Microsatellite Research Articles

Nanopore-based full-length transcriptome sequencing for understanding the underlying molecular mechanisms of rapid and slow progression of diabetes nephropathy.

Diabetic nephropathy (DN) has been a major factor in the outbreak of end-stage renal disease for decades. As the underlying mechanisms of DN development remains unclear, there is no ideal methods for the diagnosis and therapy. We aimed to explore the key genes and pathways that affect the rate progression of DN. Nanopore-based full-length transcriptome sequencing was performed with serum samples from DN patients with slow progression (DNSP, n = 5) and rapid progression (DNRP, n = 6). Here, transcriptome proclaimed 22,682 novel transcripts and obtained 45,808 simple sequence repeats, 1,815 transcription factors, 5,993 complete open reading frames, and 1,050 novel lncRNA from the novel transcripts. Moreover, a total of 341 differentially expressed transcripts (DETs) and 456 differentially expressed genes (DEGs) between the DNSP and DNRP groups were identified. Functional analyses showed that DETs mainly involved in ferroptosis-related pathways such as oxidative phosphorylation, iron ion binding, and mitophagy. Moreover, Functional analyses revealed that DEGs mainly involved in oxidative phosphorylation, lipid metabolism, ferroptosis, autophagy/mitophagy, apoptosis/necroptosis pathway. Collectively, our study provided a full-length transcriptome data source for the future DN research, and facilitate a deeper understanding of the molecular mechanisms underlying the differences in fast and slow progression of DN.

Complete chloroplast genomes of Desmidorchis penicillata (Deflers) plowes and Desmidorchis retrospiciens Ehrenb.: comparative and phylogenetic analyses among subtribe Stapeliinae (Ceropegieae, Asclepiadoideae, Apocynaceae)

The succulent shrubs Desmidorchis penicillata and D. retrospiciens, part of the taxonomically challenging genus Desmidorchis, are well‐known for their ecological resilience and medicinal significance. This study sequences the first complete chloroplast genomes of these species, shedding light on their genomic characteristics and evolutionary relationships. The circular genomes of D. penicillata (161 776 bp) and D. retrospiciens (162 277 bp) display a quadripartite structure typical of Angiosperms. Gene content, order, and GC content are consistent, featuring 114 unique genes, including 80 protein‐coding, 30 transfer RNAs, and four ribosomal RNAs genes. Codon usage analysis underscores A/U‐rich preferences, while RNA editing sites, predominantly in ndhB and ndhD genes, suggest post‐transcriptional modifications. Analysis of long repeated sequences reveals a predominance of forward and palindromic repeats. Simple sequence repeats (SSRs), particularly A/T motifs, are abundant, with high presence of mononucleotide, offering potential molecular markers. Comparative analysis with their relatives in subtribe Stapeliinae identifies mutational hotspots such as ycf1, ndhF, trnG(GCC)‐trnfM(CGA) and ndhG‐ndhI that could be potential DNA barcoding markers. The inverted repeat (IR) boundaries analysis revealed an expansion of IR on the small single copy region, leading to the formation of a pseudogene. Overall, substitution rate analysis indicated purifying selection, with a few genes (rpl22, clpP and rps11) showing signatures of positive selection. Additionally, the phylogenetic analysis positioned Desmidorchis within the Stapeliinae clade and strongly supported the sister relationship between D. penicillata and D. retrospiciens. This study provides comprehensive molecular data for future research in Desmidorchis.

Differentiation of wild accessions within the Panax bipinnatifidus complex using newly developed polymorphic microsatellite markers

Abstract Panax L., renowned as ginseng genus, is a famous medicinal group of family Araliaceae. Within this genus, the taxa of Panax bipinnatifidus complex are mainly distributed in Himalayas and Hengduan Mountain areas. Due to the complex evolutionary history and short-term rapid radiation, the relationships among species within the complex have not been clearly resolved, and the taxa identification is difficult due to the intermediate morphological traits. This study aimed to use the available restriction-site associated DNA sequence data from 29 individuals of P. bipinnatifidus complex to mine high-polymorphic simple sequence repeat (SSR) markers, with the goal of evaluating their utility in taxa identification. Eleven polymorphic SSR loci were ultimately selected and validated through polymerase chain reactions amplifying across 63 individuals of P. bipinnatifidus complex and 13 individuals of three outgroup species. The subsequent genetic diversity analysis uncovered 76 alleles in total, ranging from 5 to 15 per locus. Observed heterozygosity spanned 0.241–0.512, while expected heterozygosity ranged between 0.345 and 0.644. The genetic kinship analysis revealed a sister relationship between Panax zingiberensis and Panax vietnamensis. The analysis result also supported the classification of samples from Hunan and Hubei provinces into a single genetic unit within the P. bipinnatifidus complex. These newly developed SSR markers will facilitate the identification of wild ginseng plants.

Self-assembly and condensation of intermolecular poly(UG) RNA quadruplexes.

