Validated multiplex PCR of microsatellites for breeding purposes, clonal fidelity and controlled crosses in teak

Herbicide-free plantations of oaks and ashes along a gradient of open to forested mesic environments

BackgroundPreviously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research.ResultsThe cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of FST, DA, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations.ConclusionsThis novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.

The International Society for Animal Genetics (ISAG) currently advocates for a transition towards single nucleotide polymorphism (SNP) markers as a potential alternative for equine parentage verification. To ascertain the efficacy of this transition, it is imperative to evaluate the performance of parentage testing using SNPs in juxtaposition with short tandem repeats (STRs). As per ISAG's recommendation, we used an equine genotyping-by-sequencing panel with 144 SNPs for this purpose. Equine parentage is currently realized using 16 microsatellites (STRs) excluding the LEX3 marker. In this study, 1074 horses were genotyped using the 144 SNPs panel, including 432 foals, 414 mares, and 228 stallions, from five different breeds: 293 Arabians, 167 Barbs, 189 Thoroughbreds, 73 Anglo-Arabians, and 352 Arabian-Barbs. As a result, two SNPs markers were eliminated from the panel system due to inconsistent amplification across all examined individuals leaving 142 SNPs markers for analysis. A comparative analysis between SNPs and STRs markers revealed that the mean expected heterozygosity was 0.457 for SNPs and 0.76 for STRs, while the mean observed heterozygosity stood at 0.472 for SNPs and 0.72 for STRs. Furthermore, the probability of identity was calculated to be 5.722 × 10-57 for SNPs and 1.25 × 10-15 for STRs markers. In alignment with the Hardy-Weinberg equilibrium in polyploids test, 110 out of the total SNPs were consistent with the Hardy-Weinberg equilibrium in polyploids test (p > 0.05). Employing both SNPs and STRs markers, the mean polymorphic information content was discerned to be 0.351 for SNPs and 0.72 for STRs. The cumulative exclusion probabilities for SNP markers exceeded 99.99%, indicating that the 142 SNPs panel might be adequate for parentage testing. In contrast, when utilizing STRs markers, the combined average exclusion probabilities for one and both parents were determined to be 99.8% and 99.9%, respectively. Our comprehensive study underscores the potential of SNPs in equine parentage verification, especially when compared to STRs in terms of exclusion probabilities. As a corollary, the application of SNPs for parentage verification and identification can significantly contribute to the conservation initiative for the five Moroccan horse breeds. Nonetheless, further research is required to address and replace the deficient SNPs within the panel.

Plant tissue culture is an important tool in forestry and biotechnology with its substantial improvements in plant propagation. The development of tissue culture is started with Haberlandt’s totipotency theory and the mid-20th century discovery of plant growth regulators which transformed methods of plant multiplication. The utilization of tissue culture in the micropropagation of teak (Tectona grandis L.) is a valuable hardwood species used for building, furniture and lumber. Through the production of genetically identical seedlings with desired characteristics including disease resistance, rapid development and high wood density. Tissue culture overcomes the drawbacks of traditional teak propagation which includes low germination rates, pest susceptibility and slow growth. This method supports conservation and sustainable forestry practices by providing a few benefits such as disease-free plant production, quick multiplication and year-round availability. Highly expensive, somaclonal fluctuation, contamination dangers and acclimatization hurdles are still major drawbacks. The importance of tissue culture in genetic improvement which allows for the preservation of elite genotypes and the creation of faster-growing, pest-resistant teak varieties is also examined in the research. Tissue culture is still a vital technique for large-scale teak production, reforestation and biodiversity protection despite its difficulties. It provides a revolutionary approach to commercial forestry and environmental sustainability.

