Monomorphic Loci Research Articles

Genetic diversity of lion populations in Kenya: Evaluating past management practices and recommendations for future conservation actions.

The decline of lions (Panthera leo) in Kenya has raised conservation concerns about their overall population health and long-term survival. This study aimed to assess the genetic structure, differentiation and diversity of lion populations in the country, while considering the influence of past management practices. Using a lion-specific Single Nucleotide Polymorphism (SNP) panel, we genotyped 171 individuals from 12 populations representative of areas with permanent lion presence. Our results revealed a distinct genetic pattern with pronounced population structure, confirmed a north-south split and found no indication of inbreeding in any of the tested populations. Differentiation seems to be primarily driven by geographical barriers, human presence and climatic factors, but management practices may have also affected the observed patterns. Notably, the Tsavo population displayed evidence of admixture, perhaps attributable to its geographic location as a suture zone, vast size or past translocations, while the fenced populations of Lake Nakuru National Park and Solio Ranch exhibited reduced genetic diversity due to restricted natural dispersal. The Amboseli population had a high number of monomorphic loci likely reflecting a historical population decline. This illustrates that patterns of genetic diversity should be seen in the context of population histories and that future management decisions should take these insights into account. To address the conservation implications of our findings, we recommend prioritizing the maintenance of suitable habitats to facilitate population connectivity. Initiation of genetic restoration efforts and separately managing populations with unique evolutionary histories is crucial for preserving genetic diversity and promoting long-term population viability.

Genetic Diversity of Bananas (Musa spp.) in Pesawaran District, Lampung Province Based on Sequence Related Amplified Polymorphism (SRAP) Marker

Banana (Musa spp.) is a leading commodity that makes a major contribution to national fruit production. One of the districts in Lampung Province which has a significant contribution to Indonesia's banana production is Pesawaran Regency. The molecular marker used in this study is Sequence-Related Amplified Polymorphism (SRAP). This study aims to determine the genetic diversity of bananas, determine the suitable combination of SRAP primers to determine the genetic diversity of bananas and determine the similarity relationship of bananas in the Pesawaran District. The total genomic DNA was extracted using DNeasy Plant Mini Kit (Geneaid) according to the protocol from the manufacturer followed by their amplification using SRAP primers. Similarity analysis was carried out based on similarity index values and dendrogram construction using the NTSYS 2.0 program. Research results show that a total of 204 loci (167 polymorphic loci and 37 monomorphic loci) were obtained from 9 selected primer combinations. The genetic diversity of 19 banana cultivars originating from Pesawaran Regency resulted in low genetic diversity values (0-0.27). Banana kinship based on Sequence Related Amplified Polymorphism (SRAP) markers divided 19 banana cultivar samples and 4 wild banana samples into 2 large clusters with similarity coefficient values between 0.70-0.97.

Documentation of Genetic Diversity by Insulin-Like Growth Factor1 Receptor (Exon2) Gene for Fallow Deer (Dama dama) in Iraq.

