Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS)

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Molecular characterization of Iranian dromedaries using microsatellite markers

Considering the importance of maintaining the genetic diversity in native animals, this study conducted to analyse genetic diversity in dromedary populations in the north of Kerman province, Iran, using eight autosomal microsatellite markers. Eighty-one blood samples were collected from five different populations and DNA was extracted. The highest and the lowest allele number and effective alleles were shown in YWLL08 (21 and 4) and VOLP32 (14.97 and 3.11), respectively. The expected heterozygosity varied from 0.778 in Sahra-e Jahad population to 0.847 in Nogh population. The test for Hardy-Weinberg equilibrium showed significant deviations in most loci. The mean multilocus FST value (0.057) suggested that differentiation is moderate between populations. From total genetic diversity, only 6% were due to differentiation among populations, while the remaining 94% corresponded to differences among individuals within each population. The results of the current study indicated that the Camelus dromedarius populations in the north of Kerman province have a relativity high genetic variation and the data could be useful for designing the breeding strategies and conservation. The degree of variability demonstrated implies that studied populations are rich reservoirs of genetic diversity that must be preserved. A future direction to our study can be studying all of the Iranian Camelus dromedarius populations to better evaluate the level of inbreeding and establish the appropriate conservation strategies aimed to avoid losses of genetic diversity.

Multidrug-resistant Neisseria gonorrhoeae infection in heterosexual men with reduced susceptibility to ceftriaxone, first report in Thailand

The global rapid emergence of azithromycin/ceftriaxone resistant Neisseria gonorrhoeae threatens current recommend azithromycin/ceftriaxone dual therapy for gonorrhea to ensure effective treatment. Here, we identified the first two N. gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Among 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, two isolates (NG-083 and NG-091) from urethral swab in male heterosexual patients had reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L). Both were multidrug resistant and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). NG-083 and NG-091 remained susceptible to azithromycin (MIC of 1 mg/L and 0.5 mg/L, respectively). Reduced susceptibility to ceftriaxone was associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region. NG-083 belonged to sequence type (ST) 7235 and NG-091 has new allele number of tbpB with new ST. Molecular docking revealed ceftriaxone weakly occupied the active site of mosaic XXXIV penicillin-binding protein 2 variant in both isolates. Molecular epidemiology results revealed that both isolates display similarities with isolates from UK, USA, and The Netherlands. These first two genetically related gonococcal isolates with decreased ceftriaxone susceptibility heralds the threat of treatment failure in Thailand, and importance of careful surveillance.

Investigation of the Genetic Structure of Some Common Bean (Phaseolus vulgaris L.) Commercial Varieties and Genotypes Used as a Genitor with SSR and SNP markers

Common bean is a species belonging to the Phaseolus genus of the Leguminosae family. It has economic importance due to being rich in protein, vitamin A and C, and minerals. Being one of the most cultivated species of legumes, the determination of genetic diversity in bean genotypes or populations has an important role in terms of our genetic resources. The objective of this study was to evaluate the genetic structure of 94 genotypes which were cultivated in different parts of the world and our country with SSR and SNP markers. 10 SSR loci and 73 SNP primers were used for the determination of genetic structure in commercial cultivars and breeding lines. All of the SSR and SNP loci used in the study were found to be polymorphic. A total of 89 alleles were identified for 10 SSR loci. Mean number of alleles per locus (Na=8.9), effective allele number (Ne=3.731), Shannon information index (I=1.468), observed heterozygosity (Ho=0.023), and expected heterozygosity (He=0.654) were calculated based on SSR analysis. According to the results of Bayesian-based STRUCTURE analysis using SSR and SNP data, 94 bean genotypes were genetically divided into three main clusters. According to genetic similarity based UPGMA dendrogram obtained from SSR and SNP analysis, 94 bean genotypes were divided into 2 main clusters. The obtained results provide important information about the genetic structures of the studied bean cultivars and breeding lines. With the obtained results, it will be possible to develop breeding programs to develop new cultivars by using our gene resources.