Chapter 17 - Applications of DNA Typing

Forest trees cultivars developed by breeders have been increasingly deployed as clones, following long generations of breeding and testing. An established protocol for distinctiveness, uniformity and stability (DUS) testing becomes an essential element for protecting the intellectual property rights associated with these clones. DUS testing with morphological descriptors has, however, shown limitations in categorically distinguishing cultivars, especially with narrow genetic base. DNA fingerprinting based on microsatellite markers has been a powerful tool to discriminate clones. Teak (Tectona grandis) is an economically valuable exotic timber species planted in Brazil. We assessed the individual and combined performance of a selected set of 21 teak microsatellites for identity analysis and parentage testing in a sample of 50 clones planted in Brazil. The clones displayed high genetic diversity attributable to their wide provenance origin. The 21 microsatellites combined provide high power of individual identification with a combined probability of identity of 1.84E-23 for unrelated individuals, and 4.82E-09 for full-sibs, and a power of paternity exclusion higher than 99.99999 % in all testing scenarios. Variable subsets of these 21 markers still provide abundant power of discrimination, although a recommended set should include a minimum of 12 markers with higher information content and reliable genotyping performance. Relatedness and genetic distance analyses revealed unexpected identities or significant recent shared ancestry among otherwise considered unique clones. These results advocate for the importance of including DNA markers, at least as discretionary complementary descriptors to the 22 morphological traits currently adopted for plant variety protection of teak cultivars in Brazil.

Mahogany (Swietenia macrophylla King [Meliaceae]) is the most valuable hardwood species in the neotropics. Its conservation status has been the subject of increasing concern due to overexploitation and habitat destruction. In this work we report the development and characterization of 10 highly variable microsatellite loci for S. macrophylla. Twenty-nine percent of the 126 sequenced mahogany clones yielded useful microsatellite loci. Three high-throughput genotyping systems were developed based on polymerase chain reaction (PCR) multiplexing of these mahogany loci. We identified a total of 158 alleles in 121 adult individuals of S. macrophylla, with an average of 15.8 alleles (range 11-25) per locus. All loci showed Mendelian inheritance in open-pollinated half-sib families. The mean expected heterozygosity was 0.84 and the mean observed heterozygosity was 0.73. The combined probability of identity-the probability that two individuals selected at random from a population would have identical genotypes--was 7.0 x 10(-15), and combined probability of paternity exclusion was 0.999998 overall loci. These microsatellite loci permit precise estimates of parameters such as gene flow, mating system, and paternity, thus providing important insights into the population genetics and conservation of S. macrophylla.

Tandemly repeated DNA sequences are extensively polymorphic, due to variability in reiteration number (Jeffreys et al., 1985). There are two types of variable number of tandem repeats (VNTRs), long and short. Long VNTRs, also known as minisatellites, are generally detected by the technique of Southern hybridisation, but due to technical limitations the allele sizes of the long VNTR loci cannot be determined with precision, which complicates their application. In contrast, short trandem repeats (STRs), also known as microsatellites (Litt and Luty, 1989), are widely scattered throughout the human genome and provide a valuable source of polymorphic markers (Weber and May, 1989). These STRs can be precisely amplified by the polymerase chain reaction (PCR) and the variability of allele size can be determined faithfully in repeated experiments. Several STRs can simultaneously be studied by multiplex PCR (Chamberlain et al., 1988), making the STR analysis both rapid and less expensive. For both technical and economic reasons STR analysis provides an important and valuable tool for individual identification in paternity and forensic testing. This study presented here provides population frequency data for two tetrameric loci, one trimeric and one dimeric locus. The efficiency of these systems in paternity testing was also analysed by a retrospective analysis of ten paternity cases (trios) in which the exclusion had already been established with each of three long VNTR probes.

Tectona grandis L.F., known as teak, is a species of high commercial and ecological value for which the development of efficient clonal propagation strategies is being sought. The availability of clonally propagated materials from plus trees is scarce. The main objective of the research was to evaluate the root induction capacity of Tectona grandis L.F. plus tree sprouts using different hormone concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) selected at 30 and 60 days. A completely randomized design (CRD) with seven treatments, four replicates, and three experimental units per replicate was used to determine statistical differences between treatments. The best results for rooting of teak shoots were observed in treatment T5 (500 mg kg-1 of IBA) with an average of three roots per plant, T1 (control), and T7 (1500 mg kg-1 of IBA) with an average of four leaves per plant. The highest survival rates were observed in T2 (500 mg kg-1 of NAA) and T7 (1500 mg kg-1 of IBA) with 58.33% and in T1 (control), T2 (500 mg kg-1 of NAA) 50% rooting was obtained after 60 days. Although no differences were observed between treatments, the numerical differences allowed to observe that the use of auxins promotes rooting and survival of the shoots of adult trees of T. grandis and opens the possibility of propagation and cloning strategies for this species. This study provides valuable knowledge on vegetative propagation, breeding and conservation of forest species, which is relevant for academia, public and private sectors.

