Allele Size Research Articles

Genetic Variation among Three Species of Genus <i>Erythroculter</i>

The genus Erythroculter (Cypriniformes, Cyprinidae, Abramidinae) is one of the most commercially important freshwater fish species inhabiting China. However, very limited information is available regarding genetic variation among Erythroculter species, therefore hindering their effective management. In this regard, microsatellite markers and sequences of mitochondrial COII and D-loop were analyzed to investigate the genetic variation among three species of genus Erythroculter. Results demonstrated that 11 microsatellite loci were amplified effectually, and the size of allele was 110–350, 94–338 and 86–384 bps in E. ilishaeformis, E. dabryi and E. mongolicus respectively. Average number of allele was 5.82, 5.73 and 6.45 per locus for E. ilishaeformis, E. dabryi and E. mongolicus respectively. Fragments of COII and D-loop was 675 and 929 bps respectively. On the basis of Shannon’s information index, heterozygosity, polymorphic information content and nucleotide diversity index (P), the microsatellite and nucleotide diversity of E. ilishaeformis was most abundant, and that of E. mongolicus was least abundant. Based on the haplotype distribution of D-loop or the allele size in microsatellite loci of EI19 or HWB05, it could be distinguished perfectly among E. ilishaeformis, E. dabryi and E. mongolicus. Some historical and environmental elements might result in the high genetic divergence and promote the speciation of E. ilishaeformis, E. dabryi and E. mongolicus. These results would be of avail in the conservation and sustainable utilization of these three species.

SSR assisted identification of mango (Mangifera indica L.) hybrids and development of DNA barcodes

The importance of varietal identification has grown dramatically on a global scale, particularly in the context of plant variety protection. To address the same, 100 new hyper-variable mango simple sequence repeats (HMSSRs) markers were screened for polymorphism among 24 mango hybrids bred at ICAR- IARI, New Delhi. Out of which, 89 polymorphic HMSSRs were used for the generation of DNA barcodes of mango hybrids. A total of 1,861 alleles with an amplicon size of 130 to 450 bp have been detected. The average number of alleles was 2.60 per locus, and PIC ranged from 0.04 (HMSSR1382) to 0.72 (HMSSR1289) with an average of 0.39. UPGMA analysis grouped these mango hybrids into two major clusters broadly representing the parental influence in hybrids. Cluster II comprised of 14 hybrids and cluster I had 10 hybrids. Model-based structure analysis revealed two gene pools, and AMOVA indicated that higher molecular variation is due to individuals (82%). The generated allelic variations of these polymorphic markers were translated into DNA barcodes by separating the allele size for each polymorphic HMSSR locus. A total 11 unique and 35 rare alleles from amplified alleles have been observed. Validation of these hybrid-specific alleles was attempted on a set of four tree replicates of the same genotypes grown at different places, of which seven hybrid-specific alleles could be validated in the present study. Overall, the present set of HMSSRs convincingly revealed to be highly informative and useful for future molecular research in mango. Furthermore, identifying hybrid-specific alleles would be highly useful in identifying and protecting mango hybrids.

Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

Characterization of Microsatellite Markers in Crossbred Ducks

Background: Chicken represents a valuable genetic resource and protein source, it remains a potential threat to human health as they serve as a reservoir for diseases and food borne pathogens. Diversification of the poultry production is one of the viable options to enhance the food production with less susceptibility to the threats from the emerging diseases and changing climatic conditions. Ducks are promising species suitable for the diversification and will be complimentary with an adaption potential from small to commercial farming. Majority of ducks in the country are desi type with a meagre proportion of improved duck strains. genetic characterization by developing duck specific microsatellite markers and designing suitable improvement program are required to be undertaken at the earliest. The microsatellite markers are extensively used for assessing genetic structure, diversity, and relationships. Information about genetic diversity of indigenous duck breeds is important to design effective improvement and conservation strategies. Therefore, the current studies aim at genetic characterization and evaluation of growth, production and reproduction traits of the crosses of local duck population besides undertaking a suitable duck improvement program. Methods: Spectrophotometric evaluation of DNA extraction, Electrophoretic evaluation of DNA extraction , microsatellite primers- Presently, a panel of 10 informative duck microsatellite markers were identified from database as reported by Alyethodi and Kumar (2010) and Huang et al., (2006) and used for the studies, Polymerase chain reaction (PCR) , Documentation of PCR products by Agarose Gel Electrophoresis, Metaphor agarose gel electrophoresis(MAGE)of microsatellites alleles, Determination of molecular size of microsatellite alleles and genotypes, Statistical analysis of population genetics data, Data on genotype of all experimental birds at ten microsatellites were compiled and analyzed using POPGENE® 3.1 software (Yeh et al., 1999) for their population genetics parameters. Results: The study was conducted at College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam, India. In this study, the genomic DNA was isolated and characterized microsatellite loci in crossbred duck (Pati x White Pekin). Ten microsatellites were used to detect polymorphisms in 50 cross bred ducks. A total of 28 nos of alleles were observed and all loci were polymorphic. The number of alleles ranged from 2 to 5 with an average of 2.6 ±0.08 per microsatellite locus. The observed and expected heterozygosity of these polymorphic makers ranged from 0.00 to 0.42 with an average number of 0.047 and 0.41 to 0.82 with an average number of 0.57, respectively. The Polymorphic markers consist of observed heterozygosities of loci were less than 0.50. The polymorphism information content (PIC) of 10 loci ranged from 0.32 to 0.78 with an average of 0.477. Microsatellite markers will be useful tool for constructing the genetic linkage map of the duck as well as comparative mapping with the chicken.

Microsatellite-Based Molecular Diversity in Sour Cherry Genotypes (Prunus cerasus L.) Cultivated in Hungary

The aim of this study was to evaluate the genetic diversity of sour cherries using SSR markers, correlate the data with phenotypic traits, and investigate the suitability of Prunus-specific microsatellite markers in this species. Nineteen sour cherry genotypes from the Fruit Research Institute in Érd, Hungary, were analyzed using twelve SSR primer pairs. The number of alleles ranged from two to ten, with a mean value of 4.67 per locus. The highest number of alleles was generated with BPPCT 007. All the primers displayed a polymorphic pattern. The most informative markers, based on the highest PIC values, were CPPCT022, BPPCT041, and BPPCT030. The genotypes were grouped based on flowering time, ripening time, and fruit weight. To determine the correlation, we have performed a regression analysis association with fruit traits and molecular markers. The marker PceGA025 appeared to have an allele size that statistically significantly correlates to flowering and ripening time. Also, BPPCT002, BPPCT007 and UCDCH17 have an allele that significantly correlates to ripening time. Additionally, one of the alleles of UDP 98 410 appeared to be correlated with fruit weight.

Validation of the Microreader™ 36A ID System for forensic genetic casework and database construction

ABSTRACT Autosomal short tandem repeats (STRs) are crucial in identifying humans. Herein, a developmental validation was performed for a newly generated autosomal STR kit (Microreader™ 36A ID System). The sensitivity of this kit was fine when the input DNA dropped down to 0.02 ng/uL. Mixture analysis showed that when male:female = 200 pg: 5400 pg, full profiles of both individuals could be obtained. When inhibited substances existed, all alleles were acquired up to 2000 μM haematin, 1000 ng/μL humic acid or 250 ng/μL tannic acid. The kit exhibited a high precision level with no more than 0.29 nt standard deviation of allele size, with the accuracy (the difference of allele size between the true allele and ladder allele) no more than 0.4 nt. The total discrimination power of this system in the Chinese Han and Hui population was larger than 1–0.1 × 10−30. For the full-sibling test, the identical-by-state (IBS) score varied from 37 to 46, while it was from 21 to 28 when using the AGCU Express marker 22 STR Kit. This kit can increase the autosomal STR information of a single PCR reaction and is promising in the casework of forensic genetic as well as database construction.

