Microsatellite Research Articles

A Whole-Genome Survey and the Mitochondrial Genome of Acanthocepola indica Provide Insights into Its Phylogenetic Relationships in Priacanthiformes

Acanthocepola indica, a deep-sea snake fish, is primarily found in the Indo-west Pacific region, including India, Korea, Japan, and the South China Sea. The taxonomic classification of A. indica based on morphological characteristics remains inaccurate and unclear. In this study, we utilized next-generation sequencing to generate comprehensive genomic data for A. indica. The estimated genome size of A. indica was 422.95 Mb, with a heterozygosity ratio of 1.02% and a sequence repeat ratio of 22.43%. Our analysis suggested that A. indica is diploid, and the draft genome assembly consists of 1,059,784 contigs with a contig N50 of 1942 bp. We identified a total of 444,728 simple sequence repeats in the genome of A. indica. Furthermore, we successfully assembled the complete mitochondrial genome (16,439 bp) of A. indica, which included 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. Phylogenetic analysis based on mitochondrial genomes revealed that A. indica is closely related to Acanthocepola krusensternii and Cepola schlegelii, providing evidence that the family Cepolidae belongs to the order Priacanthiformes. Population size dynamics analysis indicated that A. indica experienced a bottleneck effect during the Pleistocene Glacial Epoch, likely due to the changes in glacial cycles and sea level fluctuations since ~800 Kya.

Genetic Features of the Bream Abramis brama from the Lake System of the Sebezhsky National Park

For the first time, the analysis of the genetic variability of microsatellite DNA for five loci in the bream Abramis brama (L.) from lakes Sebezhskoye and Necheritsa of the Sebezhsky National Park, Sebezhsky district of the Pskov region, included in the lake system, was carried out. The results obtained demonstrate the uniformity of estimates of the genetic variability of bream in the lake system. Estimates of the allelic diversity of microsatellite loci and the expected heterozygosity of local bream groups did not differ significantly. The total genetic differentiation of bream was θ = 0.004. 95% CI (-0.01; 0.01) and was significantly insignificant. The population-genetic structure based on the studied multilocus genotypes has not been revealed by the Bayesian analysis method. The result indicate a high level of gene flow between local bream groupings and make it possible to assume the presence of a genetically unified panmixed population in the Sebezh lakes system.

RXLR effector genes mediate regional adaptation of Phytophthora infestans

AbstractLocal adaptation has been a central theme of eco-evolutionary research for decades. It is generally assumed that plant pathogens are locally adapted due to their standing interactions with biotic and abiotic factors in the ecosystem. Effectors, secreted small proteins encoded by pathogens, play critical roles in host–pathogen interactions, by activating host genotype-specific resistance, suppressing plant immunity, and playing other functions. In this study, we investigated the potential involvement of RXLR effector genes in ecological adaptation by examining the simple sequence repeat (SSR), virulence, and effector profiles in Phytophthora infestans isolates collected from two geographic regions differing in ecological environments. Genotypic analyses with SSR markers and virulence assays showed that the pathogen from the two regions shared genetic background but differed in virulence spectrums. High-throughput sequencing and expression analysis of 24 selected P. infestans isolates further showed variations in the RXLR effector repertoire, ranging from 536 to 548 for each isolate and the expression of effector genes was highly associated with the accumulation of homologous sRNA. Regional specific alleles were detected at 94 RXLR effector genes, and a specific accumulation of homologous 25–26 nt sRNAs was found at 67 RXLR effector genes. Two of the regional specific RXLR effector genes were confirmed to be virulence factors. Taken together, these results suggest that genomic and epigenetic variations in RXLR effector genes contribute significantly to the ecological adaptation of P. infestans populations and that regional specific effector genes will help to understand the adaptive landscape of pathogens and efficient use of host resistance genes.

Kazakhstan tulips: comparative analysis of complete chloroplast genomes of four local and endangered species of the genus Tulipa L.

