Development of 3 Multi-PCR Based Microsatellite Instability Analysis (MSA) for Bladder Cancer Detection

Several studies have shown that microsatellite changes can be profiled in urine for the detection of bladder cancer. Use of microsatellite analysis (MSA) for bladder cancer detection requires a comprehensive analysis of up to 15 to 20 markers, which were based on amplification and interpretations of many individual MSA markers and can be technically challenging. Here, in a way to develop fast, more efficient, standardized and less costly MSA for detection of bladder cancer, we have developed 3 multiplex PCR based MSA assay, all of which were analyzed by genetic analyzer. First, we have selected 16 MSA markers based on 9 selected publications. Based on samples from Johns Hopkins University (JHU Sample, first set sample), we have attempted to develop MSA assay based on doublet, 2 tube based multiplex PCR combined with singlet, 1 tube based single plex PCR. While this assay was initially successful, we have encountered a reproducibility issue and we then developed MSA based on triplet, 3 tube based multiplex PCR (Triplet MSA assay). From second set samples (6 cancer patients’ and 14 normal individuals’ sample), our Triplet Assay with 15 MSA markers correctly predicted all of 6/6 cancer samples to be cancerous sample and 14/14 samples from normal individuals as normal samples. This result suggests that our Triplet MSA Assay combined with genetic analyzer is a potentially time and cost-effective genetic assay for bladder cancer detection and can be used potentially as a dependable assay in patient care.

PSXII-1 Tracing the historical genetic components in turano-mongolian cattle breeds based on the microsatellite analysis of modern and museum samples

In their centuries-long history, the nomads of the south of Russia developed the local cattle breeds, which are well adapted to the harsh steppe conditions. In 20th century, the population size of such cattle breeds was dramatically decreased. At the same time, intensive cross breeding with high-producing transboundary breeds had been practiced on the small remaining part of population. The aim of our study was to trace the historical genetic components in modern populations of local cattle breeds of the turano-mongolian origin. The historical specimens of Kalmyk (n = 10), Kyrgyz (n = 11) and Kazakh (n = 3) cattle dated by the first quarter of the 20th century were derived from the craniological collection of the E.F. Liskun Museum for Animal Husbandry and subjected to the study. The modern representatives of Kalmyk (n = 28), Kyrgyz (n = 20), Kazakh White-headed (n = 30), Hereford (n = 26), and Mongolian (n = 41) cattle were used for comparison. All works with historical samples were performed in dedicated facility of the L.K. Ernst research Center for Animal Husbandry. The samples were genotyped for 11 microsatellite loci (BM1818, BM2113, BM1824, ETH10, ETH225, INRA023, SPS115, TGLA53, TGLA122, TGLA126, and TGLA227). For historical samples, the PCR reactions were carried out in five replicates to determine the consensus genotypes. In total, 132 alleles were identified, including 97 alleles in historical samples and 124 alleles in modern samples. The values of unbiased expected heterozygosity were 0.767–0.776 and 0.653–0.778 for the historical and modern samples, respectively. The STRUCTURE clustering showed the visible differences in genetic structure between the historical and modern populations of Kalmyk, Kyrgyz and Kazakh cattle breeds; however, the historical genetic components were still maintained in modern representatives of all of studied breeds. The research results will be useful for the sustainable breeding and conservation of valuable local genetic resources. The study was funded by the RSF No. 19-76-20012.

Screening of Mango Landraces for Polyembryony and Confirmation of Seedling Origin using Microsatellite Markers

Background: The introduction of polyembryonic rootstocks in the area of propagation is of great importance since they produce one zygotic and several nucellar plantlets. Proper identification of sexual embryo from each hybrid seed is necessary in order to preserve only the nucellar seedlings, which would help to maintain the rootstock’s genetic characteristics as well as to overcome the major constraints in the area of fruit breeding especially in hybridization programme by eliminating the nucellar ones to advanced generations. Contrasting reports exists regarding the vigour of zygotic seedlings of polyembryonic mango genotypes. It is necessary to identify/ distinguish the zygotic seedling from the nucellar population at an early stage, for which, microsatellite analysis could be a reliable tool. Methods: The experiment was laid out in completely randomized design (CRD) with 20 treatments replicated thrice. The twenty local mango landraces from Thiruvananthapuram (Kerala) were screened for polyembryony and were geo-referenced. Germination studies were conducted. Microsatellite analysis of all the plantlets from two varieties which exhibited the highest polyembryony were done using SSR primers and their banding patterns were compared with those of their respective mother plants. Result: Out of twenty mango varieties screened, seventeen were polyembryonic. Kappa Manga recorded the highest germination, germination index and seedling vigour index-I. Kotookonam Varikka recorded the highest polyembryony and followed by Kochu Kilichundan. Microsatellite analysis revealed that all the seedlings obtained from the respective stones of Kotookonam Varikka and Kochu Kilichundan had identical SSR profile to the mother plant, which indicated nucellar origin of seedlings having similar genetic composition to the mother plant.