Validation study of 6 dye-based SF25™ PCR amplification kit

Ever since the inception of STR-based forensic DNA analysis for human identification and DNA profiling, the approach developed continuously. Most of these developments are pertaining to achieve the set of best polymorphic loci for analysis, high inhibitor tolerance capability, and increase in sensitivity, reduction in turnaround time, and cost-effectiveness. On the similar line, the SF25™ PCR amplification kit, a 6 fluorescent dye-chemistry based autosomal STR kit was developed, which analyses 25 STR loci viz., FAM fluorescent dye-labelled D3S1358, D13S317, D7S820, D16S539, D1S1656, and Penta E; HEX fluorescent dye-labelled TPOX, TH01, D2S1338, CSF1PO, and Penta D; SUM fluorescent dye-labelled D19S433, vWA, D21S11, D18S51, and D6S1043; LYN fluorescent dye-labelled Amelogenin, D8S1179, D5S818, D12S391, and FGA; PUR fluorescent dye-labelled D22S1045, Y-indel; D2S441 and D10S1248. The developmental validation of the SF25™ PCR Amplification reagents were carried out in this study to analyze its specificity, sensitivity, and concordance. This multiplex kit showed higher sensitivity with 125 pg of DNA template generating a complete profile (with 100% allele call). Out of 316 samples, 32samples showed aberrant triallelic pattern/aberrant heterozygous pattern at D16S539 locus. The possible discrepant homozygous condition at locus D1S1656 might be due to the invasion of alleles at D1S1656 locus present adjacent to D16S539 locus. Such invasion resulted due to the shortening of the allelic bin size at the D1S1656 locus. The allelic ladder of the SF25™ PCR amplification kit reveals the allelic range of D1S1656 from 11 to 19.3 which can be interpreted according to the casework as deemed by the end-users for their samples.

Polygenic risk prediction: why and when out-of-sample prediction R2 can exceed SNP-based heritability

In polygenic score (PGS) analysis, the coefficient of determination (R2) is a key statistic to evaluate efficacy. R2 is the proportion of phenotypic variance explained by the PGS, calculated in a cohort that is independent of the genome-wide association study (GWAS) that provided estimates of allelic effect sizes. The SNP-based heritability (hSNP2, the proportion of total phenotypic variances attributable to all common SNPs) is the theoretical upper limit of the out-of-sample prediction R2. However, in real data analyses R2 has been reported to exceed hSNP2, which occurs in parallel with the observation that hSNP2 estimates tend to decline as the number of cohorts being meta-analyzed increases. Here, we quantify why and when these observations are expected. Using theory and simulation, we show that if heterogeneities in cohort-specific hSNP2 exist, or if genetic correlations between cohorts are less than one, hSNP2 estimates can decrease as the number of cohorts being meta-analyzed increases. We derive conditions when the out-of-sample prediction R2 will be greater than hSNP2 and show the validity of our derivations with real data from a binary trait (major depression) and a continuous trait (educational attainment). Our research calls for a better approach to integrating information from multiple cohorts to address issues of between-cohort heterogeneity.

Impact of cross-ancestry genetic architecture on GWASs in admixed populations

Genome-wide association studies (GWASs) have identified thousands of variants for disease risk. These studies have predominantly been conducted in individuals of European ancestries, which raises questions about their transferability to individuals of other ancestries. Of particular interest are admixed populations, usually defined as populations with recent ancestry from two or more continental sources. Admixed genomes contain segments of distinct ancestries that vary in composition across individuals in the population, allowing for the same allele to induce risk for disease on different ancestral backgrounds. This mosaicism raises unique challenges for GWASs in admixed populations, such as the need to correctly adjust for population stratification. In this work we quantify the impact of differences in estimated allelic effect sizes for risk variants between ancestry backgrounds on association statistics. Specifically, while the possibility of estimated allelic effect-size heterogeneity by ancestry (HetLanc) can be modeled when performing a GWAS in admixed populations, the extent of HetLanc needed to overcome the penalty from an additional degree of freedom in the association statistic has not been thoroughly quantified. Using extensive simulations of admixed genotypes and phenotypes, we find that controlling for and conditioning effect sizes on local ancestry can reduce statistical power by up to 72%. This finding is especially pronounced in the presence of allele frequency differentiation. We replicate simulation results using 4,327 African-European admixed genomes from the UK Biobank for 12 traits to find that for most significant SNPs, HetLanc is not large enough for GWASs to benefit from modeling heterogeneity in this way.

Genetic characterisation of indigenous duck of Tripura state in India using microsatellite markers.