Species of Tulipa are important ornamental plants used for horticultural purposes in various countries, across Asia, Europe, and North Africa. The present study is the first report on typical features of the complete chloroplast genome sequence of four local and endangered species including T. alberti, T. kaufmanniana, T. greigii, and T. dubia from Kazakhstan using Illumina sequencing technology. The comparative analyses revealed that the complete genomes of four species were highly conserved in terms of total genome size (152. 006 bp - 152. 382 bp), including a pair of inverted repeat regions (26. 330 bp - 26. 371 bp), separated by a large single copy region (82.169 bp - 82,378 bp) and a small copy region (17.172 bp -17.260 bp). Total GC content (36.58-36.62 %), gene number (131), and intron length (540 bp - 2620 bp) of 28 genes. The complete genomes of four species showed nucleotide diversity (π =0,003257). The total number of SSR loci was 159 in T. alberti, 158 in T. kaufmanniana, 174 in T. greigii, and 163 in T. dubia. The result indicated that ten CDS genes, namely rpoC2, cemA, rbcL, rpl36, psbH, rps3, rpl22, ndhF, ycf1, and matK, with effective polymorphic simple sequence repeats (SSRs), high sequence variability (SV) ranging from 2.581 to 6.102, and high nucleotide diversity (Pi) of these loci ranging from 0,004 to 0,010. For all intergenic regions longer than 150 bp, twenty one most variable regions were found with high sequence variability (SV) ranging from 4,848 to 11,862 and high nucleotide diversity (Pi) ranging from 0,01599 to 0,01839. Relative synonymous codon usage (RSCU) analysis was used to identify overrepresented and underrepresented codons for each amino acid. Based on the phylogenic analysis, the sequences clustered into four major groups, reflecting distinct evolutionary lineages corresponding to the subgenera Eriostemons, Tulipa, and Orithyia. Notably, T. greigii was distinctively grouped with species from Orithyia and Eriostemons rather than with other Tulipa species, suggesting a unique evolutionary history potentially shaped by geographical isolation or specific ecological pressures. The complete chloroplast genome of the four Tulipa species provides fundamental information for future research studies, even for designing the high number of available molecular markers.

Complete Chloroplast Genomes and Phylogenetic Analysis of Woody Climbing Genus Phanera (Leguminosae)

Background: Phanera Lour., a genus in the subfamily Cercidoideae of the family Leguminosae, is characterized by woody liana habit, tendrils, and distinctive bilobate or bifoliolate leaves. The genus holds important medicinal value and constitutes a complex group characterized by morphological diversity and unstable taxonomic boundaries. However, limited information on the chloroplast genomes of this genus currently available constrains our understanding of its species diversity. Hence, it is necessary to obtain more chloroplast genome information to uncover the genetic characteristics of this genus. Methods: We collected and assembled the complete chloroplast genomes of nine representative Phanera plants, including Phanera erythropoda, Phanera vahlii, Phanera aureifolia, Phanera bidentata, Phanera japonica, Phanera saigonensis, Phanera championii, Phanera yunnanensis, and Phanera apertilobata. We then conducted a comparative analysis of these genomes and constructed phylogenetic trees. Results: These species are each characterized by a typical quadripartite structure. A total of 130–135 genes were annotated, and the GC content ranged from 39.25–42.58%. Codon usage analysis indicated that codons encoding alanine were dominant. We found 82–126 simple sequence repeats, along with 5448 dispersed repeats, mostly in the form of forward repeats. Phylogenetic analysis revealed that 16 Phanera species form a well-supported monophyletic group, suggesting a possible monophyletic genus. Furthermore, 10 hypervariable regions were detected for identification and evolutionary studies. Conclusions: We focused on comparing chloroplast genome characteristics among nine Phanera species and conducted phylogenetic analyses, laying the foundation for further phylogenetic research and species identification of Phanera.