Comparative Study of the Genetic Diversity of Local Steppe Cattle Breeds from Russia, Kazakhstan and Kyrgyzstan by Microsatellite Analysis of Museum and Modern Samples

The comparative molecular genetic study of museum and modern representatives of cattle breeds can help to elucidate the origin and maintenance of historical genetic components in modern populations. We generated the consensus genotypes for 11 microsatellite loci for 24 museum samples of Kalmyk, Kyrgyz, and Kazakh cattle, dated from the first quarter of the 20th century, and compared them with those of modern Kalmyk, Kyrgyz, and Kazakh white-headed breeds. The level of genetic diversity of the modern Kalmyk and Kyrgyz cattle (uHe = 0.771–0.778) was similar to those observed in the museum samples (uHe = 0.772–0.776), while a visible decrease in genetic variability in the modern Kazakh white-headed breed compared to museum Kazakh cattle was detected (uHe = 0.726 and 0.767, respectively). The PCA plot, FST- and Jost’s D-based networks, and STRUCTURE clustering provided strong evidence of the maintenance of the historical genetic background in modern populations of Kalmyk and Kyrgyz cattle. In spite of the allele pool of Kazakh white-headed cattle having undergone great changes compared to the museum Kazakh cattle, several animals still carry the visible aspect of the historical genetic components. Our results can be used for the selection of individuals for the creation of gene banks and may significantly improve the efficiency of conservation programs aimed at preserving genetic diversity in the national genetic resources of cattle.

Prediction of Genetic Groups within Brettanomyces bruxellensis through Cell Morphology Using a Deep Learning Tool

Brettanomyces bruxellensis is described as a wine spoilage yeast with many mainly strain-dependent genetic characteristics, bestowing tolerance against environmental stresses and persistence during the winemaking process. Thus, it is essential to discriminate B. bruxellensis isolates at the strain level in order to predict their stress resistance capacities. Few predictive tools are available to reveal intraspecific diversity within B. bruxellensis species; also, they require expertise and can be expensive. In this study, a Random Amplified Polymorphic DNA (RAPD) adapted PCR method was used with three different primers to discriminate 74 different B. bruxellensis isolates. High correlation between the results of this method using the primer OPA-09 and those of a previous microsatellite analysis was obtained, allowing us to cluster the isolates among four genetic groups more quickly and cheaply than microsatellite analysis. To make analysis even faster, we further investigated the correlation suggested in a previous study between genetic groups and cell polymorphism using the analysis of optical microscopy images via deep learning. A Convolutional Neural Network (CNN) was trained to predict the genetic group of B. bruxellensis isolates with 96.6% accuracy. These methods make intraspecific discrimination among B. bruxellensis species faster, simpler and less costly. These results open up very promising new perspectives in oenology for the study of microbial ecosystems.

Genealogical structure of the Belarusian black-and-white breed based on the microsatellite analysis of the lines bred at gene pool enterprises

The methods of molecular genetic analysis are widely used to assess the genetic structure and to study the dynamics of population-genetic processes in populations of domestic animals. I n particular, these are the highly polymorphic genetic markers – the microsatellites that are the most common molecular markers in genetic and genomic research. The aim of the research was to develop the genealogical structure of the Belarusian black-and-white breed based on the microsatellite analysis of the lines bred at a gene pool enterprise. The research was carried out with the animals bred at the JSC “SGC “Zarechye”. For the microsatellite analysis of boars of the Belarusian black-and-white breed, the DNA testing was carried out at 9 STR-loci (SО155, SО355, SО386, SО005, SW72, SW951, SО101, SW240, and SW857). In all studied lines, the largest number of effective alleles was at the SО005 locus – 4.00–5.14. The lines of animals with heterozygosity can be used in various breeding schemes, both in purebred breeding (in pedigree pig breeding) and in crossing (in industrial pig breeding). Polymorphism determines the degree of genetic variation in populations. The locus is classified as polymorphic when the frequency of the most common allele of this locus does not exceed 95% (р ≤ 95). The revealed polymorphism of loci in the lines Maket, Tik, and Veseliy indicates the presence of genetic variability in them, which allows using them in breeding and breed-forming processes. Based on the studies made, the genealogical structure of the Belarusian black-and-white breed, consisting of one subcluster and two branches, has been developed, which makes it possible to assess the state and degree of relation of structural units (lines), and reflects the prospect of their further use.