsatellite loci will facilitate further conservation genetics studies for rhesus monkeys and can be further applied to develop novel genetic markers for other species.

     

Designing new microsatellite markers for linkage and population genetic analyses in rhesus macaques and other nonhuman primates

Identification of polymorphic microsatellite loci in nonhuman primates is useful for various biomedical and evolutionary studies of these species. Prior methods for identifying microsatellites in nonhuman primates are inefficient. We describe a new strategy for marker development that uses the available whole genome sequence for rhesus macaques. Fifty-four novel rhesus-derived microsatellites were genotyped in large pedigrees of rhesus monkeys. Linkage analysis was used to place 51 of these loci into the existing rhesus linkage map. In addition, we find that microsatellites identified this way are polymorphic in other Old World monkeys such as baboons. This approach to marker development is more efficient than previous methods and produces polymorphisms with known locations in the rhesus genome assembly. Finally, we propose a nomenclature system that can be used for rhesus-derived microsatellites genotyped in any species or for novel loci derived from the genome sequence of any nonhuman primate.     

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。     

Polymorphic microsatellite loci for population studies of <Emphasis Type="Italic">Bullacta exarata</Emphasis>

Nine polymorphic microsatellite loci were isolated and characterized from an AC-enriched genomic library of Bullacta exarata. They were polymorphic in 30 individuals from Qidong in Jiangsu Province, China. The number of alleles per polymorphic loci varied from 7 to 14 and the values of observed heterozygosity and expected heterozygosity ranged from 0.2593 to 0.9667 and from 0.7090 to 0.9164, respectively. Six of the nine microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These informative microsatellite markers will be useful in studies of population genetic structure for this species.     

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>)

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2–10), and average heterozygosity was 0.63 (range 0.10–0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.     

Toward developing a genome-wide microsatellite marker set for linkage analysis in the rhesus macaque (Macaca mulatta): identification of 76 polymorphic markers

Linkage analysis can be problematic in humans because of the lack of large, multigenerational pedigrees and the difficulties in obtaining phenotypic data on all family members. In contrast, large, captive colonies of rhesus macaque are a potentially valuable resource for linkage studies because detailed phenotypic and genealogical data are kept, inbreeding is avoided, and DNA samples can usually be obtained. Microsatellite marker sets for genome-wide screening are available in a number of species, but not for the rhesus macaque. We tested primers to 400 human microsatellite markers from a genome-wide mapping set using DNA from nine unrelated female rhesus macaques. We found that 76 (19%) of the primers amplified a polymorphic product using the standard protocols for human DNA. The average heterozygosity of the markers in humans was 0.80, compared to 0.65 in the rhesus macaques. This study provides preliminary data, which could be used toward the development of a linkage mapping set in this species. There would be a need, however, to confirm the Mendelian inheritance of the markers.     

Conserved Microsatellites in Ants Enable Population Genetic and Colony Pedigree Studies across a Wide Range of Species

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.     

Genetic variation and identification of promising sour cherries inferred from microsatellite markers

The aim of this study was to identify the group of highly polymorphic microsatellite markers for identification of promising sour cherries. From among 30 tested microsatellite (SSR) markers, 19 were selected to profile genetic variation in sour cherries due to high polymorphisms. Results indicated a high level of polymorphism of the accessions based on these markers. Totally 148 alleles were generated at 19 SSR loci which 122 alleles were polymorphic. The number of total alleles per locus ranged from 2 to 15 with an average of 7.78 and polymorphism percentage varied from 50 to 100% with an average of 78.76%. Also, PIC varied from 0.47 to 0.89 with an average of 0.79 and heterozygosity ranged from 0.35 to 0.55 with a mean of 0.45. According to these results, these markers specially PMS3, PS12A02, PceGA34, BPPCT021, EMPA004, EMPA018, and Pchgms3 produced good and various levels of amplifications and showed high heterozygosity levels. By the way, the genetic similarity showed a high diversity among the sour cherries. Cluster analysis separated improved cultivars from promising sour cherries, and the PCoA supported the cluster analysis results. Since the studied sour cherries were superior to the improved cultivars and were separated from them in most groups, these sour cherries can be considered as distinct genotypes for further evaluations in the framework of breeding programs and new cultivar identification in cherries. Results also confirmed that the set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of sour cherry genotypes.     

Polymorphic microsatellite loci for the common marmoset (Callithrix jacchus) designed using a cost- and time-efficient method

We describe a cost- and time-efficient method for designing new microsatellite markers in any species with substantial genomic DNA sequence data available. Using this technique, we report 14 new polymorphic dinucleotide microsatellite loci isolated from the common marmoset. The relative yield of new polymorphisms was higher with less labor than described in previous marmoset studies. Of 20 loci initially evaluated, 14 were polymorphic and amplified reliably (70% success rate). The number of alleles ranged from 3 to 9 with heterozygosity varying from 0.48 to 0.83.     

Characterization of nine novel microsatellite loci for the Venus clam (Cyclina sinensis)

The Venus clam, Cyclina sinensis, is one of the most important bivalves in China marine aquaculture. Using (CA)(15)-enriched genomic libraries of this species, nine novel polymorphic microsatellite loci were isolated and characterized. The mean number of observed alleles per locus was 16 (range 8-24). The observed and expected heterozygosity ranged from 0.119 to 0.872 and from 0.626 to 0.931, respectively. Three loci had significant departure from Hardy-Weinberg equilibrium and non-significant linkage disequilibrium was found among all nine loci. These highly informative microsatellite markers should be useful for population genetic analyses of C. sinensis.     

Isolation and characteristics of eight novel polymorphic microsatellite loci in Lippia alba (Verbenaceae)

? Premise of the study: A set of eight microsatellite (simple sequence repeat [SSR]) markers for Lippia alba, an important medicinal and cosmetic plant, was developed to aid studies of genetic diversity and to define efficient strategies for breeding programs. ? Methods and Results: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 11 SSR loci were developed and optimized in L. alba. Of the 11 loci, eight were found to be polymorphic after screening 61 accessions from two populations. The parameters used to characterize loci were expected heterozygosity (H(e)) and number of alleles. A total of 44 alleles were identified, with an average of 5.5 alleles per loci, which were moderately to highly informative according to H(e). ? Conclusions: These new SSR markers have potential for informing genetic diversity, allele mining, and mapping studies and will be used to generate information for breeding programs of L. alba.     

Variation of short tandem repeats within and between species belonging to the Canidae family

Frequency distribution and allele size in 20 canine microsatellite loci were analyzed in 33 flat-coated retrievers, 32 dachshunds, 10 red foxes, and 10 Arctic foxes. Overall, the major difference between the two dog breeds was the relative allele frequencies rather than the size ranges of alleles at the individual locus. The average heterozygosity within the two dog breeds was not significantly different. Since the average heterozygosity at several polymorphic loci is a relative measure of heterogeneity within the population, analysis of heterozygosity within microsatellite loci is suggested as a measure for the diversity of populations. Eighty percent (16 of 20) of the canine microsatellite primer pairs amplified corresponding loci in the two fox species. This reflects a very high sequence conservation within the Canidae family relative to findings in, for instance, the Muridae family. This indicates that it will be possible to utilize the well-characterized fox karyotype instead of the dog karyotype as a step towards physical mapping of the dog genome. Analysis of exclusion power and probabilities of genetic identity between unrelated animals by use of the seven most informative loci demonstrated that it will be possible to assemble a panel of microsatellite loci that is effective for parentage analysis in all breeds.