Combined Non-exclusion Probability Research Articles

Conventional Gel Electrophoresis-Resolvable Insertion/Deletion Markers for Individual Identification and Analysis of Population Genetics in Red-Crowned Cranes in Eastern Hokkaido, Japan.

Simple SummaryThe red-crowned crane is an endangered bird species in the Far East Eurasian continent and Hokkaido, Japan. Individual identification has been achieved with banding and installed transmitters in only a few cranes, though individual identification is essential for various assessments of cranes on their behavioral characteristics, for example. An insertion/deletion (InDel) mutation is a mutation ranging from 1 to 50 bp and is very useful for genetic studies. If primer sets across InDels (>20 bp) can be designed, an InDel polymorphism can be determined with conventional agarose gel electrophoresis, as reported in some plant species. We found a sufficient number of InDel primer sets to be used for individual identification and analysis of population genetics of red-crowned cranes in Hokkaido. The method has additional advantages, such as convenience and low cost, without sequencing and an expensive apparatus.Red-crowned crane Grus japonensis is an endangered species in two separate populations: the mainland population in the Eurasian continent and the island population in eastern Hokkaido, Japan. We found 11 insertion/deletion (InDel) markers in the genome of the red-crowned crane and designed primer sets across these InDels that can be analyzed with conventional agarose gel electrophoresis. Sixty-six samples of whole blood and skeletal muscle obtained from red-crowned cranes, including 12 families in eastern Hokkaido from 1994 to 2021, showed different patterns in gel images of 11 InDel PCR reactions except for two pairs. The combined non-exclusion probability of the 11 markers indicates that individuals can be determined with a probability of 99.9%. In 39 non-relative chicks, the expected heterozygosity (He) was 0.316, suggesting low genetic diversity. This might not be caused by high levels of inbreeding since the average FIS was not significantly different from zero (0.095, p = 0.075). The results suggest that the 11 InDel primer sets can be used for fairly accurate individual identification as well as genetic population analyses in red-crowned cranes in the island population.

High Frequency of Multiple Paternity in Eastern Red Bats, Lasiurus borealis, Based on Microsatellite Analysis.

Most species of bats give birth to only 1 pup each year, although Eastern red bats (Lasiurus borealis) can produce up to 5 pups per litter. Offspring in a single litter have been documented to be at different stages of development, suggesting that multiple paternity occurs. We tested the null hypothesis of genetic monogamy in red bats using 6 autosomal microsatellites and 1 X-linked microsatellite from 31 parent/offspring groups for a total of 128 bats. We sampled both pregnant females and mothers with pups that were obtained from bats submitted to departments of health in Oklahoma and Texas for rabies testing. Multiple paternity was assessed using a maximum-likelihood approach, hypothesis testing, and X-linked locus exclusion. The mean polymorphic information content of our markers was high (0.8819) and combined non-exclusion probability was low (0.00027). Results from the maximum-likelihood approach showed that 22 out of 31 (71%) parent/offspring groups consisted of half siblings, hypothesis testing rejected full sibship in 61% of parent/offspring groups, and X-linked locus exclusion suggested multiple paternity in at least 12 parent/offspring groups, rejecting our hypothesis of genetic monogamy. This frequency of multiple paternity is the highest reported thus far for any bat species. High levels of multiple paternity have the potential to impact interpretations of genetic estimates of effective population size in this species. Further, multiple paternity might be an adaptive strategy to allow for increased genetic variation and large litter size, which would be beneficial to a species threatened by population declines from wind turbines.

12-plex highly polymorphic microsatellite marker set for parentage analysis in Banija spotted pigs

Microsatellites (MS) have been, for the last two decades, widely used for parentage analysis in all types of livestock, including pigs. Conservation efforts on Banija spotted pigs included genetic characterization of the breed with MS markers. Since recent comparison of pedigree and MS data revealed some inconsistencies, the aim of this study was to develop a set of highly polymorphic and heterozygous MS markers, which could be used for parentage analysis and to prevent pedigree errors. 12 MS markers with the polymorphic information content (PIC) above 0.62 were chosen and combined into a single multiplex polymerase chain reaction (PCR). The combined non-exclusion probability for one candidate parent (NE-1P) was 0.00246149, the combined NE-1P given the genotype of a known parent of the opposite sex (NE-2P) was 0.00003739 and combined non-exclusion probability for a candidate parent pair (NE-PP) was 0.00000003. Due to a high information content of selected MS markers it was possible to obtain high accuracy in parentage assignment, which was confirmed by analyzing actual data with known genetic relationships