Genetic Characterization of Rainfed Durum Wheat Genotypes Based On Functional Markers Associated With Grain Quality Proteins

Analysis of genetic diversity provides helpful information necessary to develop the breeding and conservation strategies of crops. In this study, the genetic diversity and population structure of 90 durum wheat genotypes maintained at Sararud Dryland Agricultural Research Institute, Kermanshah, Iran, were evaluated by using 23 gene-specific markers (functional markers, FMs) encoding high and low molecular weight glutenin and gliadin alleles. Results showed that 12 out of the 23 FMs used were polymorphic and amplified 52 polymorphic loci. Primer Ax2 ⃰ had the highest discriminatory power. The population structure analysis classified the durum wheat collection into four populations. On average, population 4, consisting of 8 genotypes, had the highest allele number as well as genetic variation. Analysis of molecular variance indicated that 82% of the total variation was distributed among populations. The diversity among populations and gene flow were 0.14 and 3.03, respectively. The Jaccard distance coefficient revealed that genetic dissimilarities ranged from 0.031 between G62 and G65 to 0.725 between G36 and G51. Neighbor-joining method clustered individuals into six main groups. Results showed a remarkable level of genetic diversity among studied durum wheat genotypes which can be of interest for future breeding programs.

Evaluation of the Conservation Status of the Croatian Posavina Horse Breed Based on Pedigree and Microsatellite Data

The Croatian Posavina horse (CPH) is native Croatian breed under a conservation program and under various programs of economic use (ecosystem services, agrotourism, and meat production). The aim of this study was to analyze the status of the CPH population through an analysis of their pedigree (28,483 records), phenotype (292 licensed stallions, 255 mares), and genetic structure (292 licensed stallions). The average generation interval was 8.20 years, and the number of complete generations was 1.66. The effective number of founders and ancestors was 138 and 107, respectively, with a ratio of 1.29, and the genetic conservation index was 4.46. As for the morphometric characteristics, the average withers height of the stallions was 142.79 cm, the chest circumference was 194.28 cm, and the cannon bone circumference was 22.34. In mares, the withers height, chest, and cannon bone circumference were lower (139.71 cm, 190.30 cm, and 20.94 cm, respectively). Genetic microsatellite analysis of the 29 sire-lines showed high genetic diversity, expressed as the mean allele number (7.7), allele richness (4.0), and expected heterozygosity (0.740). There was no evidence of high inbreeding or a genetic bottleneck. The genetic and phenotypic data indicate that the CPH is an important and diverse reservoir of genetic diversity and can be conserved because of its special characteristics (adaptability).

Genetic Diversity of Cambuci (Campomanesia Phaea) Revealed by Microsatellite Markers

Campomanesia phaea (Myrtaceae), known as cambuci, is a native species from the Brazilian Atlantic Forest with great potential to be developed as a new fruit crop. Microsatellite markers were developed for cambuci to characterize the genetic diversity and to investigate the genetic structure of a group of accessions originally collected at the presumed center of diversity of the species. The work involved the collection of 145 accessions from five regional groups (Juquitiba, Paraibuna, Mogi das Cruzes, Ribeirão Pires, and Salesópolis) in São Paulo state, Brazil. Fourteen loci were identified in an enriched genomic library developed from one of these accessions. Six out of 14 loci revealed to be polymorphic, disclosing 26 alleles. Based on the allele frequencies, the calculated genetic parameters of the five groups indicated an average allele number per loci (A) of 3.83, with the expected heterozygosity (He) of 0.57 and the observed heterozygosity (Ho) of 0.54. The analysis of the genetic structure indicated that most of the genetic diversity is found within each population (HS = 0.57), whereas the genetic diversity among populations was low (GST = 0.19). The genetic diversity parameter of Nei was considered low for the cambuci analyzed populations, with no evidence of inbreeding. Based on Darwin analysis, we chose 18 accessions from the five regional populations to compose a core collection that includes most of the genetic diversity found in this study. Our findings may contribute to define better conservation strategies and genetic breeding approaches for this native species in Brazil.

ISSR-Based Genetic Diversity Assessment of Genus Jasminum L. (Oleaceae) from Pakistan