Insulin-Like Growth Factor1 Receptor (Exon2) (IGF1R) gene plays a vital role in physiological impacts, such as growth, development, reproduction, and metabolism. A significant difference was noted between the IGR1R (exon 2) gene and the body weight of Dama dama. In addition, the heterozygosity pattern (AB) was significantly higher than the other pattern (AA). There are three single nucleotide polymorphisms (SNPs; 144G>C, 147A>G, and 210A>C) within the IGF-1R (exon 2) locus. The statistical analyses indicated the presence of three different haplotypes (GAA, CAA, and GGC). The analysis of relative frequencies indicated that the most frequent haplotype in the studied Dama dama population was Hap3 (GGC) (43.4782%) out of the three observed haplotypes. The results of SSCP-PCR revealed the variability of the target gene between the genotype frequencies in Fallow deer (Dama dama) with a high level of significance (P≤0.01) with two patterns (AA and AB) and an absence of BB pattern. The allele frequency of AA record a high level (71.74%) than the other genotype (AB) (28.26%), with a high-frequency level of the A allele (0.86) than the B allele (0.14). In current findings, SSCP genotyped in the Dama dama DNA observed an estimated 72% monomorphic loci and 28% polymorphic loci approximately. Hardy Weinberg equilibrium test (HW) was applied to the SSCP-PCR data matrix, and the statistical test was based on a chi-square (χ2) test. Chi-square was (55.928%) with a highly significant level (P≤0.01) recorded in the present study. As related to AA and AB genotypes mean, a significant difference (P≤0.05) was noted between IGF1R (exon 2) gene with a body weight of Dama dama, as well as the heterozygosity pattern (AB), was significantly (P≤0.05) higher than the other pattern (AA) (30.34±3.01kg versus 24.85±1.94kg), respectively. A significant impact (P≤0.05) between IGF1R (exon2) polymorphism and heart girth was founded to be related to the AB pattern (heterozygous) (76.92 ± 3.20 cm), whereas the lower value was related to the AA pattern (71.33 ± 2.49 cm). No significant differences in effects were shown in relation to body length and height at the shoulder. The present study is also interested in genetic characterization by calculating (Ne) as a tool for genetic diversity. Therefore, the number of alleles detected (Na) indicates that two alleles only were unique in the population of the study, with (1.3204) being the number of efficient alleles (Ne). Moreover, Shannon's Information index was recorded at 0.4073. The observed homozygosity (O.Hom.) and heterozygosity (HO) were (0.7174 and 0.2826), respectively. The values of expected homozygosity (E.Hom.) and heterozygosity (HE) were 0.7547 and 0.2453, respectively. The genetic diversity of Nei was 0.2427. The results showed an unexpected influx of IGF1R diversity measured by Fis and recorded the value (- 0.1646). In this sense, the results of the current study may be considered an approximation to the total genetic diversity of the population of Dama dama in Iraq, but the information obtained is relevant to proposing the strategies of conservation for the genetic diversity observed.

Low genetic diversity among introduced axis deer: comments on the genetic paradox and invasive species

Abstract Human-mediated introductions and subsequent establishment and spread of nonnative species have the potential to create a founder effect in such populations, which typically results in low genetic diversity and potential for inbreeding. However, several exotic invasive species exhibit a “genetic paradox” in which they thrive and adapt to novel environments while also avoiding complications from low genetic diversity. Axis deer (Axis axis) were introduced into Texas, Hawaii, South America, Australia, and Croatia during the 19th and 20th centuries and successfully established large populations from a few founding individuals. Mitochondrial (Cytochrome-b, Cytb; displacement loop, D-loop) and nuclear (10 microsatellites) markers were used to assess genetic diversity within and between axis deer populations in Texas and Hawaii and then compared to other introduced (Australia and Croatia) and native (India) populations. Overall, mtDNA divergence was 0.54% (Cytb) and 1.55% (D-loop) indicating high mitochondrial similarity within the species. Further, each invasive population was composed of only one or two mtDNA haplotypes. Microsatellite allele diversity also was low within and between populations in Texas and Hawaii resulting in monomorphic loci and Hardy–Weinberg equilibrium violations in both populations. The low genetic diversity in native Indian axis deer and within and between invasive populations suggests that the introduced populations experienced founder effects following introduction, and yet overcame this potential handicap by undergoing successful establishment and expansion. Axis deer appear to be another successful invasive species characterized by the genetic paradox where they exhibit genetic profiles that suggest inbreeding effects should be imminent, yet display no signs of inbreeding and are highly successful adapting to novel environments.