Determination of Twin Zygosity in Brazil: A DNA Validation of Two Short Questionnaires

Twin research in Brazil is expanding and should be built upon solid foundations using zygosity questionnaires validated through DNA. Our goals were to adapt and validate two short zygosity questionnaires to Brazilian Portuguese using DNA testing in Brazilian twins. One hundred same-sex twin pairs (27 males and 73 females; M age = 30.92, SD = 12.87) answered socio-demographic and two short zygosity questionnaires. Blood samples were collected for DNA extraction, followed by a forensic analysis on 22 autosomic short tandem repeat plus amelogenin and DYS391 markers to obtain an individual genetic profile. According to DNA tests, 17% of twin pairs were classified as dizygotic and 83% as monozygotic. Both questionnaires correctly distinguished the zygosity with 96.7% accuracy, with a high Kappa index, comparable with each original validation. These DNA validated zygosity questionnaires may foster twin research in Brazil with stronger methodology.

Comparative analysis of chloroplast genome of Lonicera japonica cv. Damaohua

Abstract Lonicera japonica is a well-known medicinal plant, and the Damaohua cultivar is one of the oldest known honeysuckle cultivars in China. The 155,151 bp chloroplast genome of this cultivar was obtained through Illumina sequencing. The genome includes a pair of inverted repeats (IRa and IRb; 23,789 bp each), a large single-copy region (88,924 bp), and a small single-copy (SSC) region (18,649 bp). In total, 127 unique genes were identified: 80 protein-coding, 39 tRNA, and 8 rRNA genes. Only ycf3 contained two introns. Eighty-nine large repetitive sequences and 54 simple sequence repeats were detected. Fifty potential RNA editing sites were predicted. Adaptive evolution analysis revealed that infA, matK, petB, petD, rbcL, rpl16, rpl2, rps3, ycf1, and ycf2 were positively selected, possibly reflecting the specific environmental adaptations of this cultivar. Sequence alignment and analysis revealed several candidate fragments for Lonicera species identification, such as the intergenic regions rpoB-petN, rbcL-accD, and psaA-ycf3. The IR region boundary and phylogenetic analysis revealed that the L. japonica cv. Damaohua chloroplast genome was most closely related to the L. japonica genome, but there were five distinct differences between the two. There are four sites with high variability between L. japonica and L. japonica cv. Damaohua with nucleotide variability (Pi) greater than 0.002, including rps2-rpoC2, atpB-rbcL, ycf1, and ycf1-trnN GUU. The differences between L. japonica and L. japonica cv. Damaohua were further confirmed by the single nucleotide polymorphism sites between these two species. Therefore, this study revealed that the chloroplast genome can serve as a universal super barcode for plant identification, which can identify differences and help distinguish Lonicera japonica from related species. An understanding of Lonicera japonica cv. Damaohua chloroplast genomics and a comparative analysis of Lonicera species will provide a scientific basis for breeding, species identification, systematic evolution analysis, and chloroplast genetic engineering research on medicinal honeysuckle plants.

Assessment of Complete Plastid Genome Sequences of Tulipa alberti Regel and Tulipa greigii Regel Species from Kazakhstan

Background. Tulipa species are economically, culturally, scientifically, and ecologically important. Tulips present taxonomic complexities that cannot be adequately resolved by examining their morphological characteristics alone or by relying on a limited selection of genetic markers. Methods. In the present study, we assessed the complete plastid sequences of Tulipa alberti Regel and Tulipa greigii Regel collected from Kazakhstan. Additionally, 14 previously published plastomes were obtained from GenBank for comparison and phylogenetic analysis. Results. The plastid genome sizes of T. alberti and T. greigii were 152,359 bp and 152,242 bp, respectively. In the plastid genomes of T. alberti and T. greigii, 136 genes were annotated, 114 of which were unique. These unique genes comprised eighty protein-coding, thirty transfer RNA, and four ribosomal RNA genes. Additionally, 415 simple sequence repeats were identified, comprising 107 tandem, 40 forward, 49 palindromic, 8 reverse, and 1 complementary repeat. Notably, the region containing ycf1 exhibited high variability and may serve as an informative DNA barcode for this genus. Conclusion. Phylogenetic analysis showed strong support for the relationships among Tulipa species, indicating the utility of plastid genome data for further taxonomic studies within the genus.