Exploring genetic variability by microsatellite markers is essential for genetic improvement, preservation of indigenous germplasm and production of high-quality offspring. Lack of information on microsatellite profiling of Indian indigenous ducks (Tripura state) has stoked curiosity in this work. Genomic DNA samples from randomly selected 36 native ducks were analysed at 25 duck-specific microsatellite loci. Alleles were separated through 3.4% MetaPhore™ agarose gel electrophoresis. Allele sizes were determined using Image Lab 6 software of GelDoc™ EZ System. Allelic data were analysed by POPGENE version 1.31. Total 112 alleles were resolved and all the loci were found polymorphic with 2 to 15 alleles across the loci. Average number of allele (Na) was 4.480 ± 0.659. Allele sizes and frequencies ranged from 96 to 357bp and 0.014 to 0.819, respectively. Average heterozygosity of Nei, effective number (Ne) of alleles and Shannon's information index (I) were 0.617 ± 0.036, 3.538 ± 0.527 and 1.184 ± 0.112, respectively. The estimates of Ne were less than the Na at all the loci, indicating prevalence of heterozygosity. Polymorphic information content (PIC) ranged from 0.252 (CAUD020) to 0.911 (CAUD019) with an average of 0.562 ± 0.040. Sixteen loci were moderate to highly polymorphic and informative (PIC ˃ 0.5). Chi-square and G-square statistics revealed Hardy-Weinberg disequilibrium at all the loci. Moderate to high level of polymorphism of the studied microsatellites indicated that these markers might be helpful for genetic characterisation and adoption of appropriate conservation strategies to exploit optimum genetic potentiality of indigenous ducks of Tripura.

Analysis of genome instability and implications for the consequent phenotype in Plasmodium falciparum containing mutated MSH2-1 (P513T).

Malarial parasites exhibit extensive genomic plasticity, which induces the antigen diversification and the development of antimalarial drug resistance. Only a few studies have examined the genome maintenance mechanisms of parasites. The study aimed at elucidating the impact of a mutation in a DNA mismatch repair gene on genome stability by maintaining the mutant and wild-type parasites through serial in vitro cultures for approximately 400 days and analysing the subsequent spontaneous mutations. A P513T mutant of the DNA mismatch repair protein PfMSH2-1 from Plasmodium falciparum 3D7 was created. The mutation did not influence the base substitution rate but significantly increased the insertion/deletion (indel) mutation rate in short tandem repeats (STRs) and minisatellite loci. STR mutability was affected by allele size, genomic category and certain repeat motifs. In the mutants, significant telomere healing and homologous recombination at chromosomal ends caused extensive gene loss and generation of chimeric genes, resulting in large-scale chromosomal alteration. Additionally, the mutant showed increased tolerance to N-methyl-N'-nitro-N-nitrosoguanidine, suggesting that PfMSH2-1 was involved in recognizing DNA methylation damage. This work provides valuable insights into the role of PfMSH2-1 in genome stability and demonstrates that the genomic destabilization caused by its dysfunction may lead to antigen diversification.

Medium and large alleles of the PGC gene are risk factors for gastric cancer.

A 100-bp insertion/deletion polymorphism in the pepsinogen C gene has been associated with the risk of gastric cancer (GC). We analyzed the relationships of the 100-bp insertion/deletion polymorphism with GC, atrophic gastritis (AG), and intestinal metaplasia (IM) in the Mexican general population (MGP). We studied the genomic DNA of subjects with GC n = 80, AG and IM n = 60, controls n = 110, and the MGP n = 97. PGC gene insertion/deletion polymorphism was identified by means of PCR, capillary electrophoresis and GeneScan software. Different allele sizes of PGC polymorphism were observed in the studied groups, from 266 bp to 499 bp, which were grouped for the analysis as short alleles of 266-399 bp, medium alleles of 400-433 bp and large alleles of 434-499 bp. Carriers of one or two medium alleles, had an increased risk of GC, with OR of 1.99 (CI95% 1.08-3.67 p = 0.026) compared to homozygotes (no medium/no medium). Previous studies have related PGC short alleles to risk for or protection against GC depending on the ethnic origin of the population. In our study, medium alleles were related to risk for GC. Further studies are required to establish the importance of this polymorphism in the origin of gastric neoplasia.

