Parentage Identification Research Articles

Assemble and comparative analysis of the mitochondrial genome of Rhododendron delavayi: Insights into phylogenetic relationships and genomic variations

Rhododendron delavayi, a notable ornamental plant primarily found in regions of China like Yunnan and Guizhou provinces, holds substantial horticultural value. To elucidate the systematic phylogenetic relationships and organelle genomic differences within R. delavayi and related Rhododendron species, we conducted sequencing and assembly of the complete mitochondrial genome of R. delavayi. The full-length mitochondrial genome of it was a singular circular molecule spanning 1,009,263 bp, comprising 53 protein-coding genes, including 18 transfer RNA (tRNA) genes, 3 ribosomal RNA (rRNA) genes, and 32 protein-coding genes. A total of 1,182 simple sequence repeats (SSRs) loci were identified in the R. delavayi mitochondrial genome, primarily consisting of single nucleotide, dinucleotide, and trinucleotide repeats. Nucleotide diversity analysis highlighted five genes (atp6, atp9, cox2, nad1, and rpl10) with the highest diversity within the mitochondrial genomes of Rhododendron genus. Comparative analysis of the mitochondrial genome of R. delavayi with those of four other Rhododendron species indicated complex rearrangements in 21 genes, including rps4, nad6, rps3, atp6, cob, atp9, nad7, among others. The mitochondrial phylogenetic tree revealed a close relationship between R. delavayi and R. decorum, forming a sister clade to R. × pulchrum and R. simsii. Furthermore, 126 plastid-to-mitochondrial gene transfers in R. delavayi were identified, ranging from 30 bp to 19,385 bp. These fragments collectively constituted 47.54 % and 9.52 % of the chloroplast and mitochondrial genomes (202,169 bp), respectively. Complex mitochondrial-to-mitochondrial transfers were also observed, with 843 identified fragments totaling 312,036 bp (30.92 % of the mitochondrial genome). Segments exceeding 10 kb may mediate homologous recombination within the mitochondrial molecules. Remarkably, our study underscores that the mitochondrial genome of R. delavayi was the largest reported within the Rhododendron genus to date. The intricate rearrangements observed in the mitochondrial genomes of Rhododendron species, alone with the identification of five potential molecular marker sites, provided valuable insights for species classification and parentage identification within the Rhododendron genus.

The development of novel genome-SSRs, multiplex PCR panels, and allelic ladders for parentage identification in Tachypleus tridentatus

Tachypleus tridentatus, one of the oldest animal species on Earth, with significant biological and ecological value, its population sizes have declined due to overfishing. Stock enhancement technology has been used to protect T. tridentatus, however, its efficiency needs to be addressed. In the present study, a parentage identification system was developed. We identified 235,336 SSRs and 182,394 primer pairs were designed. Out of 321 synthesized primers, 14 highly polymorphic primers were selected to reveal the genetic diversity of 110 T. tridentatus. SSRs were amplified with the 14 developed primers, which were fluorescently labeled, and the resulting PCR products were analyzed using the ABI DNA Analyzer 3730xl. The number of alleles per locus, the polymorphism information content, the observed heterozygosity, and the expected heterozygosity ranged from 7 to 48, 0.581 to 0.963, 0.636 to 0.909, and 0.645 to 0.969, respectively. The cumulative exclusion probability with both unknown parents, with only unknown parents, or with both known parents was >99.9999% for the 14 loci. Furthermore, to facilitate data exchange across laboratories and platforms, multiplex PCR detection, SSR fragment size ladders, and parentage identification were employed. Out of the 14 primers, 6 optimal primers were selected for multiplex PCR validation, and two sets of triplex systems were established. Three thousand clones for 160 alleles of the six primers were selected for plasmid extraction and sequencing, and 600 correct plasmids were obtained for new T. tridentatus SSR fragment size ladder construction. Two pairs of parents with 12 offspring each were used for parentage identification by the multiplex PCR systems and ladders, the cumulative average exclusion probability of these 6 primers reached 99.9999% among parents and their offsprings, and was 0% in nonparent-offspring group. The parentage identification system constructed in this study can serve as a tool for monitoring the survival rates of T. tridentatus in stock enhancement programs.

Establishment of Parentage Identification Method for Sea Urchin Strongylocentrotus intermedius Based on SSR-seq Technology.