Impact of population size on population genetic analysis of Short Tandem Repeat (STR) allelic data, forensic and paternity parameters and its effect on forensic DNA analysis.

The selection of an appropriate STR allelic frequency database is the prerequisite for assessing the evidentiary value of DNA evidence. Four data sets comprising 50, 100, 200, and 500 samples were evaluated in 21 autosomal STR markers in the Indian and the Bahrain population. Allelic richness showed an increasing trend with the increase in sample size i.e., 193 and 201 (50 samples), 217 and 221 (100 samples), 255 and 238 (200 samples), and 292 and 285 (500 samples) in both the populations. TPOX and D13S317 markers did not show any increase in allele number, whereas SE33 markers showed the highest increase in both populations. With the increase in sample size, 70 (Bahrain population) and 100 (Indian population) alleles having < MAF were detected. Similarly, 37 and 47 previously undetected alleles could be detected when the sample size was increased from 50 to 500 in the Indian and Bahrain populations respectively. In the Indian population, Match probability, decreased with a 500-sample size, whereas, the PIC, PE, Heterozygosity, and PI increased with the increase in sample size. Further, database size did not show any statistical difference in the outcome of the Paternity Index value in the 50 paternity trio cases studied.

Patellae as a source of DNA in forensic and archaeological analysis.

Analysing genetic material from skeletonised human remains has become valuable in forensic and archaeological contexts. While the petrous bone is often preferred for DNA extraction, its availability is not guaranteed, and because of destructive sampling, it is not frequently used in forensic cases. This study explores the potential of patellae as an alternative source of bone material for genetic investigations. Forty-five patellae were sampled from a post-World War II mass grave and an archaeological Christian cemetery dated from the 13th to 19th centuries. A full demineralisation extraction method was used to obtain the DNA, and real-time PCR quantification was used to determine the quantity and quality of DNA. To evaluate the suitability of patellae for forensic and archaeological analyses, short tandem repeat (STR) typing was performed using the ESI17 Fast PCR amplification kit (Promega). To explore the difference in DNA yield, DNA degradation and STR typing success between the post-World War II and archaeological patellae, statistical analysis was performed. The results revealed significantly higher DNA yield and STR typing success in WWII patellae and higher degradation of DNA in archaeological patellae, highlighting the impact of environmental exposure time on genetic material preservation. Almost all WWII patellae achieved a high success rate in STR typing with full profiles generated. More than half of the archaeological patellae showed high STR typing performance and highly informative partial profiles were obtained, indicating the suitability of patellae not only for forensic purposes but also for archaeological genetic analyses.

Assembly and characterization of the complete chloroplast genome of the Colombian coffee varieties Caturra Chiroso, Bourbon Chiroso and Chiroso, Coffea arabica L. (Rubiaceae), with insights on their phylogenetic relationships

AbstractVarieties represent a defined group with differentiated characteristics derived through natural selection and/or selective breeding from within a species. In the Central-Andean region of Colombia (Urrao) there are three endemic varieties of the species Coffea arabica L [“Caturra Chiroso” (CCH), “Bourbon Chiroso” (BCH), and “Chiroso” (CHCH)], known as “Chiroso” group, globally renowned for their high quality and distinctive cup profile. Despite its significance, there is a lack of reported genomic resources or basic biological information for these. In this study, we conducted the first assembly and characterization of the complete chloroplast (Cp) genomes of these varieties and reconstructed their ancestry relationships. The Cp genomes were 155,188 bp in length (A = 30.93%; C = 19.06%; G = 18.37%; T = 31.64%); containing 131 genes, comprising 86 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. They consisted of four subregions: the large single-copy (LSC) region (85,159 bp; 83 genes), the short single-copy (SSC) region (18,136 bp; 12 genes), and the inverted repeats IRA (25,944 bp; 18 genes) and IRB (25,945 bp; 18 genes). Likewise, among 26 intraspecific varieties analyzed, CCH + BCH formed a unique haplotype, and CHCH + Bourbon + Caturra formed another. CCH and BCH featured an exclusive Cytosine mutation (SNP: C/A), position 47,413 bp (intergenic spacer region trnT(UGU)-trnL(UAA)]. Likewise, a total of 445 short tandem repeats were found in the Cp genomes (dinucleotides: 370; trinucleotides: 71; tetranucleotides: 1). Finally, the three formed a well-supported monophyletic group with conspecific varieties, being more closely related to Eastern Ethiopian-origin varieties [e.g. Berbere region], as well as with traditional ones like Typica, Bourbon, and Caturra. These coffee varieties are a valuable new genetic resource for use as a gene source for genetic improvement, biotechnology, direct exploitation and cultivation worldwide.