Evaluating the genetic consequences of population subdivision as it unfolds and how to best mitigate them: A rare story aboutkoalas.

The genetic consequences of the subdivision of populations are regarded as significant to long-term evolution, and research has shown that the scale and speed at which this is now occurring is critically reducing the adaptive potential of most species which inhabit human-impacted landscapes. Here, we provide a rare and, to our knowledge, the first analysis of this process while it is happening and demonstrate a method of evaluating the effect of mitigation measures such as fauna crossings. We did this by using an extensive genetic data set collected from a koala population which was intensely monitored during the construction of linear transport infrastructure which resulted in the subdivision of their population. First, we found that both allelic richness and effective population size decreased through the process of population subdivision. Second, we predicted the extent to which genetic drift could impact genetic diversity over time and showed that after only 10 generations the resulting two subdivided populations could experience between 12% and 69% loss in genetic diversity. Lastly, using forward simulations we estimated that a minimum of eight koalas would need to disperse from each side of the subdivision per generation to maintain genetic connectivity close to zero but that 16 koalas would ensure that both genetic connectivity and diversity remained unchanged. These results have important consequences for the genetic management of species in human-impacted landscapes by showing which genetic metrics are best to identify immediate loss in genetic diversity and how to evaluate the effectiveness of any mitigation measures.

Biomphalaria pfeifferi (Gastropoda: Planorbidae) in Lake Malawi and Upper Shire River, Mangochi District, Malawi: Distribution, Genetic Diversity and Pre-Patent Schistosome Infections.

In November 2017, Biomphalaria pfeifferi, the key intermediate host for Schistosoma mansoni in Africa, was first reported in Lake Malawi, Mangochi District. Two subsequent malacological surveys in 2018 and 2019 confirmed its lacustrine presence, as well as its presence along the Upper Shire River. These surveys provided sufficient specimens for analyses of the genetic structure and a transmission assessment for intestinal schistosomiasis. A total of 76 collected snails were characterized by a DNA sequence analysis of a 650 bp fragment of the mitochondrial cytochrome oxidase subunit 1 (cox1); by size fractionation of six fluorescently labelled microsatellite loci (Bgμl16, Bgμl, Bpf8, rg6, U-7, and rg9);by denaturing PAGE; and by detection of pre-patent Schistosoma infection by real-time PCR with a TaqMan® probe. Five closely related cox1 haplotypes were identified, all present within a single location, with only one haplotype common across all the other locations sampled. No allelic size variation was detected with the microsatellites and all loci were monomorphic. Overall, the pre-patent prevalence of Schistosoma spp. was 31%, with infected snails found at several sampling locations. In this part of Lake Malawi, Bi. pfeifferi exhibits low genetic diversity and is clearly being exposed to the miracidia of S. mansoni, which is likely facilitating the autochthonous transmission of this parasite.

Genome-wide association study finds multiple loci associated with intraocular pressure in HS rats.

Elevated intraocular pressure (IOP) is influenced by environmental and genetic factors. Increased IOP is a major risk factor for most types of glaucoma, including primary open angle glaucoma (POAG). Investigating the genetic basis of IOP may lead to a better understanding of the molecular mechanisms of POAG. The goal of this study was to identify genetic loci involved in regulating IOP using outbred heterogeneous stock (HS) rats. HS rats are a multigenerational outbred population derived from eight inbred strains that have been fully sequenced. This population is ideal for a genome-wide association study (GWAS) owing to the accumulated recombinations among well-defined haplotypes, the relatively high allele frequencies, the accessibility to a large collection of tissue samples, and the large allelic effect size compared to human studies. Both male and female HS rats (N = 1,812) were used in the study. Genotyping-by-sequencing was used to obtain ∼3.5 million single nucleotide polymorphisms (SNP) from each individual. SNP heritability for IOP in HS rats was 0.32, which agrees with other studies. We performed a GWAS for the IOP phenotype using a linear mixed model and used permutation to determine a genome-wide significance threshold. We identified three genome-wide significant loci for IOP on chromosomes 1, 5, and 16. Next, we sequenced the mRNA of 51 whole eye samples to find cis-eQTLs to aid in identification of candidate genes. We report 5 candidate genes within those loci: Tyr, Ctsc, Plekhf2, Ndufaf6 and Angpt2. Tyr, Ndufaf6 and Angpt2 genes have been previously implicated by human GWAS of IOP-related conditions. Ctsc and Plekhf2 genes represent novel findings that may provide new insight into the molecular basis of IOP. This study highlights the efficacy of HS rats for investigating the genetics of elevated IOP and identifying potential candidate genes for future functional testing.