To establish a parentage identification method for Strongylocentrotus intermedius, 15 microsatellite loci and simple sequence repeat sequencing (SSR-seq) technology were used to perform SSR sequencing and typing of the validation population with known pedigree information and the simulation population. Cervus v3.0 was used for gene frequency statistics, simulated analysis, and parentage identification analysis. The results showed that, in validation population, using 15 microsatellite loci, the highest success rate of parent pairs identification was 86%, the highest success rate of female parent identification was 93%, and the highest success rate of male parent identification was 90%. The simulated population was analyzed using 12-15 loci, and the identification rate was up to 90%. In cases where accurate parentage was not achieved, individuals could exhibit genetic similarities with 1-3 male or female parents. Individuals identified as lacking a genetic relationship can be selected as parents to prevent inbreeding. This study shows that parent pairs or single parents of most offspring can be identified successfully using these 15 selected loci. The results lay a foundation for the establishment of a parentage identification method for S. intermedius.

Microsatellite-based Parentage Assignment in Tiger Trout (Salmo trutta × Salvelinus fontinalis)

Hybridization plays a crucial role in fish species, often resulting in unique traits and enhanced performance. The present study focuses on the genetic analysis of tiger trout, a sterile hybrid species resulting from the cross between brown trout, Black Sea strain (Salmo trutta), and brook trout (Salvelinus fontinalis), to gain insights into parentage identification, species differentiation, genetic diversity, and population structure. Parentage assessment analyses were performed by employing two software programs, COLONY and CERVUS. Results indicatest, varying levels of accuracy, with COLONY exhibiting better performance for hybrid specimens, and CERVUS performing well with intraspecific offspring. The results highlight challenges in differentiating hybrids from parental species based on the selected microsatellites. Our analysis revealed the presence of allele deletion in hybrids, affecting their genetic structure and clustering patterns. In conclusion, the present work contributes significantly to the field by emphasizing the critical need for the development of a more precise and comprehensive array of genetic markers; in particular İn species in which hybrids and parental species may potentally co-occur. The findings of this study offer promise for enhancing breeding programs and managing aquatic ecosystems, providing insights into the genetic complexities of hybrid species.

Identification of the efficacy of parentage testing based on bi-allelic autosomal single nucleotide polymorphism markers in Taiwanese population.

Parentage testing is crucial for forensic DNA analysis, using short tandem repeats (STRs). Single nucleotide polymorphisms (SNPs) with high minor allele frequency (MAF) are promising for human identification. This study aimed to develop SNP markers for parentage testing in the Taiwanese population and compare their accuracy with STRs. The TPMv1 SNP microarray (714,457 SNPs) was used to screen 180,000 Taiwanese individuals and analyze the SNP data using PLINK. After quality control, allelic distribution, and MAF considerations, a set of SNPs with significant inheritance information was selected. Parentage testing was conducted on 355 single parent-child pairs using both STRs and SNPs, employing three kinship algorithms: identity by descent, kinship-based inference for genome-wide association studies, and the combined paternity index/probability of paternity (CPI/PP). An Affymetrix signature probe for kinship testing (ASP) was also used. Based on the quality control and selection criteria, 176 SNPs with MAF > 0.4995 were selected from the Taiwanese population. The CPI/PP results calculated using SNPs were consistent with the STR results. The accuracy of the SNPs used in the single-parent-child parentage testing was > 99.99%. The set of 176 SNPs had a higher identification rate in the single parent-child parentage test than in the ASP. The CPI/PP value calculated using 176 SNPs was also more accurate than that calculated using ASP. Our findings suggest that these 176 SNPs could be used for single-parent-child parentage identification in the Taiwanese population.

Efficiency evaluation of common forensic genetic markers for parentage identification involving close relatives

To explore the efficacy of commonly used forensic identification panels in complex paternity testing of trios that involved close relatives, we wrote a code by R to generate 10,000 pedigrees at 20 CODIS STR, 21 non-CODIS STR and 30 InDel loci in Chinese five ethnic groups based on their allele frequencies. Parentage identification index——cumulative paternity index (CPI) value was set as output and was further analyzed to evaluate the performance of the aforementioned panels in complex paternity testing when the alleged parent is a random individual, biological parent, grandparent, sibling of biological parent, half-sibling of biological parent, etc. The results showed that the false inclusion of parent sibling posed as parent demonstrated no statistically significant difference from that of grandparent posed as parent. The scenarios where both biological parent and alleged parent were consanguineous to the other parent were also simulated. The results revealed that the complexity of paternity testing would raise when biological parents were consanguineous and the alleged parent was a close relative of theirs. Despite the values of non-conformity number could vary in different genetic relationships, populations and panels, 20 CODIS STRs and 21 non-CODIS STRs performed satisfactorily in most simulated scenarios. However, the joint use of 20 CODIS STRs and 21 non-CODIS STRs is more recommendable when resolving the paternity testing of the incest mating case. Overall, the current study could be regarded as a worthwhile reference in complex paternity testing of trios that involved close relatives.