Unraveling the genetic and physiological potential of donkeys: insights from genomics, proteomics, and metabolomics approaches.

Donkeys (Equus asinus) have played a vital role in agriculture, transportation, and companionship, particularly in developing regions where they are indispensable working animals. The domestication of donkeys marked a significant turning point in human history, as they became essential for transportation, agriculture, and trade, especially in arid and semi-arid areas where their resilience and endurance were highly valued. In modern society, donkeys are indispensable due to their diversified applications, including meat, dairy, medicine, and functional bioproducts, supporting economic, cultural, and medical industries. Despite their critical importance, research on donkeys has historically been overshadowed with studies on horses. However, recent advancements in high-throughput sequencing and bioinformatics have significantly deepened our understanding of the molecular landscape of donkey genome, uncovering their unique adaptations, genetic diversity, and potential therapeutic applications. Microsatellite and mitochondrial DNA (mtDNA) markers have proven effective in assessing the genetic diversity of donkeys across various regions of the world. Additionally, significant strides have been made in characterizing differentially abundant genes, proteins, and metabolic profiles in donkey milk, meat, and skin, and in identifying specific genes/proteins/metabolites associated with sperm quality, motility, and reproduction. Advanced genomic technologies, such as genome-wide association studies and the identification of selection signatures, have also been instrumental in delineating genomic regions associated with phenotypic and adaptive traits. This review integrates data from diverse studies, including those on genetic diversity, transcriptomics, whole genome sequencing, protein analysis, and metabolic profiling, to provide a comprehensive overview of donkey biology. It underscores the unique characteristics of donkeys and emphasizes the importance of continued research to improve their genetic management, conservation, and agricultural use, ensuring their ongoing contribution to human societies.

Comparative Analyses of the Complete Mitogenomes of Two Oxyria Species (Polygonaceae) Provide Insights into Understanding the Mitogenome Evolution Within the Family

Oxyria (Polygonaceae) is a small genus only comprising two species, Oxyria digyna and O. sinensis. Both species have well-documented usage in Chinese herbal medicine. We sequenced and assembled the complete mitogenomes of these two species and conducted a comparative analysis of the mitogenomes within Polygonaceae. Both O. digyna and O. sinensis displayed distinctive multi-branched conformations, consisting of one linear and one circular molecule. These two species shared similar gene compositions and exhibited distinct codon preferences, with mononucleotides as the most abundant type of simple sequence repeats. In the mitogenome of O. sinensis, a pair of long forward repeat sequences can mediate the division of molecule 1 into two sub-genomic circular molecules. Homologous sequence analysis revealed the occurrence of gene transfer between the chloroplast and mitochondrial genomes within Oxyria species. Additionally, a substantial number of homologous collinear blocks with varied arrangements were observed across different Polygonaceae species. Phylogenetic analysis suggested that mitogenome genes can serve as reliable markers for constructing phylogenetic relationships within Polygonaceae. Comparative analysis of eight species revealed Polygonaceae mitogenomes exhibited variability in gene presence, and most protein-coding genes (PCGs) have undergone negative selection. Overall, our study provided a comprehensive overview of the structural, functional, and evolutionary characteristics of the Polygonaceae mitogenomes.