The genus Jasminum L., of the family Oleaceae, includes many species occurring in the wild, or cultivated worldwide. A preliminary investigation based on inter-simple sequence repeats (ISSR) was performed to assess the genetic diversity among 28 accessions, representing nine species of Jasminum from various regions, representing a range of altitudes in Pakistan. A total of 21 ISSR primers were used, which produced 570 amplified bands of different sizes, with a mean polymorphic band percentage of 98.26%. The maximum resolving power, polymorphism information content, and index values of the ISSR markers recorded for primers 6, 16, and 19 were 0.40, 12.32, and 24.21, respectively. Based on the data of the ISSR markers, the resulting UPGMA dendrogram with the Jaccard coefficient divided the 28 accessions into two main clades. At the species level, the highest values for Shannon’s information index, polymorphism percentage, effective allele number, Nei’s genetic variations, and genetic unbiased diversity were found in Jasminum sambac L. and J. humile L., while the lowest were observed in J. mesnyi Hance and J. nitidum Skan. Based on Nei’s unbiased genetic identity pairwise population matrix, the maximum identity (0.804) was observed between J. elongatum Willd and J. multiflorum (Burm. f.) Andrews, and the lowest (0.566) between J. nitidum Skan. and J. azoricum L. Molecular variance analysis displayed a genetic variation of 79% among the nine populations. The study was aimed to established genetic diversity in Jasminum species using ISSR markers. With the help of this technique, we were able to establish immense intra- and interspecific diversity across the Jasminum species.

Simple Sequence Repeat and S-Locus Genotyping to Assist the Genetic Characterization and Breeding of Polyploid Prunus Species, P. spinosa and P. domestica subsp. insititia

Polyploid Prunus spinosa (2n = 4 ×) and P. domestica subsp. insititia (2n = 6 ×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programs. In Hungary, 16 cultivar candidates and a recognized cultivar ‘Zempléni’ were selected from wild-growing populations including ten P. spinosa, four P. domestica subsp. insititia and three P. spinosa × P. domestica hybrids (2n = 5 ×) were also created. Genotyping in eleven simple sequence repeat (SSR) loci and the multiallelic S-locus was used to characterize genetic variability and achieve a reliable identification of tested accessions. Nine SSR loci proved to be polymorphic and eight of those were highly informative (PIC values ˃ 0.7). A total of 129 SSR alleles were identified, which means 14.3 average allele number per locus and all accessions but two clones could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified and the complete and partial S-genotype was determined for 10 and 7 accessions, respectively. The DNA sequence was determined for a total of 17 fragments representing 11 S-RNase alleles. ‘Zempléni’ was confirmed to be self-compatible carrying at least one non-functional S-RNase allele (SJ). Our results indicate that the S-allele pools of wild-growing P. spinosa and P. domestica subsp. insititia are overlapping in Hungary. Phylogenetic and principal component analyses confirmed the high level of diversity and genetic differentiation present within the analysed accessions and indicated putative ancestor–descendant relationships. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species but non-related accessions sharing common S-alleles may distort phylogenetic inferences.

Genetic Diversity and Population Structure of Metaphire vulgaris Based on the Mitochondrial COI Gene and Microsatellites

The earthworm species Metaphire vulgaris (a member of the Clitellata class) is widely distributed across China, and has important ecological functions and medicinal value. However, investigations into its genetic diversity and differentiation are scarce. Consequently, we evaluated the genetic diversity of five populations of M. vulgaris (GM, HD, NYYZ, QDDY, and QDY) in Yancheng, China via the mitochondrial COI gene and the novel microsatellites developed there. A total of nine haplotypes were obtained by sequencing the mitochondrial COI gene, among which NYYZ and QDDY populations had the greatest number of haplotypes (nh = 5). Further, the nucleotide diversity ranged from 0.00437 to 0.1243. The neighbor-joining trees and the TCS network of haplotypes indicated that earthworm populations within close geographical range were not genetically isolated at these small scale distances. Results of the identification of microsatellite molecular markers revealed that the allele number in 12 microsatellite loci ranged from 4 to 13. The observed heterozygosity ranged from 0.151 to 0.644, whereas the expected heterozygosity ranged from 0.213 to 0.847. The polymorphism data content of most sites was >0.5, which indicated that the designed sites had high polymorphism. Structural analysis results indicated that GM, HD, and NYYZ had similar genetic structures across the five populations. The Nei’s genetic distance between HD and NYYZ populations was the smallest (Ds = 0.0624), whereas that between HD and QDY populations was the largest (Ds = 0.2364). The UPGMA tree showed that HD were initially grouped with NYYZ, followed by GM, and then with QDDY. Furthermore, cross-species amplification tests were conducted for Metaphire guillelmi, which indicated that the presented markers were usable for this species. This study comprised a preliminary study on the genetic diversity of M. vulgaris, which provides basic data for future investigations into this species.