ASSESSMENT OF ECOLOGICAL AND GENETIC DIVERSITY OF THE GENUS VIGNA SAVI USING BIOTECHNOLOGICAL METHODS - ISSR MARKERS

The world's genetic resources of plants are the main source of improving crops for decades to come. The gene pool of plants has a hidden resource of new genes, or their combinations, including - selection-important features. The study of the potential of the plant gene pool with the main biological and economic characteristics allows to expand the genetic base of crops for the successful implementation of breeding programs in various areas. The genus Vigna has about 200 species that are grown in warm regions of the planet. The centers of origin of the species are in Africa, but mung beans, urd, azuki and rice beans are of Asian origin. The collection includes 20 specimens belonging to 7 species of the genus Vigna: V. aconitifolia (Jacq.) Marechal (willow aconitolista, mott) – 3 specimens, V. angularis (Willd.) Ohwi et Ohashi (adzuki) – 4, V. radiata (L.) R. Wilczek (mash) – 4, V. umbellata (Thunb.) Ohwi et Ohashi (vigna rice) – 4, V. unguiculata (L.) Walp. (Chinese) – 5. These are mainly local varieties, about 10 % are breeding varieties and 1% are forms that grow in nature.
 The multifaceted use of cultivated species of the genus Vigna contributed to their spread throughout the tropical, subtropical and temperate zones of the globe. They are economically important crops in many developing countries. Geographical analysis showed that most of the samples came from the areas of world agriculture and crop formation. most samples of V. radiata, V. mungo, V. aconitifolia, V. trilobata, V. umbellata were obtained from India and Pakistan, V. angularis – from East Asia and China, V. unguiculata – East Africa (Ethiopia, Kenya), V unguiculata – from China. However, the exact place of domestication of this culture has not been established, and in numerous sources of literature can be found different opinions of scientists on the origin of cowpea and the centers of its diversity. Recently, using the methods of molecular genetics (RAPD, AFLP and others) it was confirmed that the northern part of Africa is the center of origin of the cultured, because the wild types of West Africa are closer to cultural forms than the wild types of East and South Africa. It should be noted that the species of cowpea have a significant intraspecific polymorphism. The samples showed strong variability of morphological and economically valuable features. Such a wide range of variable variability is due to the places of cultivation of samples, different environmental conditions (plains, mountains, climate).
 According to the results of the evaluation of the DNA polymorphism of cowpea using molecular genetic markers, it was found that the species of cowpea involved in the study are characterized by a high level of DNA polymorphism, which averaged 78.6 %. 145 loci were identified, including 31 unique, specific to a particular sample, and 31 monomorphic, characteristic of all samples. Monomorphic loci are conserved regions of DNA that indicate the common origin of the species of cowpea involved in the work, and can be used as genus and species-specific markers. Unique loci indicate genetic divergence of the studied material and can serve as markers of certain samples. The average level of intrapopulation polymorphism of cowpea DNA (37.2–93.8 %, depending on the genotype) was established, which indicates the existence of significant variability in the studied samples of cowpea. It shows a high level of genetic divergence of cowpea species and testifies in favor of the polyphyletic theory of their origin.

Study of genetic variability of umbilical cord blood using RAPD assay

Background: The genetic variability of Umbilical Cord Blood (UCB) is most important for newborn screening, therapeutic possibility of haematological disorders as well as for the establishment of cord blood banking and stem cell research.
 Method: Genetic variability of umbilical cord blood (UCB) of 22 human subjects was evaluated first time by applying Random Amplified Polymorphic DNA (RAPD) assay using six decamar primers (B-14, OPB-05, OPB-08, OPB-12, OPB-19 and UBC-122).
 Result: A total number of bands were recorded 312 from 116 polymorphic loci and single monomorphic locus. All the markers showed highest polymorphism (100%) except the primer OPB 08 (92.31%) among tested individuals. The genetic distance was observed with highest 1.0 and lowest 0.72 respectively whereas mean genetic distance was recorded 0.90. Considering Shannon-Wiener index average diversity was recorded 0.139365. The mean Nei genetic similarity was found 0.17 which was found opposition to genetic distances. A phylogenetic relationship among the individual subjects was also observed between the linkage distances of 11 to 27 with 8 clades, 3 subclusters and a cluster. In addition, average allele frequency p and q was observed 0.08156 and 0.948751 respectively whereas highest intra locus gene diversity and average gene diversity were found 3.323817 and 0.144572 respectively.
 Conclusion: Considering different parameters, higher genetic variability was found among the experimental subjects, probably due to the mixture DNA of parents and newborn.
 Bangladesh Journal of Medical Science Vol.20(4) 2021 p.848-854

Allelic polymorphism in exon 1 of GDF9 and exon 2 of BMP15 genes and its impact on litter size at lambing in Iran-Black sheep