Genetic diversity assessment of cucumber landraces using molecular signatures

Genetic profiling of the biodiversity in cultivated crop plants is necessary to preserve important genes and utilize them in a breeding program. Cucumber is used as a model plant to study various characteristics of Cucurbitaceae. Its adaptation to a wide range of climatic conditions suggested analyzing the landraces. The present study was conducted to evaluate the differences, at the genetic level, among landraces spanning five continents. DNA extracted from fifty-six landraces selected from USDA germplasm bank to cover a global representative sample of world cucumber landraces was used for polymerase chain reaction using twenty-eight polymorphic expressed sequence tags simple sequence repeat (EST-SSR) markers. Twenty-eight EST-SSR markers covering all seven chromosomes yielded 98 bands with an average of 3.42 bands per marker. Polymorphic information content ranged from 0.00 (EC35) to 0.74 (EC17) with an average of 0.34. Six clusters provided an appropriate summary of the variation among the landraces, with the two largest groups including 32 (Asiatic) and 17 (European and American) landraces, respectively. Four small groups, three with two members, and one with one member (PI 525155-Egypt) were dissimilar to the two main groups. Landraces from the same region were often clustered together. Genetic similarity of the landraces was revealed by marker banding patterns. The locations of genetic diversity for cucumber landraces can be identified from this study.

Generation of genomic resources and phylogenetic contributions in Oxalis from desert fog oases in Peru

AbstractSeveral species in the genus Oxalis occupy Peruvian fog oases (Lomas) with a significant habitat‐adapted and endemic diversity. Acknowledging this aspect, the genus Oxalis is a conceivable group for evolutionary and biogeographic hypothesis testing; however, molecular resources for the genus still need improvement. We conducted a genome skimming approach to assemble new plastomes from 18 accessions (six species) of Oxalis collected in Lomas locations in Peru. These complete plastomes of Oxalis species (several reported for the first time) present a highly conserved composition. Our phylogenetic results were congruent with previous section‐based backbone phylogenies of Oxalis; however, a closer look at the phylogeny of sect. Carnosae revealed nonmonophyletic arrangements involving Oxalis megalorrhiza and Oxalis bulbocastanum individuals. We also propose a set of three hypervariable plastid regions as potential molecular markers. Likewise, an array of primers for nuclear simple sequence repeat markers based on the most widely distributed species, O. megalorrhiza, were listed and evaluated for their transferability to the other species under examination. These new genomic resources represent a significant development for future population, phylogenetic, and biogeographic studies in Oxalis.

Identification of a novel SNP mutation causing drop-out alleles in a paternity test using combined nest and touch-down PCR with Sanger sequencing

Background Short tandem repeat (STR) markers are widely used in forensic DNA analysis due to their ability to provide automated and standardised typing. However, incorrect STR typing can have a significant impact on forensic outcomes. Aim In this study, we detected drop-out alleles at the SE33 locus in a putative father-son pair using the Microreader™ 28 A ID System. This result could lead to a false conclusion of non-paternity. Subjects and methods To investigate the cause of the drop-out alleles, we developed a nest and touch-down PCR program for Sanger sequencing of the SE33 locus. Subsequently, we investigated the mutation frequency in 300 unrelated individuals and reviewed the results of 429 paternity tests. Results The results showed that the frequency of the G > T mutation at this locus was less than 0.01, which is a novel and rare mutation. Our analysis revealed a novel G > T mutation in the primer-binding region of both samples, which was a rare single-nucleotide mutation site in the Chinese population. This variation was found to be responsible for the drop-out alleles observed in the samples. Conclusion Our findings have important implications for optimising primer design and constructing DNA databases for forensic analysis.

Uniparental IsoDisomy: a case study on a new mechanism of Friedreich ataxia.