We report the genetic analysis of 100 individuals of an elite breeding flock of Afshari sheep with a selected set of eighteen microsatellite markers. A full characterization of this set of eighteen loci was carried out generating allele frequency distributions that were used to estimate the genetic information content of these loci, including genetic variability, inbreeding, individual and parent verification parameters. Disregarding MCMA26 monomorphic pattern, microsatellite loci showed moderate level of polymorphism, as such totally 102 alleles were detected with a mean number of 6 alleles per locus. The average expected heterozygosity was 0.72 (SD = 0.07) and the average Polymorphism Information Content (PIC) was 0.67 (SD = 0.08). Total value of inbreeding based on marker data was estimated as -0.02 so it indicates that inbreeding occurred less than would be expected at random. The overall probability of identity considering all twelve independent loci combined was 3.148E-13 meaning lower than 1 in 31 trillions. The results of this study indicate, despite the selective breeding and closed flock system over a number of generations, a relatively high level of heterozygosity still exists in the representative sheep flock. The high degree of multiallelism and the clear and simple codominant Mendelian inheritance of the set of microsatellites used provide a powerful system for the unique identification of Afshari sheep individuals for fingerprinting purposes and parentage testing.

서울대공원에서 사육중인 Miniature horse 10두의 혈통정립을 목적으로 PCR에 의한 성별 판정 및 16개 microsatellite marker를 사용하여 친자감정을 실시하였다. 성별 판정에서 430 bp의 SRY band가 관찰된 7두는 숫말로 판정되었고, 친자감정에서는 멘델의 유전양식에 부합된 3두가 친자관계가 확인되었다. 향후 Miniature horse의 혈통보존 및 관리에 유용한 자료가 될 것으로 사료된다. The aim of this study was to construct a correct pedigree of miniature horses (MH). The sex of MH was detected by PCR amplification of the sex determining region of the Y chromosome gene (SRY) prior to parentage testing. Ten random MH samples for parentage testing were genotyped by using 16 micro satellite markers. Since the SRY band (430 bp) was detected in horses No.1, 2, 6, 7, 8, 9, 10, these are male. However, the DNA segment was not identified in horses No.3, 4, and 5, which therefore are female. After genotyping, parentage testing was performed according to Mendelian fashion and International Society for Animal Genetics (ISAG) guideline. Of the 10 MH, 3 were qualified by the compatibility of 16 markers according to Mendelian fashion in the present DNA typing for parentage verification. These results can provide basic information for developing parentage verification and an individual identification system in MH.