The main purpose of this study was to assess the presence of the previously reported single nucleotide polymorphisms (SNPs) in the sheep growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes and their association with litter size at lambing in Iran-Black sheep breed. Blood samples were taken from 120 Iran-Black ewes. DNA extraction was conducted using a modified salting-out method. DNA fragments with sizes of 462bp and 141bp for the GDF9 and BMP15 genes were amplified using PCR with specific primers, respectively. The PCR-RFLP approach was adopted for detecting the genotypes. The results indicated that the SNP in the exon 2 of BMP15 is a monomorphic locus in Iran-Black sheep. However, the substitution of G to A nucleotide was determined in the GDF9 locus. Digestion of the 462bp PCR product from exon 1 of theGDF9 using the HhaI restriction enzyme produced fragments of 52, 156, and 254bp. However, DNA fragments containing the A nucleotide yielded only two fragments (52 and 410bp). The heterozygous animals for this mutation in GDF9 locus had fragments of all four sizes (52, 156, 254, and 410bp). The frequency (0.75) of the wild type allele (+) in GDF9 locus was higher than the frequency (0.25) of mutant allele (G). The observed frequencies for the GG, G+ and ++ genotypes were 0.05, 0.40 and 0.55, respectively. The association results indicated that the mutation of GDF9 gene has a substantial impact on lambing rate and the Iran-Black ewes with the GG and G+ genotypes had higher lambing rate than those with the ++ genotype. Thus, a gene assisted selection program to improve lambing rate in this breed can be designed based on the GDF9 gene mutation. Key words: BMP15, GDF9, Litter Size, Iran-Black Sheep

Genetic structure of deltamethrin-resistant populations of Triatoma infestans (Hemiptera: Reduviidae) in the Gran Chaco.

The genetic structure of natural populations offers insight into the complexities of their dynamics, information that can be relevant to vector control strategies. Microsatellites are useful neutral markers to investigate the genetic structure and gene flow in Triatoma infestans, one of the main vectors of Chagas disease in South America. Recently, a heterogeneous pyrethroid-resistant hotspot was found in the Argentine Gran Chaco, characterized by the highest levels of deltamethrin resistance found at the present time. We applied population genetics analyses to microsatellite and village data and search for associations between the genetic variability and the heterogeneous toxicological pattern previously found. We genotyped 10 microsatellite loci in 67 T. infestans from 6 villages with no, low, and high pyrethroid resistance. The most genetically diverse populations were those susceptible or with low values of resistance. In contrast, high-resistance populations had lower herozygosity and some monomorphic loci. A negative association was found between variability and resistant ratios. Global and pairwise FSTs indicated significant differentiation between populations. The only susceptible population was discriminated in all the performed studies. Low-resistance populations were also differentiated by a discriminant analysis of principal components (DAPC) and were composed mostly by the same two genetic clusters according to STRUCTURE Bayesian algorithm. Individuals from the high-resistance populations were overlapped in the DAPC and shared significant proportions of a genetic cluster. These observations suggest that the resistant populations might have a common origin, although more genetic markers and samples are required to test this hypothesis more rigorously.

Molecular Characterization of Seedlings Derived from Two Polyemberyonic Egyptian Mango Cultivars Using SSR Markers

The molecular characterization and genetic variability between two of commercial and polyemberyonic Egyptian mango cultivars, namely: Zebda and Ewais, with thirty seedlings of their offspring were analyzed using 8 Simple Sequences Repeat (SSR) markers. LMMA _15 marker was discarded in data analysis because of producing only one band (monomorphic locus). Other seven markers produced total of 22 alleles with a high level of Polymorphism (~100 percent). The effective number of alleles ranged from 1.7, to 3.4 with average value of 1.47. Heterozygosity per locus varied from 0.00 to 0.75 with an average of 0.36. Polymorphic Information content (PIC) value scored from 0.41 to 0.70 with average of 0.57. The discrimination power (Dp) ranged between 0.11 and 0.72 with an average of 0.50 per locus. Generally, the genetic similarity values varied between 0.12 and 100% over 32 genotypes. A cluster analysis was used to determine genetic similarities. The dendrogram can be grouped into two major clusters (I and II). Cluster I consists of Ewais seedlings exhibiting 94-100% genetic similarity among them. Cluster II consists of all seedlings of Zebda cultivar exhibiting 52-100% genetic similarity and divided to two sub clusters. Seedling (Z_C_S2) was the most divergent in first sub cluster and second sub cluster exhibited less distance and consists of all other Zebda seedlings. This study additionally indicates that SSR markers are useful for distinguishing and characterizing mango genotypes. The genetic relatedness among these genotypes could provide useful information for conservation and selection of cross parents in breeding.

Microsatellite marker development in the crop wild relative Linum bienne using genome skimming.

PremiseNuclear microsatellite markers were developed for Linum bienne, the sister species of the crop L. usitatissimum, to provide molecular genetic tools for the investigation of L. bienne genetic diversity and structure.Methods and ResultsFifty microsatellite loci were identified in L. bienne by means of genome skimming, and 44 loci successfully amplified. Of these, 16 loci evenly spread across the L. usitatissimum reference nuclear genome were used for genotyping six L. bienne populations. Excluding one monomorphic locus, the number of alleles per locus ranged from two to 12. Four out of six populations harbored private alleles. The levels of expected and observed heterozygosity were 0.076 to 0.667 and 0.000 to 1.000, respectively. All 16 loci successfully cross‐amplified in L. usitatissimum.ConclusionsThe 16 microsatellite loci developed here can be used for population genetic studies in L. bienne, and 28 additional loci that successfully amplified are available for further testing.

Development of microsatellite markers for an extremely limited distributed rare diving beetle species, Acilius kishii, and a widely distributed species, Acilius japonicus (Coleoptera: Dytiscidae)

AbstractAcilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga‐Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next‐generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00–1.00 and 0.00–0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.

Comparative Genetic Characteristics of the Russian and Belarusian Populations of Wisent (Bison bonasus), North American Bison (Bison bison) and Cattle (Bos taurus)

A comparative study of the allele pool and the genetic structure of two Russian and one Belarusian populations of the wisent Bison bonasus against the representatives of the species of Bison bison and the genus Bos taurus was carried out. Russian populations were represented by samples of the Oka State Natural Biosphere Reserve (n = 42) and the Prioksko-Terrasny Nature Biosphere Reserve (n = 69), the Belarusian population was represented by samples of the Reserve “Belovezhskaya Pushcha” (n = 42), bison samples (n = 8) and cattle (n = 55) were used as an outgroup. The analysis of the mtDNA D-loop 630 bp fragment polymorphism for the presence of bison and cattle haplotypes, was performed. It was shown that there was a single haplotype for all wisent (Bison bonasus) which was different from the sequences of bison (Bison bison) and cattle. The analysis of population genetic parameters, calculated using 11 microsatellite markers, showed reduced diversity in wisent (Bison bonasus) groups compared to bison (Bison bison) and cattle. The largest number of monomorphic loci as well as the absence of private alleles were found in the group of Belarusian wisent. The analysis of the DJost pairwise genetic distances allowed us to establish clear genetic differentiation of the wisent from the outgroups. This fact was also confirmed by PCA analysis, carried out in the context of population identity and the analysis of population structure which demonstrated some proximity of bison (Bison bison) to wisent (Bison bonasus), which is due to their belonging to the same genus. The data obtained from this study showed differentiation between wisent (Bison bonasus) from bison (Bison bison) and cattle. It can be used in assessing the population genetic parameters of wisent, identifying and eliminating hybrid individuals. It also can be used in developing strategies and measures for the preservation and improvement of the wisent genetic resources.

Biochemical and molecular studies on effect of polymer seed coating of soybean

The present study was conducted at the Research Farm Adhartal, Department of Plant Breeding and Genetics, JNKVV, Jabalpur (M.P.) during Kharif season of 2016-17. Research experiment was laid out in a Randomized block design with five replications and four treatments i.e T0 No treatment or (Water + Thiram), T1- Polymer seed coating (Disco AG SP Red L-200) + Thiram + Carboxine, T2- Polymer seed coating (Disco AG SP Red L-200) + Thiram + Genius coat, T3- Polymer seed coating (Disco AG SP Red L-200) + Thiram + Mycorhiza comprised of soybean genotype JS JS 20-29. Five plants were randomly selected from each treatment and replications for Phenological and Physiological Parameters. Maximum protein % was registered in Treatments Polymer seed coating (Disco AG SP Red L-200) + Thiram + Genius coat (T2) (40.64), Treatments T3 (20.04) possessed the maximum fat % and showed differed significantly among them. The DNA amplification pattern revealed that a total number of 42 RAPD loci were amplified with an average of 7.0 loci per RAPD primer comprising 30 polymorphic loci (71.43%) and 12 monomorphic loci (28.57 %). The range of polymorphism was 50 % (OPA-01&OPA 02) to 89.0 % (OPA 07). Amplified product (band) size ranged from 300 bp (OPA-03) to 2800 bp (OPA-05). The no of bands ranged from 04 (OPA-02) to 09 (OPA -07).

Genetic diversity, population structure and runs of homozygosity in Ethiopian short fat-tailed and Awassi sheep breeds using genome-wide 50k SNP markers

Improved Awassi sheep from Israel has been used for the improvement of the productivity of Ethiopian short-fat tailed Menz and Wollo sheep breeds through crossbreeding. High density (~50,000) single nucleotide polymorphism (SNP) markers distributed along the ovine genome were analyzed for Menz (n = =34), Wollo (n = =18), local Awassi (n = =24) and improved Awassi (n = =23) sheep breeds to assess genetic diversity and population structure. Proportion of polymorphism was highest for local Awassi (96.2%) followed by the Ethiopian breeds (91.7 to 93.0%). Lowest proportion of polymorphic SNP was found in improved Awassi (84.3%). Expected heterozygosity was high (0.37) for local Awassi followed by the local Ethiopian breeds (0.35 and 0.36) and the improved Awassi (0.35). Highest genetic differentiation measured as fixation index (FST) appeared between local Ethiopian breeds and improved Awassi (FST ~ 0.14) while the two Ethiopian breeds were closely related (FST=0.004). Surprisingly, local Awassi sheep differentiated from improved Awassi at a higher level (FST = 0.09) than it differentiated from Wollo (FST = 0.058) and Menz (FST = 0.065). Principal component, model based structure and neighbor net analysis revealed clustering of populations according to their geographic location and breed development history. Strong selection pressure on the Awassi sheep resulted in significant differentiation from local Awassi, less within population variation, excess monomorphic loci and high level of runs of homozygosity (ROH) based genomic inbreeding. The high level of differentiation between local Ethiopian and improved Awassi supported the use of improved Awassi sheep to improve Menz and Wollo sheep breeds. However, importation of Awassi sheep and mating should consider strategies of controlling the level of inbreeding.

Genetic assessment of the internal transcribed spacer region (ITS1.2) in Mangifera indica L. landraces.

Mango (Mangifera indica) is one of the most important tropical fruits in the world. Twenty-two genotypes of native mangoes from different regions of southern Iran (Hormozgan and Kerman) were collected and analyzed for the ribosomal genes. GC content was found to be 55.5%. Fu and Li's D* test statistic (0.437), Fu and Li's F* test statistic (0.500) and Tajima's D (1.801) were positive and nonsignificant. A total of 769 positions were identified (319 with insertion or deletion including 250 polymorphic and 69 monomorphic loci; 450 loci without any insertion or deletion including 35 Singletons and 22 haplotypes). Nucleotide diversity of 0.309 and a high genetic differentiation including Chi square of 79.8; P value of 0.3605 and df value of 76 was observed among mango genotypes studied. The numerical value of the ratio dN/dS (0.45) indicated a pure selection in the examined gene and the absence of any key changes. Cluster analysis differentiated the mango used in this research (M. indica L.) into two genotypes but could not differentiate their geographical locations. The results of this study indicated that a high genetic distance exists between HajiGholam (Manojan) and Arbabi (Rodan) genotypes and showed higher genetic diversity in mango of Rodan region. Results of present study suggested that for successful breeding, the genotypes of Rodan region mango especially Arbabi mango can be used as a gene donor and ITS can be a suitable tool for genetic evaluationsof inter and intra species.

Genetic diversity of three sunu groupers species (Plectropomus spp)

The quality of fish seed may be decreased as a result of inbreeding that is a marriage between two individuals who have the same genetic trait. Fish seed quality improved with the genetic approach to parent selection and crossbreed. Quality of fish seeds is taking from natural populations are determined by the genetic diversity of a population. Inbreeding can decrease the quality of seed because it can increase the number of homozygosity and decrease the number of heterozygosity. This study aims to determine differences in genetic variation three species of sunu groupers (Plectropomus maculatus, Plectropomus leopardus, and Plectropomus oligacanthus) and know the kinship of the three species of grouper. While the benefits of this research can provide information on genetic diversity in natural coral trout grouper, expected inbreeding can be avoided. Muscle and liver tissue was used as a sample for electrophoresis analysis with twelve kinds of enzymes namely aspartate aminotransferase (AAT), Alcohol dehydrogenase (ADH), Estrase (EST), -Gliserofosfat dehydrogenase (-GPD), Glukofosfat isomerase (GPI), Isocitrate dehydrogenase (IDH), Lactate dehydrogenase (EST), Malic dehydrogenase (MDH), Malic Enzyme (ME), 6-phosphogluconate dehydrogenase (6- PGD), phosphoglucomutase (PGM), and sarcoplasmic enzymes (SP). Three polymorphic loci in Plectropomus oligacanthus species are 6-PGD, GPI-2, and PGM. In Plectropomus maculatus, and Plectropomus leopardus all monomorphic loci. The three polymorphic loci on Plectropomus oligacanthus meet the Hardy-Weinberg balance with X2 value for 6-PGD value is 0.123, GPI-2 is 0.028 and PGM is 0.028. Plectropomus oligacanthus has a polymorphism level of 0.188, the total number of the focusing allele is 1,154, and the average heterozygosity is 0.023. The genetic distance between the sepsis population based on twelve enzymes showed between Plectropomus maculatus, and Plectropomus leopardus had a spacing of 0.134, between Plectropomus leopardus and Plectropomus oligacanthus of genetic distance 0.196 and between Plectropomus maculatus and Plectropomus oligacanthus the genetic distance was 0.200. The larger the genetic distance between the species, the kinship relationship will be farther away. The distant kinship is owned by Plectropomus oligacanthus.

Profile of secondary metabolites and genetic stability analysis in new lines of Echinacea purpurea (L.) Moench micropropagated via somatic embryogenesis

Echinacea purpurea (L.) Moench is a plant species important for the phytopharmaceutical industry and in horticulture. Currently, there is lack of standardized plant material with an increased content of secondary metabolites in purple coneflower. The following research meets the expectations of the industry, as new selected lines of purple coneflower were micropropagated by somatic embryogenesis. The plant lines were analyzed both in terms of the content of main secondary metabolites by High Performance Liquid Chromatography (HPLC), as well as the genetic stability within the line and the genetic distance between lines using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) genetic markers. Significant differences were found in the relative percentage composition of individual phenolic acids in the tested plant material. Among six selected lines of Echinacea purpurea, three were characterized by a higher content of cichoric acid in relation to the other lines studied. A higher mean polymorphism rate (>90%) was found with the RAPD technique, with a total of 1427 scorable bands produced (142.7 products per one primer). Unlike the RAPD analysis, ISSRs detected mostly monomorphic loci (63.4%), followed by polymorphic ones (36.6%), while there were no specific loci present. Cluster analysis of both marker systems showed that the tested genotypes were grouped according to their respective lines.

Isolation and characterization of genic microsatellites from de novo assembly transcriptome in the bivalve Ruditapes philippinarum

The marine bivalve Ruditapes philippinarum (Veneridae) has always been an economically important aquaculture species. In this study, 106 831 unigenes and 2 664 SSR loci (1 locus/40 sequences) were achieved from the de novo assembly transcriptome. Among all the SSRs, tri-nucleotides (46.40%) was the most, followed by di-nucleotides (32.43%). Meanwhile, AAC/GTT (19.82%) was the most common SSR loci searched. After polymorphism detection using 32 wild R. philippinarum individuals, 34 polymorphic and 3 monomorphic SSR loci were screened, and the genetic index of them was calculated. The results show that PIC of 30 polymorphic SSR loci was at medium and high levels (PIC>0.25). However, there were five SSR polymorphic loci (e.g. MG871423, MG871428, MG871429, MG871434, MG871435) deviating from the Hardy-Weinberg equilibrium after the Bonferroni correction (adjusted P=0.001 471). The Na value (number of alleles per locus) ranged from 2 to 7. In addition, the Ho (observed heterozygosities) and He (expected heterozygosities) were 0.100 0-1.000 0 and 0.191 3-0.723 6, respectively. Therefore, RNA-Seq was shown as a fast and cost-effective method for genic SSR development in non-model species. Meanwhile, the 37 loci from R. philippinarum will further enrich the genetic information and advance the population conservation and restoration.