Friedreich's Ataxia (FRDA) is the most common hereditary ataxia and is mainly caused by biallelic GAA repeat expansion in the FXN gene. Rare patients carrying FXN point mutations or intragenic deletions are reported. We describe the first FRDA patient with a chromosome 9 segmental Uniparental isoDisomy (UPiD) unmasking a homozygous FXN expansion initially undetected by TP-PCR. The child presented with a progressive proprioceptive ataxia associated with peripheral sensory neuronopathy and severe scoliosis. Whole genome sequencing (WGS) identified a maternal segmental Uniparental Isodisomy (UPiD) encompassing FXN. Short tandem repeats analysis on WGS showed a biallelic FXN expansion. The identification of a deletion in the primer-annealing region of the TP-PCR explained the initial TP-PCR failure. This is the first documented case of FRDA caused by segmental UPiD. This case highlights the complexity of the molecular diagnosis of FRDA, and emphasises the importance of integrating results from various technical diagnostic approaches.

Comparison of the Distribution Patterns of Microsatellites Across the Genomes of Reptiles

ABSTRACTMicrosatellites or simple sequence repeats (SSRs) are prevalent across various organisms' genomes. However, their distribution patterns and evolutionary dynamics in reptile genomes are rarely studied systematically. We herein conducted a comprehensive analysis of SSRs in the genomes of 36 reptile species. Our findings revealed that the total number of SSRs ranged from 1,840,965 to 7,664,452, accounting for 2.16%–8.19% of the genomes analyzed. The relative density ranged from 21,567.82 to 81,889.41 bp per megabase (Mbp). The abundance of different SSR categories followed the pattern of imperfect SSR (I‐SSR) &gt; perfect SSR (P‐SSR) &gt; compound SSR (C‐SSR). A significant positive correlation was observed between the number of SSRs and genome size (p = 0.0034), whereas SSR frequency (p = 0.013) or density (p = 0.0099) showed a negative correlation with genome size. Furthermore, no correlation was found between SSR length and genome size. Mononucleotide repeats were the most common P‐SSRs in crocodilians and turtles, whereas mononucleotides, trinucleotides, or tetranucleotides were the most common P‐SSRs in snakes, lizards, and tuatara. P‐SSRs of varying motif sizes showed nonrandom distribution across different genic regions, with AT‐rich repeats being predominant. The genomic SSR content of the squamate lineage ranked the highest in abundance and variability, whereas crocodilians and turtles showed a slowly evolving and reduced microsatellite landscape. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that genes harboring P‐SSRs in the coding DNA sequence regions were primarily involved in the regulation of transcription and translation processes. The SSR dataset generated in this study provides potential candidates for functional analysis and calls for broader‐scale analyses across the evolutionary spectrum.

Morphological and genetic characterization of mutants of Gladiolus cultivar prince of orange for two successive vegetative generations

Gladiolus mutants developed using gamma irradiations and EMS treatments were characterized using morphological, genetic and Polymerase Chain Reactions (PCR) based Inter-Simple Sequence Repeats (ISSR) for designing future breeding strategies. As many as 7 mutants based on phenotypic variations such as colour and shape modifications were subjected to molecular characterization using 11 ISSR primers that confirmed variations. ISSR markers showed 13.88 to 36.00 percent polymorphism among the mutants was recorded, signifying variability among different mutants. UPGMA (unweighted pair group method with arithmetic mean) based analysis of cluster separated gladiolus variety prince of orange mutants into four clusters. ISSR markers were subjected for comparative analysis like effective multiplex ratio, marker index, polymorphic information content and resolving power wherein, highest EMR (3.24), MI (1.32) were found for UBC 810, Rp (4.37) values were found for UBC 827, and maximum PIC value (0.46) for ISSR 22218 UBC 812. The results clearly indicated the reproducibility of ISSRs and their ability to detect variability among the mutants. A significant level of genetic variation was depicted by ANOVA which suggested the usefulness of studies in gladiolus breeding and unique bands obtained could be utilized for mutant/ varietal identification.

Analysis of Tandem Repeats in Short-Read Sequencing Data: From Genotyping Known Pathogenic Repeats to Discovering Novel Expansions.

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genotyping known pathogenic tandem repeat loci Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy Support Protocol 1: Installation of tools and ExpansionHunter catalog modification Basic Protocol 2: Performing genome-wide genotyping of tandem repeats Basic Protocol 3: Discovering de novo tandem repeat expansions Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles.