Genetic diversity analysis and fingerprints of Chrysanthemum×morifolium based on SSR molecular markers / 生物工程学报

The present study aims to explore the genetic diversity of germplasm resources of Chrysanthemum×morifolium (hereinafter, C.×morifolium) at the molecular level and to establish a fingerprint database of C.×morifolium varieties. We employed 12 pairs of primers with high levels of polymorphism, clear bands, and high degrees of reproducibility to analyze the SSR molecular markers and genetic diversity of 91 C.×morifolium materials and 14 chrysanthemum- related materials. With regard to constructing the fingerprints of the tested materials, we chose 9 pairs of core primers. The findings revealed that 12 primer pairs detected 104 alleles in 105 samples, ranging from 2 to 26. The average number of observed alleles (Na) per site was 9.25. The average number of effective alleles (Ne) per site was 2.745 6, with its range being 1.276 0 to 4.742 5. Shannon genetic diversity index (I) values ranged between 0.513 3 and 2.239 9 (M=1.209 0). Nei's gene diversity index (H) ranged between 0.216 3 and 0.789 1 (M=0.578 0). The observed heterozygosity (Ho) ranged between 0.223 3 and 0.895 2 (M=0.557 5). The expected heterozygosity (He) ranged between 0.217 4 and 0.793 3 (M=0.580 8). The polymorphism information content (PIC) ranged between 0.211 5 and 0.774 0 (M=0.532 9). The genetic similarity (GS) ranged between 0.228 5 and 1.000 0 (M=0.608 3). Cluster analysis revealed that when the genetic distance (GD) equals to 0.30, the tested materials can be classified into 2 groups. When the GD equals to 0.27, the first group can be divided into 6 subgroups; accordingly, 105 tested materials can be divided into 7 subgroups. The cophenetic correlation test was carried out based on the cluster analysis, and the corresponding results showed that the cluster map correlated with the genetic similarity coefficient (r=0.952 73). According to the results of Structure population analysis, we obtained the optimal population number, with the true number of populations (K) being 3 and the population being divided concerning Q≥0.5. Three subgroups, i.e., Q1, Q2 and Q3, included 34, 33 and 28 germplasms, respectively, and the remaining 10 germplasms were identified as the mixed population. During the experiment, 9 pairs of core primers were screened among the total of 12 for a complete differentiation regarding 105 tested materials, and the fingerprints of 91 C.×morifolium materials and 14 chrysanthemum-related materials were further constructed. Overall, there were significant genetic differences and rich genetic diversity among C.×morifolium materials, which would shed light on the garden application and variety selection fields of C.×morifolium. The fingerprint database of 105 C.×morifolium varieties and chrysanthemum-related species may provide technical support for future research regarding the identification and screening system of C.×morifolium varieties.

Evaluation of Detection Efficiency for Trio Full Sibling Testing / 法医学杂志

OBJECTIVES@#To study the detection efficiency of trio full sibling with another known full sibling reference added under different number of autosomal STR typing systems.@*METHODS@#Based on 43 detection systems consisting of 13 to 55 representative autosomal STR loci, 10 000 true families (full sibling group) and 10 000 false families (unrelated individual group) were randomly simulated. The full sibling index (FSI) was calculated based on the method of family reconstruction. The cumulative sibling relationship index (CFSI) of 0.000 1 and 10 000 were used as the evaluation thresholds, and the detection efficiency parameters were calculated and compared with the identification of the duo full sibling testing.@*RESULTS@#With the increasing number of STR loci, the error rate and inability of judgement rate gradually decreased; the sensitivity, specificity, correct rate of judgment and other parameters gradually increased, and the system efficiency gradually improved. Under the same detection system, trio full sibling testing showed higher sensitivity, specificity, system efficiency and lower inability of judgement rate compared with duo full sibling testing. When the system efficiency was higher than 0.85 and inability of judgement rate was less than 0.01%, at least 20 STRs should be detected for trio full sibling testing, which was less than 29 STRs required by duo full sibling testing.@*CONCLUSIONS@#The detection efficiency of trio full sibling testing is superior to that of duo full sibling testing with the same detection system, which is an effective identification scheme for laboratories with inadequate detection systems or for materials with limited conditions.

A robust microsatellite instability detection model for unpaired colorectal cancer tissue samples / 中华医学杂志(英文版)

BACKGROUND@#Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples.@*METHODS@#From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR.@*RESULTS@#We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes ( MLH1 , MSH2 , MSH6 , and PMS2 ).@*CONCLUSION@#MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection / 法医学杂志

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

Establishment of Multiplex Amplification System of STR Loci in Felis Catus and Its Forensic Application / 法医学杂志

OBJECTIVES@#To construct a Felis catus STR loci multiplex amplification system and to evaluate its application value by testing the technical performance.@*METHODS@#The published Felis catus STR loci data were reviewed and analyzed to select the STR loci and sex identification loci that could be used for Felis catus individual identification and genetic identification. The fluorescent labeling primers were designed to construct the multiplex amplification system. The system was validated for sensitivity, accuracy, balance, stability, species specificity, tissue identity and mixture analysis, and investigated the genetic polymorphisms in 145 unrelated Felis catus samples.@*RESULTS@#Sixteen Felis catus autosomal STR loci and one sex determining region of Y (SRY) were successfully selected, and constructed a multiplex amplification system containing the above loci. The complete profile of all alleles could still be obtained when the amount of DNA template was as low as 0.25 ng. There was no specific amplification peak in other common animal samples. Population genetic surveys showed that total discrimination power (TDP) of the 16 STR loci was 1-3.57×10-20, the cumulative probability of exclusion (CPE) was 1-6.35×10-5 and the cumulative probability of matching was 3.61×10-20.@*CONCLUSIONS@#The Felis catus STR multiplex amplification system constructed in this study is highly sensitive, species-specific, and accurate in typing results, which can provide an effective solution for Felis catus species identification, individual identification and kinship identification in the field of forensic science.

GGAA-Microsatellites of NR0B1 Promoter Region in Ewing's Sarcoma Patients and Healthy Individuals of a Southern Brazilian Population / Microssatélites GGAA na Região Promotora de NR0B1 em Pacientes com Sarcoma de Ewing e Indivíduos Saudáveis de uma População no Sul do Brasil / Microsatélites GGAA en la Región Promotora de NR0B1 en Pacientes con Sarcoma de Ewing y Individuos Sanos de una Población del Sur de Brasil

Introduction: The very aggressive soft tissue and bone pediatric tumor Ewing's sarcoma (ES) is caused in most cases by the chromosomal translocation t(11;22)(q24;q12), which encodes an aberrant chimeric transcription factor (EWS-FLI1) that regulates target genes, including the critical oncogene NR0B1 (Xp21.2),via GGAA-microsatellites. Objective: Analyze the GGAA-microsatellites of NR0B1promoter region of ES patients and healthy subjects in the population investigated. Method: Ten male ES patients and 71 adult healthy males from Rio Grande do Sul state, Brazil, were included in this study. DNA from peripheral blood samples was extracted, amplified by PCR, sequenced by the Sanger method and analyzed by capillary electrophoresis. Total number of GGAA-motifs, length of microsatellite in base pairs, number of segments separated by "A" insertions, and the greatest number of consecutive GGAA-motifs were analyzed as well. Statistical analyses were performed in the SPSS statistical software and p-value <0.05 was considered significant. Results: A total of 21 different alleles was identified in the 81 subjects, with 24.2 allele [(GGAA)7A(GGAA)7A(GGAA)10] being the most frequent, but when comparing the data between the two groups, no significant difference was found. Conclusion: The sample investigated had a wide variation of microsatellite structure, including the presence of rare alleles, allowing the opportunity to describe this population as an essential step to identify genetic implications in ES tumorigenesis

Introdução: O sarcoma de Ewing (ES) é um tumor pediátrico de ossos e partes moles muito agressivo, causado, na maioria das vezes, pela translocação cromossômica t(11;22)(q24;q12), codificando um fator de transcrição quimérico aberrante (EWS-FLI1) que regula genes-alvo, incluindo o oncogene NR0B1 (Xp21.2), via microssatélites GGAA. Objetivo: Analisar os microssatélites GGAA da região promotora de NR0B1 em pacientes com ES e indivíduos saudáveis da população em investigação. Método: Foram incluídos dez pacientes do sexo masculino com diagnóstico de ES e 71 indivíduos adultos hígidos do sexo masculino do Estado do Rio Grande do Sul, Brasil. O DNA foi extraído de sangue periférico e amplificado por PCR, sequenciado pelo método de Sanger e analisado por eletroforese capilar. Foram analisados o número total de repetições GGAA, comprimento total do microssatélite em pares de bases, número de segmentos separados por inserções "A" e maior número de repetições GGAA consecutivas. As análises estatísticas foram realizadas no software estatístico SPSS e o valor de p<0,05 foi considerado significativo. Resultados: Um total de 21 alelos diferentes foi identificado nos 81 indivíduos, com o alelo 24,2 [(GGAA)7A(GGAA)7A(GGAA)10], sendo o mais frequente; mas, ao comparar os dados entre os dois grupos, nenhuma diferença significativa foi encontrada. Conclusão: A amostra estudada é altamente variável em termos de estrutura de microssatélites, incluindo a presença de alelos raros, dando a oportunidade de descrever essa população, o que é uma etapa fundamental na identificação de implicações genéticas na tumorigênese do ES

Introducción: El sarcoma de Ewing (ES) es un tumor pediátrico de huesos y tejidos blandos muy agresivo, que se presenta con mayor frecuencia por translocación cromosómica t(11;22)(q24;q12), que codifica un factor de transcripción quimérico aberrante (EWS-FLI1) que regula los genes diana, incluido el oncogén NR0B1 (Xp21.2), a través de microsatélites GGAA. Objetivo: Analizar los microsatélites GGAA de la región promotora de NR0B1en pacientes con ES y personas sanas de la población investigada. Método: Este estudio incluyó a diez pacientes varones con diagnóstico de ES y 71 varones adultos del estado de Rio Grande do Sul, Brasil. El ADN se extrajo de sangre periférica y se amplificó por PCR, secuenciado por el método de Sanger y analizado por electroforesis capilar. El número total de repeticiones GGAA, longitud total de microsatélites en pares de bases, número de segmentos separados por inserciones "A" y el mayor número de repeticiones GGAA consecutivas fueran analizados. Los análisis estadísticos se realizaron con el software estadístico SPSS y se consideró significativo un valor de p<0,05. Resultados: Se identificaron un total de 21 alelos diferentes en los 81, siendo el alelo 24,2 [(GGAA)7A(GGAA)7A(GGAA)10] el más frecuente, pero al comparar los datos entre los dos grupos, no hubo diferencia estadísticamente significativa. Conclusión: La muestra estudiada es muy variable en cuanto a estructura de microsatélites, incluyendo la presencia de alelos raros, lo que nos permite la oportunidad de describir la población estudiada, lo cual es un paso fundamental en la identificación de implicaciones genéticas en la tumorigénesis de ES

Genetic polymorphism of 23 autosomal STR loci in Han population from Yuncheng, Shanxi Province / 中南大学学报(医学版)

OBJECTIVES@#Due to the genetic feature of high diversity than other DNA markers, short tandem repeat (STR) plays key roles in forensic, anthropology, and population genetics. Newly introduced multiple STR kit is more valuable because of the greatly improved discriminatory power with the increase in the number of STR loci. The genetic polymorphic data are essential for the application and research in specific population. This study aims to investigate the genetic polymorphism of Han population residing in Yuncheng district, Shanxi Province, to evaluate the application of 23 STR loci in forensic personal identification and paternity test, and to explore the genetic relationship of Han population between Yuncheng and other populations.@*METHODS@#A total of 23 STR loci were amplified from 525 healthy unrelated individuals from the Han nationality in Yuncheng, Shanxi Province using the AGCU EX25 amplification kit. The products were detected and separated by ABI 3500 Genetic Analyzer. Alleles were genotyped by GeneMapper ID (Version 3.2) software, and corresponding frequencies and forensic parameters were calculated. We calculated the genetic distance and plotted the neighboring-joining tree with other 13 population.@*RESULTS@#The allele frequency of the 23 STRs ranged from 0.0010 to 0.5090. No deviation from Hardy-Weinberg equilibrium (@*CONCLUSIONS@#These 23 STRs are highly genetic polymorphic and informative in the Han population of Yuncheng, Shanxi Province, which can provide basic data for forensic personal identification, paternity testing, and population genetic research.

Genetic polymorphisms of 21 non-combined of DNA index system short tandem repeat loci in Hainan Li population / 中华医学遗传学杂志

OBJECTIVE@#To investigate the genetic polymorphisms of 21 non-combined DNA index system short tandem repeat (STR) loci in Hainan Li population.@*METHODS@#DNA samples from 339 unrelated healthy individuals of Li population from Hainan Province were extracted and amplified with fluorescence labeled multiplex PCR system. PCR products were electrophoresed on an ABI3130 Genetic Analyzer following the manufacturer's instructions. Allele designation was performed with a GeneMapper ID-X by comparison with the allele ladder provided by the corresponding kit.@*RESULTS@#A total of 173 alleles and 489 genotypes were observed for the 21 STR loci, respectively. The frequencies of alleles and genotypes were 0.0010-0.5434 and 0.0020-0.3274, respectively. The heterozygosity varied from 0.639 to 0.833. Discrimination power (DP) was 0.803-0.948, power of exclusion for trio-paternity was 0.416-0.584, power of exclusion for duo-paternity was 0.140-0.238, the polymorphism information content(PIC) was 0.57-0.81, respectively. The total discrimination power (TDP), cumulative probability of exclusion for trio-paternity testing(CPE-trio) and cumulative probability of exclusion for duo-paternity testing (CPE-duo) were 0.999 999 999 999 99, 0.999 999 883 211 752, and 0.987 266, respectively.@*CONCLUSION@#The 21 STR loci are highly polymorphic and informative in the studied population and can be employed as supplementary loci in duo-paternity testing or cases with variant circumstances.

Microsatellite loci development for three catfish species from northwestern South America

The Neotropical catfish species Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus are important fishery resources in Colombia that show historical declines in their capture. This study used next-generation sequencing with 454 FLX technology (Roche Applied Science) and bioinformatics analysis to develop between 18 and 24 microsatellite loci for these species. The novel microsatellite loci showed high values of polymorphic information content -PIC (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876), and the average number of alleles/locus ranged from 7-15 for A. pardalis, 9-30 for P. grosskopfii and 5-14 for S. cuspicaudus. The average observed and expected heterozygosities were respectively, 0.757 ± 0.035 and 0.834 ± 0.015 for A. pardalis; 0.596 ± 0.040 and 0.881 ± 0.009 for P. grosskopfii; and 0.747 ± 0.031 and 0.757 ± 0.025 for S. cuspicaudus. For future studies, these loci can be useful to estimate the genetic diversity and population structure in these three Neotropical catfishes.(AU)

Las especies de bagres neotropicales Ageneiosus pardalis, Pimelodus grosskopfii y Sorubim cuspicaudus, son importantes recursos pesqueros en Colombia y han mostrado disminuciones históricas en sus capturas. En este estudio se empleó la secuenciación genómica de próxima generación y análisis bioinformático para desarrollar entre 18 y 24 loci microsatélites para estas especies. Los loci microsatélites mostraron altos valores del contenido de información polimórfica CIP (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876) y el número promedio de alelos/locus mostró un rango de 7-15 para A. pardalis, 9-30 para P. grosskopfii y 5-14 para S. cuspicaudus. Los valores promedio de heterocigosidad observada y esperada fueron respectivamente 0.757 ± 0.035 y 0.834 ± 0.015 para A. pardalis; 0.596 ± 0.040 y 0.881 ± 0.009 para P. grosskopfii; y 0.747 ± 0.031y 0.757 ± 0.025 para S. cuspicaudus. Los loci microsatélites desarrollados en este trabajo pueden ser útiles para estimar la diversidad genética y la estructura poblacional de estos tres bagres neotropicales en estudios futuros.(AU)

Electric eels galore: microsatellite markers for population studies

Fourteen novel microsatellite loci are described and characterized in two species of electric eels, Electrophorus variiand E. voltaifrom floodplains and rivers of the Amazon rainforest. These loci are polymorphic, highly informative, and have the capacity to detect reliable levels of genetic diversity. Likewise, the high combined probability of paternity exclusion value and low combined probability of genetic identity value obtained demonstrate that the new set of loci displays suitability for paternity studies on electric eels. In addition, the cross-amplification of electric eel species implies that it may also be useful in the study of the closely related E. electricus, and to other Neotropical electric fishes (Gymnotiformes) species as tested herein.(AU)

Catorze novos loci microsatélites são descritos e caracterizados em duas espécies de poraquês, Electrophorus varii e E. voltai de planícies alagadas e rios da floresta amazônica. Esses loci são polimórficos, altamente informativos e têm a capacidade de detectar níveis confiáveis de diversidade genética. Da mesma forma, o alto valor de exclusão de paternidade combinado com a baixa probabilidade de identidade genética demonstra que o novo conjunto de loci exibe adequação para estudos de paternidade em poraquês. Além disso, a amplificação cruzada de espécies de peixes elétricos implica que também pode ser útil no estudo da espécie intimamente relacionada E. electricus, e de outras espécies de peixes elétricos neotropicais (Gymnotiformes).(AU)

Development and characterization of nuclear microsatellite markers for Eremanthus erythropappus and their transferability across related species

BACKGROUND: We developed simple sequence repeats (SSR) for Eremanthus erythropappus (DC.) MacLeish, an endangered tree species endemic to the Brazilian Savanna and Atlantic Forest biomes, and tested their transferability to two closely related Eremanthus species. RESULTS: Using a genomic library enriched with tandem repeat motifs, we identified 16 primer pairs, and characterized them in two populations. Nine primers amplified the expected size fragments and seven SSRs were polymorphic, providing a total of 38 alleles and an average of 4.22 alleles per marker. The polymorphic information content (PIC) ranged from 0.44 to 0.94 with an average of 0.65. The average observed heterozygosity across all loci varied from 0.61 to 1.00. The observed ( HO ) and expected ( HE ) heterozygosity within the two populations varied from 0.65 to 1.00 and from 0.31 to 1.00, respectively. CONCLUSIONS: These newly developed SSR markers are a powerful tool for population genetic analyses and may be useful in studies on species ecology, evolution, and taxonomy.

Variabilidade genética de 12 loci de microssatélites em galinhas crioulas Canela-Preta / Genetic variability of twelve microsatellite loci in native Canela-Preta chickens

Esta pesquisa foi realizada com o objetivo de se conhecer a variabilidade genética de 12 loci de microssatélites em galinhas crioulas Canela-Preta. Foram coletadas amostras de sangue de 118 galinhas crioulas Canela-Preta, provenientes de três municípios do estado do Piauí (Oeiras, Queimada Nova e Teresina). Após extração do DNA, foram utilizados marcadores para 12 loci de microssatélites: LEI0192, LEI0209, LEI0212, LEI0217, LEI0221, LEI0234, LEI0237, LEI0248, LEI0258, MCW0081, MCW0183 e MCW0213, que foram amplificados pela técnica de reação em cadeia da polimerase (PCR). Foram obtidos 408 alelos (somando os alelos dos 12 loci), com os fragmentos variando entre 50 e 460 pares de bases. O número de alelos variou de 15 (MCW0081) a 52 (LEI0212), com média de 31,5 alelos por locus. A média de heterozigosidade esperada e o conteúdo de informações polimórficas foram, respectivamente, 0,887 e 0,909. Foram observados desvios no equilíbrio de Hardy-Weinberg e valores positivos do índice de fixação com excesso de homozigotos. Os microssatélites utilizados mostraram-se polimórficos e podem ser usados para investigações genéticas em galinhas Canela-Preta. As galinhas dos plantéis avaliados apresentam grande variabilidade gênica, o que as qualifica como importante fonte de recursos genéticos e, consequentemente, faculta a utilização delas em programas de melhoramento genético animal.(AU)

The aim of this study was to analyze the genetic variability of twelve microsatellite loci in native Canela-Preta chickens. Blood samples were collected from 118 chickens of the breed from five properties in three cities (Oeiras, Queimada Nova and Teresina) of Piauí state. After the DNA extraction, markers were used for twelve microsatellite loci: LEI0192, LEI0209, LEI0212, LEI0217, LEI0221, LEI0234, LEI0237, LEI0248, LEI0258, MCW0081, MCW0183, and MCW0213 that were amplified by polymerase chain reaction technique (PCR). The results showed a total of 408 alleles (adding alleles from the 12 loci) with the fragments ranging between 50 and 460 base pairs, the number of alleles ranged from 15 (MCW0081) to 52 (LEI0212) with an average of 31,5 alleles per locus. The average expected heterozygosity and PIC were, respectively, 0.887 and 0.909. Deviations were observed in the Hardy-Weinberg equilibrium and positive values of the fixation index with excess of homozygotes. It is concluded that the used microsatellites are polymorphic and can, therefore, be used for genetic research in Canela-Preta chickens. The birds of the analyzed cores present great genetic variability, which qualifies them as an important source of genetic resources, which could be used for future animal breeding programs.(AU)

Application of miniSTR Loci and Its Detection System for Degraded Materials in Forensic Medicine / 法医学杂志

OBJECTIVES@#To establish multiplex system of 16 miniSTR loci, and explore its application value for the degraded materials in forensic medicine.@*METHODS@#The multiplex system of 16 miniSTR loci was established using a six-dye fluorescence labeling technology and its application value in forensic medicine was assessed.@*RESULTS@#A six-dye fluorescence labeling miniSTR amplification kit was developed, which enabled 15 autosomal STR loci, Amelogenin locus and DYS391 to be typed simultaneously. This method showed good specificity and could provide stable and accurate typing results with a sensitivity of 50 pg. This system also provided a good test result for the normal biological sample of actual cases.@*CONCLUSIONS@#The multiplex system of 16 miniSTR loci has application value for degraded and trace materials with the advantages of high sensitivity and database compatibility, which can be used for forensic casework.

Development and characterization of microsatellite markers in Gaultheria pumila Lf. (Ericaceae)

BACKGROUND: Polymorphic microsatellite markers were developed for Gaultheria pumila (Ericaceae) to evaluate genetic diversity and population structure within its native range in Chile. This is a very important Ericaceae endemic to Chile with a large commercial potential. Its resistance to different abiotic conditions makes it a valuable target for genetic improvement. RESULTS: Ten polymorphic simple sequence repeat (SSR) loci were isolated from Gaultheria pumila using new-generation 454 FLX Titanium pyrosequencing technology. The mean number of alleles per locus ranged from 2 to 4. Observed and expected heterozygosity ranged from 0.00 to 1.0 and 0.00 to 0.64, respectively. CONCLUSIONS: From 10 SSR markers developed for G. pumila, 9 markers are promising candidates for analyzing genetic variation within or between natural populations of G. pumila and other species from the same genus.

Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

Background: Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq™ 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac. Results: A total of 3168 microsatellite loci were identified in 39,078 unigenes, and 1977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters. Conclusions: The present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.

Individual Identification of Cartilage by Direct Amplification in Mass Disasters / 法医学杂志

OBJECTIVES@#To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification.@*METHODS@#Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method.@*RESULTS@#In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent.@*CONCLUSIONS@#Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters.

Identification and genetic diversity analysis of Memecylon species using ISSR, RAPD and Gene-Based DNA barcoding tools

Background: Memecylon species are commonly used in Indian ethnomedical practices. The accurate identification is vital to enhance the drug's efficacy and biosafety. In the present study, PCR based techniques like RAPD, ISSR and DNA barcoding regions, such as 5s, psbA-trnH, rpoC1, ndh and atpF-atpH, were used to authenticate and analyze the diversity of five Memecylon species collected from Western Ghats of India. Results: Phylogenetic analysis clearly distinguished Memecylon malabaricum from Memecylon wightii and Memecylon umbellatum from Memecylon edule and clades formed are in accordance with morphological keys. In the RAPD and ISSR analyses, 27 accessions representing five Memecylon species were distinctly separated into three different clades. M. malabaricum and M. wightii grouped together and M. umbellatum, M. edule and Memecylon talbotianum grouped in the same clade with high Jaccard dissimilarity coefficient and bootstrap support between each node, indicating that these grouped species are phylogenetically similar. Conclusion: Data from the present study reveals that chloroplast psbA-trnH region could be used as a potential candidate region for identifying Memecylon species, and ISSR marker system could be used for estimating genetic diversity since it has high percent polymorphism compared to RAPD marker.

Population Difference Analysis of Allele Frequencies of 24 Y-STR Loci / 法医学杂志

OBJECTIVES@#To investigate the population genetic polymorphisms of 24 Y-STR loci in unrelated individuals in Eastern Chinese Han population, and to compare the difference of Han group between Eastern China and Guangdong.@*METHODS@#The population genetics of 24 Y-STR loci in 268 unrelated Han individuals from Eastern China were analyzed by GFS 24 Y-STR amplification kit. The allele frequencies in Eastern Chinese Han population were compared with the data in Guangdong Han population, and the difference analysis between two groups was performed.@*RESULTS@#Among the 24 Y-STR loci of 268 unrelated Han individuals from Eastern China, 235 alleles and 267 haplotypes were observed. GD value ranged from 0.564 9 to 0.966 8. The difference between 12 loci （DYS622, DYS552, DYS443, et al.） of Han population in Eastern China and in Guangdong was statistically significance.@*CONCLUSIONS@#GFS 24Y STR amplification system shows favorable polymorphisms, which can be used in patrilineal genetic relationship identification.

Construction of a 15-plex Rapid STR Multiplex Amplification System / 法医学杂志

OBJECTIVE@#To establish a 15-plex rapid STR multiplex amplification system.@*METHODS@#Fourteen auto-chromosome loci and one sex-chromosome were selected to compare the situations of allelic losses and nonspecific amplication under different conditions. FastStart Taq DNA polymerase and DNA standard sample 9947A were used during amplification and optimization process.15-plex rapid STR amplification system was achieved by performing various experiments including selection of amplification conditions and the volume of DNA polymerase, adjustment of inter-locus balance, optimization of rapid amplification, screening of reaction buffers, selection of reaction volume, and a variety of additives.@*RESULTS@#Using 10 μL rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10xFastStart high fidelity reaction buffer, a complete and well-balance DNA profile of 15 STR loci for standard genomic DNA was obtained in 32 minutes, without the allele drop-out and non-specific amplicons. Meanwhile, 5% glycerinum, 0.01% gelatin, 0.05% gelatin and 5 mmol/L ammonium sulfate could be used as the reactive additive during the amplification procedure.@*CONCLUSION@#The 15-plex rapid STR multiplex amplification system can be used to decrease reaction time and enhance sample throughput.

Genetic structuring of Salminus hilarii Valenciennes, 1850 (Teleostei: Characiformes) in the rio Paraná basin as revealed by microsatellite and mitochondrial DNA markers

Genetic variation of Salminus hilarii was assessed by screening microsatellite loci and mitochondrial D-loop DNA across four sampling in the upper rio Paraná basin of Brazil. Genetic diversity - measured as mean expected heterozygosity (0.904) and mean number of alleles across populations (13.7) - was reasonably high. Differentiation of microsatellite allele frequencies among populations was shown to be low but significant by AMOVA Φ ST (0.0192), and high by D EST (0.185). D-loop variation was high, with haplotypic diversity of 0.950 and nucleotide diversity of 0.011. Mitochondrial DNA-based estimates for population differentiation were high, with an overall Φ ST of 0.173. The results of tests of nuclear and mitochondrial variation yielded no unequivocal inference of historical demographic bottleneck or expansion. Genetic differentiation observed among S. hilarii populations in the rio Grande may be caused by a combination of historical differentiation and recent gene-flow disruption caused by the dams followed by reproduction of isolated spawning assemblages in mid-sized tributaries of the respective reservoirs. We present spatially more intensive sampling of S. hilariipopulations across the rio Paraná basin in order to more effectively distinguish between historical and contemporary differentiation.

A variabilidade genética de Salminus hilarii foi avaliada por lócus microssatélites e sequências D-Loop do DNA mitocondrial em quatro populações da região da bacia do Alto Paraná. A diversidade genética - medida pela heterozigosidade média (0,904) e número de alelos médios das populações (13,7) - foi razoavelmente alta. A diferenciação das frequências alélicas entre as populações foi baixa, mas significativa pela AMOVA Φ ST (0,0192), e alta pelo D EST (0,185). A variação mitocondrial foi alta com uma diversidade haplotípica de 0,950 e uma diversidade nucleotídica de 0,011. Estimativas de diferenciação populacional baseadas no DNA mitocondrial foram altas, com um valor global de Φ ST de 0,173. Os resultados dos testes da variação nuclear e mitocondrial demonstram nenhuma inequívoca inferência histórica de contração e expansão demográfica. A diferenciação genética observada entre as populações de S. hilarii no rio Grande pode ter sido causada pela combinação de diferenciação histórica e interrupção recente do fluxo gênico causada pela construção de barragens seguida por um isolamento reprodutivo de populações em tributários de médio porte dos respectivos reservatórios. Nós apresentamos uma amostragem mais ampla e intensiva de populações de S. hilarii ao longo da bacia do alto rio Paraná para se efetivamente distinguir se a diferenciação genética das populações encontrada é histórica ou contemporânea.