Tetranucleotide Microsatellite Markers for Molecular Testing in Thai Domestic Elephants (Elephas maximus indicus)
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Eleven microsatellite loci were evaluated for genetic profiles of Thai domestic elephants (Elephas maximus indicus) and their suitability as genetic markers for molecular testing. A total of 66 animals were tested. 10 out of 11 loci could be amplified and they demonstrated polymorphism with allelic numbers per locus ranging from 7 (LaT06) to 39 (LaT18). Values of expected heterozygosity (He) and Polymorphic Information Content (PIC) were between 0.6449 (LaT17) – 0.9593 (LaT05) and 0.5934 (LaT17) – 0.9578 (LaT05), respectively. Analysis of the ten microsatellite markers revealed Combined Exclusion Probability (CEP) of 99.99998783% or 1.2167 x 10-7 and 99.91% confident for individual verification, suggesting that using all these loci together as a set of genetic markers is an extremely powerful tool for the unique identification. In addition, this set of microsatellite markers provides a qualified system for fingerprinting purposes and parentage testing in Thai domestic elephants.Keywords:
Null allele
Microsatellites are tandem duplications with a simple motif of one to six bases as the repeat unit. Microsatellites provide an excellent opportunity for developing genetic markers of high utility because the number of repeats is highly polymorphic, and the assay to score microsatellite polymorphisms is quick and reliable because the procedure is based on the polymerase chain reaction (PCR). We have identified 404 microsatellite-containing clones of which 219 were suitable as microsatellite markers. Primers for 151 of these microsatellites were developed and used to detect polymorphisms in DNA samples extracted from the parents of two reference populations and three resource populations. Sixty, 39, 46, 49, and 61% of the microsatellites exhibited length polymorphisms in the East Lansing reference population, the Compton reference population, resource population No. 1 (developed to identify resistance genes to Marek's disease), resource population No. 2 (developed to identify genes involved in abdominal fat), and resource population No. 3 (developed to identify genes involved in production traits), respectively. The 91 microsatellites that were polymorphic in the East Lansing reference population were genotyped and 86 genetic markers were eventually mapped. In addition, 11 new random amplified polymorphic DNA (RAPD) markers and 24 new markers based on the chicken CR1 element were mapped. The addition of these markers increases the total number of markers on the East Lansing genetic map to 273, of which 243 markers are resolved into 32 linkage groups. The map coverage within linkage groups is 1,402 cM with an average spacing of 6.7 cM between loci. The utility of the genetic map is greatly enhanced by adding 86 microsatellite markers. Based on our current map, approximately 2,550 cM of the chicken genome is within 20 cM of at least one microsatellite marker.
Genetic linkage
Dinucleotide Repeat
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Microsatellite Instability
Myeloid leukaemia
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Objective To study the loss of heterozygosity(LOH) and the microsatellite instability (MSI) involved in the development of human testicular tumor. Methods 22 primary testicular tumor DNA samples were examined for loss of heterozygosity(LOH) on chromosomes 3, 5, 9, 17, 18 and X and alteration of microsatellite repeats marker by means of polymerase chain reaction, 8 microsatellite repeats markers being used for the analysis of MSI. Results LOH on chromosome 9q33 34, 5q and 18q21 were observed in 45%, 43% and 25%, respectively. Difference between unrelated microsatellites for tumor and control DNA was detected in 8 of 22(36%) . LOH and/or MSI were observed in 17 of 22(77%) cases. Two cases showed alterations on 5 microsatellite loci, three cases showed alterations on 2 microsatellite loci and three cases on 1 microsatellite locus. 5 of 22(23%) patients showed replication error(RER). Conclusions Tumor suppressor genes on chromosome 9q and 5q, and microsatellite instability might play an important role in the development of human testicular tumor.
Microsatellite Instability
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Pyrosequencing
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Eleven microsatellite loci were evaluated for genetic profiles of Thai domestic elephants (Elephas maximus indicus) and their suitability as genetic markers for molecular testing. A total of 66 animals were tested. 10 out of 11 loci could be amplified and they demonstrated polymorphism with allelic numbers per locus ranging from 7 (LaT06) to 39 (LaT18). Values of expected heterozygosity (He) and Polymorphic Information Content (PIC) were between 0.6449 (LaT17) – 0.9593 (LaT05) and 0.5934 (LaT17) – 0.9578 (LaT05), respectively. Analysis of the ten microsatellite markers revealed Combined Exclusion Probability (CEP) of 99.99998783% or 1.2167 x 10-7 and 99.91% confident for individual verification, suggesting that using all these loci together as a set of genetic markers is an extremely powerful tool for the unique identification. In addition, this set of microsatellite markers provides a qualified system for fingerprinting purposes and parentage testing in Thai domestic elephants.
