Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta

Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.

Sociogenetic Organization of the Red Honey Ant (Melophorus bagoti)

Kin selection and inclusive fitness are thought to be key factors explaining the reproductive altruism displayed by workers in eusocial insect species. However, when a colony’s queen has mated with <2 males, workers may increase their fitness by producing their own male offspring. Conversely, when the queen has mated with ≥2 males, workers are expected to increase their inclusive fitness by eschewing the production of their sons and preventing other workers from reproducing as well. Here, we investigated sociogenetic structure and worker reproduction in the red honey ant, Melophorus bagoti. Morphometric analyses revealed that workers belong to one of two distinct subcastes: they are either majors or minors. Using DNA microsatellite markers, we showed that all the colonies had a single, multiple-mated queen and that there was no relationship between worker patriline and worker subcaste. Furthermore, we found that workers were producing males in the presence of the queen, which contrasts with the predictions of inclusive fitness theory. Although our results are based on a small sample, they can serve as the foundation for future research examining worker reproduction in M. bagoti.

Genetic Variation at Microsatellite Loci in Odorrana hosii (Boulenger, 1891)

The true frog species, Odorrana hosii, found in West Sumatera Indonesia, has high morphological differentiation and also estimated has high genetic variation. A total of 35 O. hosii at a seven location have analyzed using DNA microsatellite markers. Genetic variation of five microsatellite loci provided the highest value of expected heterozygosity (He) for the population in Padang (He = 0.618), while the lowest was the population in Merapi (He = 0.427). There are genetic differences in moderate levels among populations of O. hosii in West Sumatra (FST = 0.108) with inbreeding intrapopulation value (FIS = -0.559), and high value of gene flow among the populations (Nm = 2.061). This study becomes the first molecular data for establishing effective population management conservation.

Short Communication: Molecular characteristic of taro white cattle based on DNA microsatellite markers

Heryani LGSS, Wandia IN, Suarna IW, Puja IK, Susari NNW, Agustina KK. 2019. Short Communication: Molecular characteristic of taro white cattle based on DNA microsatellite markers. Biodiversitas 20: 671-675. This research was conducted to assess and characterize the genetics of Taro White cattle. Genetic characterizations of this cattle are essential to conservation and breeding program. A total of 18 samples and 4 pairs of microsatellite DNA markers (BM2113, BM1824, INRA023, and ETH225) were amplified by PCR and the products were run on 8% bis-Acrylamide gels. All microsatellite markers were successfully amplified with a mean allelic number of 3.25. Means of observed and expected heterozygosity were found to be 0,25 and 0,628. The Polymorphism Information Content (PIC) values ranged from 0.448 (BM1824) to 0.627 (BM2113) and fixation index were 0.620. The deviation from Hardy-Weinberg equilibrium revealed that Taro White cattle population exhibited significant deviations from Hardy-Weinberg equilibrium (HWE) and possessed a possibility of inbreeding. The microsatellite loci used or focused in the present study further validate their use for evaluation of genetic diversity of Taro White

Genetic analysis reveals Finnish Formica fennica populations do not form a separate genetic entity from F. exsecta

Coptoformica Müller, 1923 is a subgenus of Formica Linnaeus, 1758 that consists of c. a dozen species of ants that typically inhabit open grassy habitats and build small nest mounds. The most recent addition to the group is Formica fennica Seifert, 2000. The description was based on morphological characters, but the species status has not been confirmed by molecular methods. In this study, we use thirteen DNA microsatellite markers and a partial mitochondrial COI gene sequence to assess the species status of F. fennica, by comparing the genetic variation among samples identified as F. fennica and six other boreal Formica (Coptoformica) species. Most of the species studied form separate, discontinuous clusters in phylogenetic and spatial analyses with only little intraspecific genetic variation. However, both nuclear and mitochondrial markers fail to separate the species pair F. exsecta Nylander, 1846 and F. fennica despite established morphological differences. The genetic variation within the F. exsecta/fennica group is extensive, but reflects spatial rather than morphological differences. Finnish F. fennica populations studied so far should not be considered a separate species, but merely a morph of F. exsecta.

Demography and behaviour of polygyne nests of the supercolonial ant Cataglyphis niger: Does kinship matter?

The basic ant colony is presumed to have evolved through kin selection. However, ants show a remarkable diversity in their social organization, from a monogynous-monandrous queen to the more derived states of polygyny with polyandrous queens. The existence of polygyny is an evolutionary enigma, since kin selection theory predicts that while queens should strive for reproductive monopoly, workers are predicted to favor their own matriline in rearing gynes. Using a barcoding system that enables tracking of individual interactions, along with polymorphic DNA microsatellite markers that indicate the matriline and patriline of all individuals, we demonstrate the complex social interactions in polygyne nests of Cataglyphis niger. C. niger is not only polygyne but also constitutes a supercolony at the study site. Our pioneering findings that both queens and workers are not necessarily related to each other support the supercolony structure of the population. Also in line with supercoloniality, we demonstrate that the workers contribute equally to the nest production and rearing of the queens. Unlike invasive supercolonial species, C. niger is native to Israel, raising questions about the driving forces, apart from kin selection, that stabilize this society.

Genetic diversity and structure of native Egyptian cattle populations and French-Egyptian Cross via DNA-microsatellite

This study investigates genetic diversity and structure of native Egyptian cattle populations, called Baladi, as Saidi from Southern Egypt, Menufi from Mid-Delta and their F1 crosses with the French Tarentaise breed using DNA Microsatellite markers. A total of unrelated 97 individuals were genotyped utilizing eight SSR primers (ETH10, ETH225, BM1818, BM1824, BM2113, SPS115, TGLA53 and TGLA126). All utilized SSR were found to be polymorphic. The highest and lowest numbers of alleles detected were 16 and 6 at TGLA53 and SPS115 loci, respectively. Baladi-Tarentaise crosses (Bal-Tar) had the highest number of alleles over all. The PIC values of 7 loci were higher than 0.5, indicating high allelic variation of utilized markers. Estimated PIC values were up to 0.898, 0.866 and 0.873 for TGLA53 genotyped in Saidi, Menufi and Bal-Tar, respectively. Hobs values were lower than the expected ones in the native populations accompanied with positive values for Fis and significant deviation from HWE indicating inbreeding trend in native populations. Structure analysis indicated three ancestral genetic backgrounds. The native populations share two main backgrounds in almost equal percentages, while the Bal-Tar had the third one. The three populations showed low percentage of admixture. The studied Mediterranean cattle populations that belong to Egypt and France seem to have differentiated from each other with only little genetic exchange between the geographically isolated populations so local cattle is very similar.

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae)

The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis,A. oratrix,A. pretrei,A. rhodocorytha,Anodorhynchus leari,Ara rubrogenysandPrimolius couloni). From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides) in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species.