ANALYSIS OF GENETIC STRUCTURE OF DOGS OF FRENCH BULLDOG BREED USING MICROSATELLITE DNA MARKERS

Genetic variability of domestic dogs is a source for effective process of breed formation and creating unique gene complexes. In the world, for preservation of genetic resources of dogs, there are dog training associations which have great confidence: American Club Dog Breeders (AKC), the British Kennel Club (KC) and the Federation Cynologique Internationale (FCI), aimed at protecting breeding dogs, standards creation, registration of a breed, and issuance of accurate pedigrees.
 Evaluation of the genetic diversity of dog breeds is able to significantly complement and improve their breeding programs. Since breeds of dog differ in morphological and economic characteristics, the problem of finding of the breed features in the genome of animals is becoming more topical. From this point of view, French Bulldog is an interesting breed of dog (FRANC.BULLDOGGE, FCI standard number 101) which belongs by the classification of breeds, adopted in FCI, to the group IX – a dog-companion for health and fun, but to a subgroup of fighting dogs of a small format. French Bulldog breed has been researched slightly not only in Ukraine and also abroad, as the main work of all dog association is focused on solving theoretical and practical issues of breeding, keeping, feeding, veterinary protection and others.
 The study was conducted at Research Department of Molecular Diagnostic Tests of Ukrainian Laboratory of Quality and Safety of Agricultural Products. 33 animals of French Bulldog breed, admitted to use in dog breeding of Ukrainian Kennel Union (UKU), were involved for the genetic analysis using DNA markers. The materials of the research were buccal epithelial cells, selected before the morning feeding of animals by scraping mucous membrane of oral cavity with disposable, dry, sterile cotton swab. Genomic DNA was extracted using KIT-set of reagents for DNA isolation according to the manufacturer's instructions.
 PEZ1, PEZ3, PEZ6, PEZ8, FHC 2010, FHC 2054 markers, recommended by International Society for Animal Genetics (ISAG), ACN, КC and FCI, were used for research.
 As a result of research 25 alleles for all the loci were detected in the experimental sample of dogs. The average number of alleles at the locus Na, obtained by direct counting, was 4.16. The most polymorphic loci for this breed were PEZ6 and PEZ3 with 8 and 6 allelic variants. Monomorphic loci were PEZ8 and FHC 2054 which had 4 and 3 alleles and the lowest level of polymorphism was observed for PEZ 1 and FHC 2010 loci in which only 2 alleles were identified.
 On analyzing the molecular genetic characteristics of dogs of French Bulldog breed, we found a high variability of genotype on rare alleles, which included alleles: M, C, D, E, J, K, L, O, N and representing 60% of the total number of the identified alleles. C, D, E alleles for PEZ3 locus and O allele at PEZ6 locus are unique to the sampling of dogs because they are not repeated in other loci. Typical alleles: N, F, R, I, P, K, M are 40% of the total. But F, R alleles for PEZ3 locus and P allele for locus PEZ6 are not repeated either in standard allelic variants or in rare one, indicating a high information content of these alleles and loci to be used for further monitoring of allele pool, genetic certification and identification of dogs.
 Microsatellite DNA loci were analyzed as a result of investigations of French Bulldogs and the most informative: PEZ3, PEZ6 and PEZ8 were found, which have high efficiency in individual and breed certification of dogs due to high variability. These data allow further monitoring of the state of genetic diversity of the breed and the development of measures for improvement of breeding to preserve the structure of breeding material. The study of individual and population genetic variability is advisable to continue for breeding of French Bulldogs "in purity" and preserving valuable gene complexes.
 The results are the basis for further monitoring of the proposed informative panels of microsatellite DNA markers for genotyping dog of French Bulldog breed and their complex evaluation.

Microsatellite marker development for the tetraploid Veronica aragonensis (Plantaginaceae) using next-generation sequencing and high-resolution melting analyses.

Premise of the StudyThe tetraploid Veronica aragonensis (Plantaginaceae) is a narrow endemic to the Iberian Peninsula. Specific microsatellite markers were developed to investigate genetic structure and diversity.Methods and ResultsA total of 15 polymorphic markers were characterized on three populations of V. aragonensis, using a microsatellite‐enriched library on an Ion Torrent sequencer and high‐resolution melting (HRM) analyses to rapidly discard nonreliable, multicopy, and/or monomorphic loci. Allele number per locus ranged from one to five, and levels of observed heterozygosity per population varied from 0.142 ± 0.301 to 0.281 ± 0.369. Most primers also amplified in the closely related species V. rosea and in three subspecies of V. tenuifolia.ConclusionsThe species‐specific microsatellite markers developed here represent an essential tool to provide genetic information on the population level for V. aragonensis. The low levels of variation detected highlight the importance of continued efforts to improve conservation of the species.