A new set of microsatellite loci for Cattleya walkeriana Gardner, an endangered tropical orchid species and its transferability to Cattleya loddigesii Lindl. and Cattleya nobilior Reichenbach

AbstractIn this study, we isolate and analyse a new set of microsatellite loci for Cattleya walkeriana. Twenty-two primer pairs were screened for C. walkeriana (n = 32) and assessed for their transferability to Cattleya loddigesii (n = 12) and Cattleya nobilior (n = 06). All loci amplified for C. walkeriana; however, for C. loddigesii and C. nobilior, four and five primers, respectively, did not present amplification. The polymorphic loci presented between 2 and 13 alleles per locus for both C. walkeriana and C. loddigesii, with respective averages of 5.1 and 4.2. For C. nobilior, we found between two and five alleles per locus, with an average of 2.6. For C. walkeriana, observed heterozygosity varied from 0.100 to 0.966, whereas expected heterozygosity ranged from 0.097 to 0.900. The observed and expected heterozygosity for C. loddigesii and C. nobilior were also estimated. We found no significant linkage disequilibrium between any pair of loci, and evidence of null alleles at four loci (Cw16, Cw24, Cw30 and Cw31) for C. walkeriana. The combined power to exclude the first parent and combined non-exclusion probability of identity were 0.999 and 2.3 × 10−20, respectively. These new loci can be used in studies of germplasm resources, and assessments of genotypic and genetic diversity and population structure, thus improving the accuracy of such analyses and their applicability in the conservation and protection of these endangered species.

An evaluation of the International Society for Animal Genetics recommended parentage and identification panel for the domestic pigeon (Columba livia domestica).

In this study, the International Society for Animal Genetics (ISAG) recommended panel for the identification of the domestic pigeon (Columba livia domestica) is characterized based on commonly used statistical parameters. The marker panel is based on 16 short tandem repeat (STR) loci (PIGN15, PIGN10, PIGN57, PIGN26, CliμD16, CliμD19, PIGN12, CliμD17, CliμT17, PIGN04, CliμD01, CliμD11, CliμD35, CliμT02, CliμT13, CliμT43). The alleles of the 16 loci consist of a mixture of tri-, tetra-, penta- and hexameric repeat patterns. A sex determination marker was included in the multiplex for quality control. The repeat sequence of the PIGN markers was previously unpublished and therefore sequenced to reveal the sequence pattern. In total, 1421 pigeons were genotyped on 16 STR loci to generate allele frequency data for each locus. For all 16 markers combined, a PE1 (combined non-exclusion probability, first parent) of 0.9986 and PE2 (combined non-exclusion probability, second parent) of >0.9999 was observed. Comparing the alleged father and mother, a PE value of >0.9999 was observed. Two of the markers, CliμD19 and PIGN12, were found to have relatively high Hardy-Weinberg equilibrium and F(null) values. Therefore these markers may be considered to be replaced by other STRs. Another point of discussion may be to add a gender identification marker to the recommended ISAG panel. Not only can this serve as an extra identification marker, but this can also confirm the sex of a sample, because it is challenging to determine the sex based on phenotypical characteristics, especially for chicks. In conclusion, the set of 16 STR markers can be used in routine parentage verification and the identification of individuals.

Development of novel tetra- and trinucleotide microsatellite markers for giant grouper Epinephelus lanceolatus using 454 pyrosequencing.

Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy-Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (PID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.

Characterization of 28 microsatellite loci in the European roller Coracias garrulus (Coracidae, AVES)

One hundred and forty-four existing microsatellite avian loci were tested for polymorphism in two to four unrelated European rollers. This allowed the identification of 28 unique polymorphic loci that we characterized in 34 unrelated individuals from a population in Guadix, Spain. The putative chromosomal locations of all the polymorphic loci in European rollers could be assigned based on the location of their orthologues in the assembled zebra finch and chicken genomes. All loci were assigned to autosomes based on predictive mapping and/or the presence of female heterozygotes, except CAM-11 that was assigned to the Z chromosome. By using a selected subset of 15 autosomal loci, the combined non-exclusion probability for identity was 4.7E-09.