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말 6개 품종 192두를 대상으로 17개의 microsatellite DNA marker를 이용하여 유전자(DNA)형을 분석하여 비교한 결과 제주마에서 각 marker별로 대립유전자의 수는 5-10개(평균 7.35개)로 분포하였고 제주마에서 관찰된 대립유전자는 총 125개가 관찰되어 평균 좌위 당 7.35개로서 몽고마의 130개(평균 7.65개)보다는 낮은 수치였다. 또한 AHT5 marker에서 대립유전자 P, ASB23 marker에서 대립유전자 Q와 R, CA425 marker에서 대립유전자 H, HMS3 marker에서 대립유전자 S, HTG10 marker에서 대립유전자 J, LEX3 marker에서 대립유전자 J 등 6개 marker에서 7개의 특이 대립유전자가 관찰되었다. 관찰된 이형접합성(observed heterozygosity)과 기대된 이형접합성(expected heterozygosity)은 각각 0.429-0.905(평균 0.703)와 0.387-0.841(평균 0.702)로 관찰되었고 다량정보량(PIC)은 0.354(HTG6)-0.816(LEX3)로서 평균 0.659로 나타났으며 17개 marker중 AHT4, AHT5, CA425, HMS2, HMS3, HTG10, LEX3, VHL20 marker 등이 다량정보량(PIC) 0.7 이상을 나타내었다. 17개 marker에 대한 전체 부권부정율(친부마 혹은 친모마 하나의 유전자형을 알고 있을 경우)을 제주마에 적용 시 99.99%로 나타났다. 말 6개 품종별로 분석하였을 때 평균 대립유전자의 수는 7.64개(몽고마)-4.23개(미니츄어 말)로 분포하였고 17개 marker 전체에서는 153개의 대립유전자가 검출되었다. 품종별로 분석한 결과 기대된 이형접합성(expected heterozygosity)과 관찰된 이형접합성(observed heterozygosity)은 각각 0.7950$\pm$ 0.0141(몽고마)-0.6751$\pm$ 0.0378(미니츄어 말), 0.7135$\pm$ 0.0180(제주경주마)-0.5621$\pm$ 0.0401(미니츄어 말)로 나타났다. 말 6개 품종을 17개 microsatellite marker로 분석한 결과 몽고마, 제주마, 제주경주마 등의 순으로 높은 유전적 다양성을 보였다. 제주마와 가장 가까운 유전적 유연 관계를 나타낸 집단은 몽고마로서 Da genetic distance에서 0.1517로 나타났고, 제주경주마와는 0.2628의 유전적 거리를 보였다. The present study was conducted to investigate the genetic characteristic and to establish the parentage verification system of the Korean native horse(KNH). A total number of 192 horses from six horse breeds including the KNH were genotyped using 17 microsatellite loci. This method consisted of multiplexing PCR procedure. The number of alleles per locus varied from 5 to 10 with a mean value of 7.35 in KNH. The expected heterozygosity and observed heterozygosity were ranged from 0.387 to 0.841(mean 0.702) and from 0.429 to 0.905(mean 0.703), respectively. The total exclusion probability of 17 microsatellite loci was 0.9999. Of the 17 markers, AHT4, AHT5, CA425, HMS2, HMS3, HTG10, LEX3 and VHL20 marker have relatively high PIC value(>0.7). This study found that there were specific alleles, P allele at AHT5, Q allele and R allele at ASB23, H allele at CA425, S allele at HMS3, J allele at HTG10 and J allele at LEX3 marker in KNH when compared with other horse populations. Also, the results showed two distinct clusters: the Korean native horse cluster(Korean native horse, Mongolian horse), and the European cluster(Jeju racing horse, Thoroughbred horse). These results present basic information for detecting the genetic markers of the KNH, and has high potential for parentage verification and individual identification of the KNH.
Molecular marker
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Marker assays based on the polymerase chain reaction (PCR) offer a far more user-friendly marker system. PCR based techniques such as RAPDs are unreliable and generally not transferable between crosses. However, microsatellites (dior tri-nucleotide repeat sequences) also known as simple sequence repeats (SSRs) are highly reliable, co-dominant markers. Microsatellites are becoming more widely used for marker assisted breeding and variety identification due to their high level of polymorphism and ease of use.
Molecular marker
Identification
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Microsatellite Instability
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Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.
Cucumis
Melon
Molecular marker
Marker-Assisted Selection
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