DNA analysis is a “gold standard” for individual identification and parentage studies as for humans, so for animals (Saks et al., 1991). First forensic testing of the rare animal species DNA occurred in 1991 in Great Britain, when the case of illegal capture of the Peregrine Falcon (Falco peregrinus) nestlings was proved by parentage testing using DNA fingerprints of birds in question and other peregrines in captivity (Shorrock, 1998). In Russia, illegal capture for foreign sales is the main threat for Saker (Falco cherrug) and Gyrfalcon (Falco rusticolus) populations (Kovács et al., 2014; Lobkov et al., 2020). In 2021, a Comprehensive action plan for creation of Reintroduction and conservation centres for falcons in the Kamchatka region and bustards in the Republic of Kalmykia was signed into law by the Vice-Prime Minister V. Abramchenko. While this statute is primarily addressed to falcon species reproduction and reintroduction, some action items there are coming from falcon species conservation to prevent illegal trade: investigating the orders of marking birds in captivity and tracing them individually from hatching to selling and transferring abroad. Genetic methods were supposed to be the heart of the orders. Accuracy of any DNA identification highly depends on genetic markers used for it. Usually, for humans and farm animals, individual identification and paternity testing are based on nuclear microsatellites (short tandem repeats) panels. These markers are codominant, multiallelic, hypervariable and allow running analyses quickly and automatically. Some studies have already shown their possibilities in forensic DNA testing of falcons (Beasley et al., 2021). We tested different microsatellite panels for genetic identification of Sakers, Gyrfalcons and Peregrines and established a simple and cheap protocol for falcon genotyping using 10 microsatellite loci primarily described for saker natural populations (Hou et al., 2018). Using a first dataset contained of 99 samples (85 Sakers, 12 Gyrfalcons and two hybids), we showed the possibility to estimate the significance of genotyping for these loci and obtained values of probability of identity (PI) less than 10-9 in total and less than 10-3 for siblings. Whereas total population number both for the Saker and Gyrfalcon over the area is estimated by the IUCN in less than 106 individuals, and the offspring of one bird pair can hardly amount to much more than a hundred, the probability of accidental appearance of two birds with same genotypes for these loci may be regarded as negligible. The accuracy of parentage testing was also shown to be sufficient as for the pair and their offspring, so for one parent and the offspring. The microsatellite panel showed specificity for Peregrines, but more investigation is needed to check if these loci are fully convenient also for Peregrine Falcon identification. Basing on these preliminary results we used this test system starting a voluntary programme of breeding stock genotyping for Russian falconries. To the date, five falconries have already provided genetic material and supporting data for their breeding stock, about 380 samples from Sakers, Gyrfalcons, Peregrines and their hybrids. Together with data from natural populations (about 300 samples including museum specimens), this dataset provides opportunities to perform species and population analysis even though our preliminary results like previous data (Nittinger et al., 2007) showed low sensitivity of microsatellites to distinguish Sakers from Gyrfalcons without supporting geographic data. For falconries, full analysis of genetic data, analysis of paternity etc. provided a probability to check the breeding stock and breeding programs and exclude occasional mistakes in identification even for old cases. But the main aim of the program is to obtain a unique genetic identifier for each bird and its offspring to show the legitimacy of rare species keeping. While our microsatellite panel showed genotype uniqueness like CODIS (Budowle et al., 2016) and other forensic test systems for human identification, we propose it as a base of the genetic passport of falcon which can be put into a pedigree sertificate as a plain text or a QR-code. For such purposes, the method should be fully reproducible using any equipment at any laboratory. Together with Russian companies specialized in forensic DNA test systems manufacturing, we plan to develop a ready-to-use kit for genetic passportization of sakers and gyrfalcons in captivity and forensic DNA testing in case these rare species illegal outtake from nature or trafficking is suspected.

To investigate the polymorphisms of 9 non-DNA combined index system (CODIS) short tandem repeats (STRs), i.e., D7S3048, D8S1132, D11S2368, D2S1772, D6S1043, D13S325, D12S391, GATA198B05, D18S1364 in Hebei Han population, and evaluate the usage of them in paternity testing. One hundred and forty-seven unrelated healthy individuals from the Han population of Hebei province were genotyped using STRtyper10G kit including 9 STR loci on ABI 3130 Genetic Analyzer. Hardy-Weinberg equilibrium and population genetic parameters were calculated. Fourteen cases of motherless paternity testing and 2 cases of standard trios with mutation in 1 locus were detected using STRtyper10G. (1) Ninety-nine alleles and 336 genotypes were observed in the 9 STR loci in the population. The cumulative discrimination power(DP) was higher than 0.999,999,999. The cumulative probability of exclusion (PE) for trios and duos were 0.999,974 and 0.998,759 respectively. Departure from Hardy-Weinberg equilibrium was not observed in any of the 9 loci. (2) The combined paternity index (PI) of the 14 cases of motherless paternity testing ranged from 10³-10⁶ for 15 STR loci in ID, whereas it reached 10⁵-10⁹ for 22 independent STR loci included in ID and STRtyper 10G. Possible mutation in FGA and vWA was observed in 2 cases of trios, and the combined PI was 5945 and 1840 respectively for 15 STR loci in ID. Adding STRtyper 10G to detect these 2 cases, the combined PI reached 2.76 × 10⁷ and 4.88 × 10⁷ respectively. The genetic polymorphism of the 9 non-CODIS STR loci included in STRtyper 10G was quite high in Chinese Hebei Han population, indicating the 9 STR loci are valuable as complement markers for ID and PP16 kit in motherless paternity testing, paternity testing with mutation and other kinds of complicated paternity testing.

BackgroundYak (Bos grunniens) is the most important domestic animal for people living at high altitudes. Yak ordinarily feed by grazing, and this behavior impacts the accuracy of the pedigree record because it is difficult to control mating in grazing yak. This study aimed to evaluate the pedigree system and individual identification in polled yak.MethodsA total of 71 microsatellite loci were selected from the literature, mostly from the studies on cattle. A total of 35 microsatellite loci generated excellent PCR results and were evaluated for the parentage testing and individual identification of 236 unrelated polled yaks. A total of 17 of these 35 microsatellite loci had polymorphic information content (PIC) values greater than 0.5, and these loci were in Hardy–Weinberg equilibrium without linkage disequilibrium.ResultsUsing multiplex PCR, capillary electrophoresis, and genotyping, very high exclusion probabilities were obtained for the combined core set of 17 loci. The exclusion probability (PE) for one candidate parent when the genotype of the other parent is not known was 0.99718116. PE for one candidate parent when the genotype of the other parent is known was 0.99997381. PE for a known candidate parent pair was 0.99999998. The combined PEI (PE for identity of two unrelated individuals) and PESI (PE for identity of two siblings) were >0.99999999 and 0.99999899, respectively. These findings indicated that the combination of 17 microsatellite markers could be useful for efficient and reliable parentage testing and individual identification in polled yak.DiscussionMany microsatellite loci have been investigated for cattle paternity testing. Nevertheless, these loci cannot be directly applied to yak identification because the two bovid species have different genomic sequences and organization. A total of 17 loci were selected from 71 microsatellite loci based on efficient amplification, unambiguous genotyping, and high PIC values for polled yaks, and were suitable for parentage analysis in polled yak populations.

Previous studies investigating donkey parentage and genetic diversity used horse-specific multiplex systems. However, several mis-allele and null-allele issues were found with some of the horse primers when used in donkeys. In 2017, the International Society for Animal Genetics (ISAG) recommended 13 dinucleotide short tandem repeats (STRs) (AHT4, ASB23, HMS2, HMS3, HMS6, HMS7, HMS18, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343) as a core panel that should be used to identify individuals and to test for parentage in donkeys. To date, no single multiplex STR typing system containing all 13 donkey STRs recommended by the ISAG has been reported. To establish a novel and donkey-specific multiplex STR typing system containing all 13 recommended STRs. Assay development and validation in field population. Primers for seven of the STRs were redesigned and conditions for polymerase chain reaction (PCR) were optimised. We analysed the allele sequences, sensitivity, species-specificity and stutter ratios of this new system. A 13-plex STR typing system for donkey was established. A full profile could be generated from a single PCR reaction using as little as 5ng of DNA template with the 13 pairs of primers labelled with fluorescent dyes. An allele ladder, containing 101 alleles from the 13 STRs, was generated. No full genotype profile was generated with these primers if DNA from humans, or 11 other commonly encountered animals, was used. Genotypes could be generated for the horse and horse-donkey hybrids (mule and hinny). Stutter ratios and population genetic parameters were calculated based on samples from 150 donkeys. The combined probabilities of paternity exclusion for this system were 0.988907326 (CPEduo) and 0.999665018 (CPEtrio). This system cannot detect sex. Our results indicate that our donkey-specific 13-plex STR typing system is sensitive, species-specific and robust for individual identification, paternity testing and population genetic analysis in donkeys, and has potential forensic applications.