Molecular analysis of Karawang local soybean genotypes with national superior varieties based on SSR analysis.

The aim of this study was to analyze the similarities between the Karawang local superior varieties and several national superior varieties based on chromosome analysis or allele size. Analysis were carried out in the laboratory of the Biogen Research Institute in 2021. There were 9 genotypes analyzed, consisting of 8 national superior varieties, namely NS Karawang, Argomulyo, Grobogan, Anjasmoro, Devon-1, Detap-1, Dena-1 and Dena-2 and 1 Karawang local soybean genotypes. The results of the analysis show that Karawang local soybean has a difference with the national superior variety. The similarity of local soybeans from Karawang to NS Karawang with varieties Agromulyo and Grobogan is 68%, with soybean varieties Anjasmoro and Devon-1 is 71%, while with soybeans Detap-1, Dena-1 and Dena-2 is 76%.

Forensic Validation Studies of a Novel 35-InDel Multiplex Polymerase Chain Reaction System

Abstract Background: A difficulty associated with forensic applications is the detection of degraded biological materials. Due to the large amplicon sizes of short tandem repeat alleles, valid genotyping results cannot be obtained from degraded biological materials. Recently, insertion/deletion (InDel) polymorphisms have been used in forensic applications for their widespread distributions in the human genome, short amplicon sizes, and low mutation rates. Purpose: Human identification InDel panels have mostly been designed for European populations. Therefore, our laboratory independently developed a multiplex polymerase chain reaction (PCR) system with 35 polymorphic InDel loci to be used for human identification in China. Forensic validation studies were conducted on this novel 35-InDel multiplex PCR system. Methods: The 35 InDel loci were screened in the database, and then used with the traditional PCR amplification and capillary electrophoresis platform combined with five-color fluorescence parallel detection technology. Validation studies were performed on this novel panel, including accuracy, repeatability and reproducibility, species specificity, sensitivity, stability, forensic case sample detection, and mixture studies. In addition, forensic efficiency assessments were conducted in populations from different continents. Results: The data of validated studies indicated that the novel 35-InDel panel was accurate, stable, and efficient for forensic purposes. For human identification, the cumulative power of discrimination values for the these 35 InDel loci in East Asian, South Asian, European, American, and African populations were 0.999999999999995, 0.999999999999995, 0.999999999999971, 0.9999999999999960, and 0.999999999998166, respectively. Conclusions: In this study, a set of 35 InDel loci were conducted in a multiplex amplification system for human identification of degraded DNA sample, and this new assay was efficient and stable. The present results suggested that the 35-InDel panel was a reliable tool for forensic use and could be efficiently used for human identification in the East Asian populations.

Genome survey sequencing and mining of genome-wide microsatellite markers in yellow-billed babbler (Turdoides affinis)

Turdoides affinis is a species of group dwelling old world passerine of family Leiothrichidae. Unavailability of genome-wide sequence and species-specific molecular markers have hindered comprehensive understanding of cooperative breeding behaviour in T. affinis. Therefore, we generated genome-wide microsatellite markers through whole genome short read sequencing of T. affinis. A total of 68.8 gigabytes of paired-end raw data were sequenced containing 195,067,054 reads. Total sequenced reads spanned a coverage of 17X with genome size of 1.18 Gb. A large number of microsatellite markers (265,297) were mined in the T. affinis genome using Krait, and 50 most informative markers were identified and validated further. In-silico PCR results validated 47 markers. Of these 47 markers, five were randomly selected and validated in-vitro in twelve individuals of T. affinis. Genotyping data on these five loci estimated observed heterozygosity (H0) and expected heterozygosity (He) ratios between 0.333 – 0.833 and 0.851–0.906, respectively. Effective allele size ranged from 6.698 to 10.667, inbreeding coefficient of the population ranged from 0.080 to 0.631 and null allele frequency was calculated at 0.055 to 0.303. Polymorphic information content of all the five loci varied between 0.850 and 0.906. Probabilities of exclusion and identity across 5 loci was estimated to be 0.95 and 0.0036, respectively. All the loci showed significant adherence to Hardy-Weinberg equilibrium. The microsatellite markers reported in this study will facilitate future population genetics studies on T. affinis and other congeneric species.

Genetic diversity assessment by microsatellite markers in summer chilli (Capsicum annuum L.) genotypes of Bangladesh

Assessment of genetic diversity among different is a prerequisite for plant breeders in choosing potential parental lines. Ten genotypes of summer growing chilli including one released variety viz., BARI morich-2; one advanced line viz., SRC- 517 and eight local cultivars viz., IAH-160, IAH-164, AHM-206, AHM-217, AC-63, AC-312, RAI-67, and RI-1(6) were characterized with a view to explore genetic diversity within these genotypes based molecular markers. Using eight microsatellite (SSR) primers across 10 genotypes, a total of 30 alleles with an average number of 3.75 alleles per locus were found. The number of alleles detected varied form three (CAMS-864, CAMS-880, CAMS-885) to five (CAMS-647). The allele size ranged from 160 (CAMS-075) to 289 bp (CAMS-864). All most all sets of primers showed high polymorphism (PIC value ≥0.6) except CAMS-880 and CAMS-885 which suggesting the greater genetic diversity in the genotypes. Gene diversity ranged from 0.460 (CAMS-885) to 0.740 (CAMS-647) and the highest and lowest value of Shannon's Information Index was registered in the same locus with their average value of 1.174. Higher level of genetic differentiation (0.971) and lower level of gene flow value (0.007) which were indicative of the presence of diversity among the genotypes. The genotypes had distinct status in the dendrogram, because of variation in genetic distance values differed from 0.138 to 0.938. SAARC J. Agric., 20(2): 69-82 (2022)

Genetic Diversity of Merozoite Surface Protein-1 and -2 Genes in Plasmodium falciparum Isolates among Asymptomatic Population in Boset and Badewacho Districts, Southern Ethiopia.

The genetic variation of Plasmodium falciparum has been studied to assess local malaria transmission genetic profile using evidence-based intervention measures. However, there are no known previous reports of P. falciparum polymorphism in Badewacho and Boset districts, Southern Ethiopia. The purpose of this study was to determine the genetic diversity of the merozoite surface protein-1 and -2 (msp-1 and msp-2) allelic families in P. falciparum isolates from an asymptomatic populations. This study was conducted from finger-prick blood samples spotted on 3 mm Whatman filter paper collected during a community-based cross-sectional study. Nested polymerase chain reaction amplification was used to type the allelic variants of msp-1 and msp-2. From 669 asymptomatic study participants, a total of 50 samples positive for P. falciparum were included for molecular analysis. Of 50 positive samples, 43 P. falciparum isolates were successfully amplified for the msp-1 and msp-2 allelic families. A total of twelve different allele sizes (75-250 bp) were identified within the three allelic families of msp-1, whereas ten different allele sizes (250-500 bp) were detected within the two allelic families of msp-2. MAD20 had a higher allelic proportion, 65% among allelic families of msp-1, whereas the 3D7 allelic family 90.7% was higher in msp-2. A slightly higher frequency of polyclonal infection 53.5% was found in msp-2 allelic family, whereas a low proportion polyclonal infection 46.5% was found in msp-1 allelic family. The overall mean multiplicity of infection (MOI) for msp-1 and msp-2 was identical (MOI = 1.56). Correspondingly, the expected heterozygosity (He) value for msp-1 (He = 0.23) and msp-2 (He = 0.22) was almost similar. The findings of this study revealed low genetic diversity of the msp-1 and msp-2 allelic families in P. falciparum isolates. However, continued monitoring status of the local genetic diversity profile in the P. falciparum population is required to support current malaria control and elimination strategies.