Isolation of tetrameric microsatellite markers and its application on parentage identification in Procambarus clarkii

Isolation of tetrameric microsatellite markers and its application on parentage identification in Procambarus clarkii

From Wisent to the Lab and Back Again—A Complex SNP Set for Population Management as an Effective Tool in European Bison Conservation

Proper management and genetic monitoring of the modern European bison (Bison bonasus) population is one of the most important responsibilities for this species’ conservation. Up-to-date, complex genetic analysis performed using a consistent molecular method is needed for population management as a tool to further validate and maintain the genetic diversity of the species. The identification of the genetic line when pedigree data are missing, as well as the identification of parentage and individuals, are crucial for this purpose. The aim of our research was to create a small but informative panel of SNP (single-nucleotide polymorphism) markers that can be used for routine genotyping of the European bison at low cost. In our study, we used a custom-designed microarray to genotype a large number of European bison, totaling 455 samples from two genetic lines. The results of this analysis allowed us to select highly informative markers. In this paper, we present an effective single nucleotide polymorphism set, divided into separate panels to perform genetic analyses of European bison, which is needed for population monitoring and management. We proposed a total of 20 SNPs to detect hybridization with Bos taurus and Bison bison, a panel of 50 SNPs for individuals and parentage identification, as well as a panel of 30 SNPs for assessing membership of the genetic line. These panels can be used together or independently depending on the research goal and can be applied using various methods.

Developing microsatellite duplex PCR reactions for sterlet (Acipenser ruthenus) and their application in parentage identification

The sterlet (Acipenser ruthenus) is one of the 27 sturgeon species and is well-known for its wide distribution and small body size in comparison to other sturgeons. For assessing the population genetics and parentage identification of sterlet, ten microsatellites developed for Chinese sturgeon and cross-amplified in sterlet were tested by 40 individuals of sterlet. The ten microsatellites were developed using transcriptome sequencing of Chinese sturgeon. The expected heterozygosity (HE), observed heterozygosity (HO), Shannon-Weiner diversity indices (H′) and polymorphic information content (PIC) of the 10 microsatellites ranged from 0.466 to 0.751, from 0.438 to 0.938, from 0.66 to 1.51 and from 0.368 to 0.716, respectively. Combined exclusion probability based on the genotype of pair parent known (CE-PP), one parent known (CE-2P), and no parent known (CE-1P) of the 10 microsatellites were 99.99%, 99.96%, and 99.49%, respectively. These result showed that the 10 microsatellites should be helpful for assessing the population genetics and parentage identification of sterlet.

The Identification of Legal Parentage in International Surrogacy

The Identification of Legal Parentage in International Surrogacy

Microsatellite Techniques in Guamandrsquo;s Specific-Pathogen-Free Penaeus vannamei Stock: Genetic Variance and Parentage Identification

A microsatellite DNA marker technique was used to monitor specific-pathogen-free Penaeus vannamei (white shrimp) stock in the hatchery of the University of Guam, mainly for assessment of genetic diversity and identification of the parentage for two consecutive generations. A panel of 16 loci was selected to analyze 36 families of P. vannamei, comprising of a total of 1,152 individual shrimp samples. The families showed high genetic variation. The average number of alleles per locus was 10.625 for the parents and 10.052 for their offspring. The average observed heterozygosity declined slightly from 0.891 in the parents to 0.813 in the offspring. Similarly the expected heterozygosity was 0.804 among the parents and 0.792 for their progeny. The inbreeding coefficient was -0.107 for the parents but -0.026 for the offspring. These two generations did not differ significantly in any foregoing measures (p>0.05). CERVUS and COLONY confirmed that any 12 loci from the panel could deliver 100% correct parentage identification when the genotyping error rate was set as 0.01.

Parentage assignment using microsatellite DNA typing for the endangered numbat (Myrmecobius fasciatus)

The numbat (Myrmecobius fasciatus) is an endangered and peculiar marsupial with a diet that consists almost exclusively of termites. This study developed a parentage-testing system for numbats using microsatellite markers. Nineteen loci detected 143 alleles, with 4–13 alleles/locus and average expected heterozygosity of 77% (range 0.665–0.855). The total parentage exclusion probability was >0.9999 (given only the genotype of the offspring), >0.9999 for excluding a candidate parent from the parentage of an arbitrary offspring (given the genotype of the offspring and parent) and the probability of identity for full-sibs was 4.6×10–9. Overall, these microsatellites offer a simple and highly informative marker-set for parentage identification in numbats.

Parentage identification of Odontobutis potamophlia based on microsatellite DNA markers

Microsatellite lociwere used for parentage identification of Odontobutis potamophlia in five full-sib families. The combined exclusion probability of the first (E-1P) and the second parent (E-2P) revealed an obvious increase with the increase of number of microsatellite loci. The combined exclusion probability based on allele frequency suggested that at least eight microsatellite loci were needed for the identification of the 150 individuals from five families supported by the genetic distance analysis of individuals of these families. The double-blind test results indicated that the candidate individuals could find their correct parents through these loci thus eight microsatellite markers can be used for pedigree analysis of O. potamophlia in breeding industry as well as for future selection studies.

Forensic genetic informativeness of an SNP panel consisting of 19 multi-allelic SNPs

Current research focusing on forensic personal identification, phenotype inference and ancestry information on single-nucleotide polymorphisms (SNPs) has been widely reported. In the present study, we focused on tetra-allelic SNPs in the Chinese Han population. A total of 48 tetra-allelic SNPs were screened out from the Chinese Han population of the 1000 Genomes Database, including Chinese Han in Beijing (CHB) and Chinese Han South (CHS). Considering the forensic genetic requirement for the polymorphisms, only 11 tetra-allelic SNPs with a heterozygosity >0.06 were selected for further multiplex panel construction. In order to meet the demands of personal identification and parentage identification, an additional 8 tri-allelic SNPs were combined into the final multiplex panel. To ensure application in the degraded DNA analysis, all the PCR products were designed to be 87–188 bp. Employing multiple PCR reactions and SNaPshot minisequencing, 511 unrelated Chinese Han individuals from Sichuan were genotyped. The combined match probability (CMP), combined discrimination power (CDP), and cumulative probability of exclusion (CPE) of the panel were 6.07 × 10−11, 0.9999999999393 and 0.996764, respectively. Based on the population data retrieved from the 1000 Genomes Project, Fst values between Chinese Han in Sichuan (SCH) and all the populations included in the 1000 Genomes Project were calculated. The results indicated that two SNPs in this panel may contain ancestry information and may be used as markers of forensic biogeographical ancestry inference.

Parentage Identification of Differentiated Achondritic Meteorites by Hand‐held Energy Dispersive X‐Ray Fluorescence Spectrometry

We evaluate the potential of a hand‐held energy dispersive XRF spectrometer for the preliminary classification of non‐chondritic differentiated meteorites. The studied achondrites include nine lunar meteorites, seventeen Martian meteorites, five angrites and eighteen meteorites from asteroid 4 Vesta. Analytical precision and accuracy was tested on thirty‐nine terrestrial igneous rock slabs with a wide range of composition. Replicate analyses, performed on the studied meteorites, show that Fe/Mn values together with Si and Ca/K ratio can be used in the discrimination of different achondrite groups. Fusion crust's Fe/Mn values of meteorites from Vesta and Mars are indistinguishable from those of the interior implying that even measurements on the fusion‐crusted external surface could be sufficient to pigeonhole non‐chondritic meteorites. Hand‐held energy dispersive XRF spectrometer is a non‐destructive but very effective technique for preliminary classification of achondrites in the field and in laboratory and for the identification of mislabelled meteorites in museum collections.

Confirm fusion cell line by STR technology of parentage identification

Objective To screen and confirm cell fusion by DNA technology of parentage identification based on detecting of short tandem repeats. Methods With 20% polyethylene glycol (PEG)-6000, human myeloma cell lines and health individual peripheral blood mononuclear cell were fused. Then selected by hypoxantin, aminopterin, thymidin (HAT) medium, and fusion cell were sub-cloned. Morphology of fusion cells was checked by regular microscope. Concentration of DNA was compared to parental cells. Allele genes, identified by short tandem repeats, of fusion cell line were sequenced and compared with each other. Results The fused cells from myeloma cell line and peripheral blood mononuclear cell (PBMC) were slightly larger than primary cells, and the proliferation cycle was not changed significantly. DNA concentration of the fused cell DNA was increased by two times. Sequences of short tandem repeats (STR) showed that the fused cell included all original genetic materials of parent cells. Conclusions DNA technology of parentage identification is a convenient and reliable method to screen and confirm fused cell. Key words: Tandem repeat sequences; Blood grouping and crossmatching; Parent-child relations/LJ; Cell fusion

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye (Scyphozoa: Rhizostomatidae). A total of 85 alleles were detected in 600 individuals. Within-population levels of observed (Ho) and expected (He) heterozygosity ranged from 0.152 to 0.839 (mean=0.464) and from 0.235 to 0.821 (mean=0.618), respectively. The polymorphism information content (PIC) of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability of ten loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.

Parentage Confirmation of Korean Bred Pear Cultivars by Simple Sequence Repeat SSR Genotyping and S-Genotypes Analysis

Identification and authentication of parentage are important for effective pear breeding. Within Korean pear cultivars discrepancies are often reported between parents and offspring in skin color of fruits and also in S-genotypes suggesting that reported parentage was often inappropriate. In Korea, the parentage of the most of pear cultivars was never confirmed at the molecular level. Simple sequence repeat (SSR) genotyping and S-genotype analysis are considered effective in identifying parents. In this study, parentage of nine Korean bred cultivars was confirmed using SSR genotyping and S-genotype analysis. A total of 53 SSR markers were used. Six different haplotype-specific endonucleases were used for restriction cleavage of S-genotypes. Most of the Korean bred cultivars had six comparatively shorter S-RNase, S(1), S(3), S(4), S(5), S(6), or S(7) of 450 bp in length whereas the Japanese control cultivars had four other comparatively longer S-RNase. Out of nine pear cultivars only ``Chuwhangbae`` and ``Whangkeumbae`` had identical SSR genotypes and S-genotype with previously reported parents. For another cultivar, ``Sujeonbae``, the parents were the mutants of reported parent, ``Niitaka``. For four other cultivars, SSR and S-genotypes of offspring matched with only one reported parent ``Niitaka`` but those of another parent did not match. For the two other pear cultivars ``Soowhangbae`` and ``Sooyoung`` none of reported parents were confirmed by SSR genotyping and S-genotype analysis. Historically, the parent ``Niitaka`` was predominant in the Korean pear breeding programs because of its high yield potential and quality. The methods have been used in this study could be used to identify pear cultivars with diverse S-genotypes to eliminate any existing obscure parent-offspring relations.

A method for single nucleotide polymorphism selection for parentage assessment in goats

Accurate pedigrees are essential to optimize genetic improvement and conservation of animal genetic resources. In goats, the use of mating groups and kidding management procedures hamper the identification of parentage. Small panels of single nucleotide polymorphisms (SNP) have been proposed in other species to substitute microsatellites for parentage assessment. Using data from the current GoatSNP50 chip, we developed a new 3-step procedure to identify a low-density SNP panel for highly accurate parentage assessment. Methodologies for SNP selection used in other species are less suitable in the goat because of uncertainties in the genome assembly. The procedure developed in this study is based on parent–offspring identification and on estimation of Mendelian errors, followed by canonical discriminant analysis identification and stepwise regression reduction. Starting from a reference sample of 109 Alpine goats with known pedigree relationships, we first identified a panel of 200 SNP that was further reduced to 2 final panels of 130 and 114 SNP with random coincidental match inclusion of 1.51×10−57 and 2.94×10−34, respectively. In our reference data set, all panels correctly identified all parent–offspring combinations, revealing a 40% pedigree error rate in the information provided by breeders. All reference trios were confirmed by official tests based on microsatellites. Panels were also tested on Saanen and Teramana breeds. Although the testing on a larger set of breeds in the reference population is still needed to validate these results, our findings suggest that our procedure could identify SNP panels for accurate parentage assessment in goats or in other species with unreliable marker positioning.

Genetic variation of 12 X-chromosomal STR loci in an East Timor sample.

East Timor (República Democrática de Timor Leste) is a country with a population around 1 million inhabitants located in Southeast Asia and composed of 13 districts, including the eastern half of the Timor Island, Ataúro and Jaco islands, and the coastal enclave of Oecusse-Ambeno located in West Timor. Examples of the importance of X-chromosomal short tandem repeats (STRs) analysis include parentage identification, namely in cases involving father/daughter relationships, paternity in close relative deficiency cases without access to the putative father, maternity testing, and in rape or incest cases. In this study, 149 saliva samples were collected from unrelated individuals from East Timor and 12 X-chromosomal STRs genotyped using Investigator® Argus X-12 kit (Qiagen). A total of 13 alleles not included in Investigator Argus X-12 allelic ladder (off-ladder alleles) were found, four of which never reported (alleles 34.1 and 38.1 at DXS10134, allele 17.2 at DXS10074, and allele 28.1 at DXS10146). Allele 27.3 at DXS10101 and alleles 26, 28, and 29 at DXS10148 have already been observed in other populations but their frequencies are considerably higher in East Timor population. Allele frequencies and population statistic parameters were calculated for East Timor population and data contextualized in Southeast Asia/Pacific Region.