Development of New Microsatellite (STR) Markers for Montagu’s Harrier (Circus pygargus) via 454 Shot-Gun Pyrosequencing

During the last decades the ground-breeding Montagu’s harrier (Circus pygargus, Linnaeus, 1758) has changed its breeding habitats in Europe to agricultural areas in which many local populations would be close to extinction without a special nest protection regime. Although Montagu’s harrier is a well-studied species in terms of ecology and breeding biology, its genetic structure and population genetics are almost unknown. As there is a lack of good genetic markers we developed a set of 19 microsatellite markers comprising 16 new STR markers which were identified by next-generation sequencing (NGS) using 454 shot-gun pyrosequencing of genomic DNA. The STR markers were arranged into three multiplex PCR sets for high throughput genotyping and characterised. The marker set provides a powerful tool for kinship analysis. The combined non-exclusion probability for parent pairs was 1.13*10-11. Only three loci showed PIC values < 0.50. In total, 121 known family relationships were compared with genetically calculated ones to test the markers suitability for parentage analysis. In 97.5% of all cases full-sibships were accurately determined and 97.6% of all mothers were assigned correctly to their chicks. The present multiplex PCR panels can be used to investigate several hypotheses concerning breeding behaviour, kinship, exchange rates between populations and phylogeography.

Development of pedigree classification using microsatellite and mitochondrial markers for Giant grouper broodstock (Epinephelus lanceolatus) management in Taiwan.

Most giant groupers in the market are derived from inbred stock. Inbreeding can cause trait depression, compromising the animals’ fitness and disease resistance, obligating farmers to apply increased amounts of drugs. In order to solve this problem, a pedigree classification method is needed. Here, microsatellite and mitochondrial DNA were used as genetic markers to analyze the genetic relationships among giant grouper broodstocks. The 776-bp fragment of high polymorphic mitochondrial D-loop sequence was selected for measuring sibling relatedness. In a sample of 118 giant groupers, 42 haplotypes were categorized, with nucleotide diversity (π) of 0.00773 and haplotype diversity (HD) of 0.983. Furthermore, microsatellites were used for investigation of parentage. Six out of 33 microsatellite loci were selected as markers based on having a high number of alleles and compliance with Hardy-Weinberg equilibrium. Microsatellite profiles based on these loci provide high variability with low combined non-exclusion probability, permitting practical use in aquaculture. The method described here could be used to improve grouper broodstock management and lower the chances of inbreeding. This approach is expected to lead to production of higher quality groupers with higher disease resistance, thereby reducing the need for drug application.

Transferability of microsatellites for studies on the social behavior of the tufted capuchin monkey (genus Sapajus).

Because of relevant results that indicated that molecular techniques can provide increased knowledge of animal social systems, they usually complement observational field studies. Despite the great utility of microsatellites, they are not available for all species. Gathering genetic information using microsatellites that were originally designed for other species is a time-saving procedure. The aim of this study was to test the transferability of microsatellites and their usefulness in studies of social behavior of black capuchin monkeys (Sapajus nigritus). We noninvasively sampled adult and subadult black capuchins of three wild groups in southeastern Brazil. Seventeen microsatellites, which were previously designed for and successfully amplified in multiple Neotropical primate species, were tested. Nine of the 17 microsatellite loci tested produced an average of 6.22 alleles (range 2-12) per locus. The allelic richness and the expected heterozygosity for all loci was 5.93 and 0.70, respectively. The combined non-exclusion probability for one candidate parent across all loci was 0.01. The nine microsatellite loci optimized in this study have a great potential for application in studies of social structure and dispersal patterns in S. nigritus populations and in other Neotropical primate species.

Development of microsatellite markers for the Asian Stubtail Urosphena squameiceps by using next-generation sequencing technology

The Asian Stubtail Urosphena squameiceps winters in Southeast Asia which is facing a serious threat from deforestation and the species is categorized as a threatened species at some of the breeding populations. Monitoring population dynamics of the species in the field is difficult because they are secretive and prefer dense undergrowth. Therefore, developing genetic markers from the species is useful for detecting appropriate conservational management units and to understanding their unique breeding system. We isolated and characterized 13 microsatellite loci of the Asian Stubtail by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 2–26 (mean 10.31) and 0.14–0.92 (mean 0.64), respectively. The combined first-parent non-exclusion probability was <0.005.

Tetranucleotide microsatellite loci from the critically endangered hawksbill turtle (Eretmochelys imbricata)

We describe isolation and characterization of 14 polymorphic tetranucleotide loci from the hawksbill turtle (Eretmochelys imbricata). We identified an average of 14.5 alleles per locus based on screening of 36 individuals captured on foraging grounds in Bocas del Toro Province, Panama, and 9.6 alleles in 13 individuals captured on foraging grounds in Bermuda. Observed heterozygosity ranged from 0.67 to 1.00, with a mean of 0.85 for the Panama foraging aggregation and 0.83 for the Bermuda foraging aggregation. This microsatellite suite has a combined non-exclusion probability of identity of 8.26 × 10–23. These markers should be informative in individual and population-focused analyses.

Tetranucleotide microsatellite loci from the endangered green turtle (Chelonia mydas)

We describe isolation and characterization of 20 polymorphic tetranucleotide loci from the green turtle (Chelonia mydas). We identified an average of 12.5 alleles per locus based on screening of 31 individuals foraging in the Indian River Lagoon, Florida, USA. Observed heterozygosity ranged from 0.53 to 1.00, with a mean of 0.85. This microsatellite suite has a combined non-exclusion probability of identity of 2.45 × 10–28. These markers will complement those already available for green turtles in individual-based as well as population level genetic analyses.

Development of microsatellite markers for the endangered grassland species Vincetoxicum pycnostelma (Apocynaceae) by using next-generation sequencing technology

Vincetoxicum pycnostelma (Apocynaceae) is distributed widely in East Asia; however, over the last several decades, the number of populations of the species in Japan has been rapidly decreasing because of the loss of its grassland habitat. Existing population sizes are extremely small. Here, we isolated and characterized 9 microsatellite loci of V. pycnostelma by using a next-generation sequencer. The number of alleles and the expected heterozygosity at each locus were 5–22 (mean, 8.7) and 0.41–0.92 (mean, 0.74), respectively. The markers described in this study will be useful for parentage analysis of V. pycnostelma (combined first-parent non-exclusion probability <0.005) and conservation genetic studies such as evaluating the genetic diversity within and between the remaining populations.

Isolation and characterization of microsatellite loci for Menziesia goyozanensis, an endangered shrub species endemic to Mt. Goyo in northern Japan

Menziesia goyozanensis, a Japanese kind of fool’s huckleberry, is a shrub species endemic only to Mt. Goyo in northern Japan. This species is listed as ‘critically endangered’ in the Red Data Book of wild plants in Japan, and only a single population has yet been discovered. We isolated and characterized 16 microsatellite loci for this species. The number of alleles ranged from 2 to 25. The range of expected heterozygosities was 0.292–0.917. Five of the 16 microsatellites were not conformed to Hardy–Weinberg equilibrium, probably due to the restricted population size, genetic drifting and the presence of null alleles. These 16 microsatellite loci gave a combined non-exclusion probability of <1.65E–15 in the sampled population. In Menziesia pentandra, which is closely related species to M. goyozanensis, the number of alleles detected and the expected heterozygosities of twelve loci ranged from 3 to 19 and 0.536 to 0.966, respectively. These informative microsatellite markers will be useful in studies of population genetic structure for these species.

Characterisation of twenty-one European badger (Meles meles) microsatellite loci facilitates the discrimination of second-order relatives

The European badger (Meles meles) breeds plurally in lowland England and is important economically due to its link with bovine tuberculosis (Mycobacterium bovis) transmission. To understand disease transmission and facilitate effective management, it is vital to elucidate the social structure of badger groups. To improve parentage assignment and the discrimination of relatives, we isolated and characterised 21 polymorphic microsatellite loci in 24 individuals from Wytham Woods, Oxfordshire, UK. These 21 loci increased the discrimination power between full-siblings and half-siblings from 71 to 88%, when added to the existing 31 loci. Similarly, the combined non-exclusion probability increased from 3.0 × 10−8 to 5.8 × 10−13. Newly isolated Mel-592 (FR745854) was X-linked, based on the genotypes of 48 known-sex individuals and will enhance the genetic sex-typing of badgers.

제주흑우 집단에서 모색 관련 유전자와 microsatellite marker의 다형현상을 이용한 수정란이식 및 인공수정 유래 후대우 검증

농가에 보급된 제주흑우 수정란이식 및 인공수정 생산축의 확인을 위하여 분자유전학적 실험기법을 이용한 개체 추척을 수행하였다. 유전자 marker 체계는 ISAG 권장 MS marker 11종, 예비시험 후 선발된 SAES marker 11종, 흑모색 관련 MC1R과 ASIP 유전자들을 조합하여 분석하였다. 분석결과 부모 정보가 없는 상태에서의 부권 부정율이 국제권장기준보다 높은 수준을 보였으며, 형매간 동일개체출현률은 <TEX>$5.3{\times}10^{-10}$</TEX>으로 조사되었다. 친자검정 결과 후보축에 대한 후보 부, 모, 부모 모두가 확인되는 경우는 각각 77.0, 54.0, 40.5%였다. 부-모-자간 trio-mismatch가 전혀 없는 수정란이식 개체는 공급 수정란 대비 14.7%로 확인되었고, 전체 후보축군 중 32.4%는 후보 부와의 mismatch가 없는 인공수정에 의해 생산된 개체들로 판정하였다. ISAG marker들만을 분석한 결과에서는 7두가 동일한 3가지 유전자형 조합을 나타내었으나, ISAG/SAES marker들을 조합했을 때에는 2두에서만 동일 유전자형 조합을 나타내었다. MS와 모색유전자 분석자료를 모두 조합했을 때는 조사된 모든 개체들이 서로 구분되었다. 현재의 제주흑우집단이 소수 핵군에서 인공수정과 수정란이식 등 생명공학 기법으로 육성된 집단이기에 제주흑우집단의 유전적 다양성은 낮게 나타났다. 본 연구는 유전자 개체식별과 혈통관리 체계의 구축을 위해서는 적어도 20개 이상의 MS marker와 모색관련 유전자형 자료가 필수적으로 활용되어야함을 제안하고 있으며, 연구결과는 향후 제주흑우의 분자육종에 있어 유용한 자료가 될 것으로 시사하고 있다. To find offspring of Jeju Black cattle (JBC) produced by embryo transfer (ET) and artificial insemination (AI), a molecular genetic study was carried out in candidate cattle populations collected from cattle farms in Jeju Island, Korea. The genetic marker set was composed of 11 ISAG microsatellite (MS) markers, 11 SAES MS markers selected by our preliminary analysis for population diversity of JBC and two major coat color related genes: MC1R and ASIP. The results showed a combined non-exclusion probability for first parent (NE-P1) that was higher than that recommended by ISAG (above 0.9995), and a combined non-exclusion probability for sib identity of <TEX>$5.3{\times}10^{-10}$</TEX>. Parentage analysis showed that the cases identified the candidate's father only (77.0%), mother only (54.0%), and both parents (40.5%) in the candidate offspring population. The ET and AI calves were identified as 14.7% in the in vitro fertilized eggs provided and 32.4% in total population, respectively. However, the result from ISAG marker analysis showed 3 identical allele-combinations in 7 calves, and that from ISAG/SAES MS marker combination also showed 1 identical allele-combination in 2 calves. Data from MS and coat-color gene analyses provided information for complete identification of all animals tested. Because the present JBC population was mostly bred using small nuclear founders through bioengineering techniques such as AI and ET, the genetic diversity levels obtained from MS analysis in the JBC population were relatively lower than those of other cattle populations, including Hanwoo. The results suggested that the more efficient marker combinations, including coat color related genotypes, should be studied and used for constructing a system for identification and molecular breeding of JBC as well.

Development and evaluation of microsatellite markers for identification of individual Greenshell™ mussels (Perna canaliculus) in a selective breeding programme

A set of 49 microsatellite loci isolated from the endemic New Zealand Greenshell™ mussel, Perna canaliculus, were evaluated for inclusion in a parentage assignment marker suite by assessing their ease of PCR amplification, allele scoring and conformity to Mendelian inheritance in hatchery-produced families. Ten polymorphic loci (mean He=0.78 and polymorphic information content (PIC)=0.72) were identified as being suitable for parentage assignment. These 10 microsatellite loci gave a combined non-exclusion probability of <0.001 (probability that an unrelated parent pair will not be excluded from parentage of an arbitrary offspring), based on allele frequencies from 16 broodstock mussels. Simulations predicted an assignment success rate of 99.9% with all 10 loci and >95% with the best 5 or more loci (mean PIC=0.84). In actual parentage assignments, 124 offspring from 8 full-sib families were assigned to the correct parent pair with 4 or more loci. We found evidence for null alleles and extensive size homoplasy in many loci, highlighting the importance of thoroughly characterizing and evaluating microsatellite markers prior to parentage assignment and other applications.