Poly(UG) or 'pUG' dinucleotide repeats are highly abundant sequences in eukaryotic RNAs. In Caenorhabditis elegans, pUGs are added to RNA 3' ends to direct gene silencing within Mutator foci, a germ granule condensate. Here, we show that pUG RNAs efficiently self-assemble into gel condensates through quadruplex (G4) interactions. Short pUG sequences form right-handed intermolecular G4s (pUG G4s), while longer pUGs form left-handed intramolecular G4s (pUG folds). We determined a 1.05 Å crystal structure of an intermolecular pUG G4, which reveals an eight stranded G4 dimer involving 48 nucleotides, 7 different G and U quartet conformations, 7 coordinated potassium ions, 8 sodium ions and a buried water molecule. A comparison of the intermolecular pUG G4 and intramolecular pUG fold structures provides insights into the molecular basis for G4 handedness and illustrates how a simple dinucleotide repeat sequence can form complex structures with diverse topologies.

Gastric Precancerous Lesions: From Progenitor Cell and Microsatellite Instability to Clinical Interpretation of Gastric Cancer Risk

Aim: to evaluate the possibility of the MMR-system status, microsatellite instability (MSI) usage in the differential diagnosis of gastric mucosa dysplasia, determination of the gastric adenocarcinoma development risk. Material and methods. The study included gastric mucosa specimens of 75 patients: 25 with high-grade dysplasia, 25 with low-grade dysplasia, 25 were indefinite for dysplasia. Gastrobiopsy specimens were examined histologically, immunohistochemically using mouse monoclonal antibodies (Diagnostic BioSystems, USA) to the MMR system proteins: MLH-1 (clone G168-15, dilution 1:50), MSH2 (clone DBM15.82, dilution 1:100), MSH6 (clone 44, dilution 1:50), PMS2 (clone A16-4, ready to use). MSI was studied with multiplex PCR evaluation of DNA microsatellites (NR-21, NR-24, NR-27, BAT-25, BAT-26) from paraffin sections, their analysis with capillary electrophoresis. The obtained data were processed with the Statistica 10.0 (StatSoft, USA), presented using descriptive, analytical statistics. VOSviewer (1.6.20) was used to visualize the bibliometric analysis. Results. MMR-deficient cases were found in low (2.8 %) and high-grade (2.8 %) dysplasia with the immunohistochemical evaluation of MMR-system proteins in gastric mucosa specimens. In all indefinite for dysplasia cases MMR-system proteins remained unaffected. Three MSI-positive cases (6.5 %) were detected by PCR with two low-grade dysplasia, one high-grade dysplasia cases. All identified cases were also immunohistochemically MSI-positive. Conclusion. Determination of MSI can be used as an auxiliary study within a panel of biomarkers aimed to support the decision-making of a pathologist in the alternative of “indefinite for dysplasia” or “definite dysplasia — obligate precancer”.

The Complete Chloroplast Genome of Meconopsis simplicifolia and Its Genetic Comparison to Other Meconopsis Species.

Background: Chloroplasts, due to their high conservation and lack of recombination, serve as important genetic resources for the classification and evolutionary analysis of closely related species that are difficult to distinguish based on their morphological features. Meconopsis simplicifolia (M. simplicifolia), an endangered herb within the Meconopsis genus, has demonstrated therapeutic potential in treating various diseases. However, the highly polymorphic morphology of this species poses a challenge for accurate identification. Methods: In this study, the complete chloroplast genome of M. simplicifolia was sequenced and assembled using Illumina sequencing technology. Simple sequence repeats (SSRs) and repetitive sequences were characterized. In addition, a comparative analysis was conducted with the chloroplast genomes of six other Meconopsis species. Results: The chloroplast genome of M. simplicifolia has a quadripartite circular structure with a total length of 152,772 bp. It consists of a large single-copy region of 83,824 bp and a small single-copy region of 17,646 bp, separated by a pair of inverted repeat sequences (IRa and IRb, 25,651 bp). The genome contains 131 genes, 33 SSRs, and 27 long repetitive sequences. Comparative analysis with six other chloroplast genomes of Meconopsis revealed that M. simplicifolia is closely related to M. betonicifolia and that the rpl2 (ribosomal protein L2) gene in the IRb region has been deleted. This deletion is of significant importance for future taxonomic studies of M. simplicifolia. Conclusions: This study provides a valuable reference for the identification of M. simplicifolia and contributes to a deeper understanding of the phylogeny and evolution of the Meconopsis genus.

Genome-Wide Characterization and Development of Simple Sequence Repeat Markers for Molecular Diversity Analyses in Yellowhorn (Xanthoceras sorbifolium Bunge).

Yellowhorn (Xanthoceras sorbifolium Bunge) is a valuable ornamental, medicinal, and woody oilseed species that is indigenous to China. The breeding improvement of yellowhorn has been hindered by a lack of suitable markers and sufficient information regarding the molecular diversity of this species. In this study, we conducted a comprehensive analysis of the yellowhorn genome to characterize the simple sequence repeat (SSR) loci. A total of 4,007,201 SSRs were successfully identified. Among these markers, mono-nucleotide SSRs were most abundant in the genome, while the tri-nucleotide SSRs accounted for the highest proportion in coding sequences. The GO and KEGG function enrichment analysis revealed that most SSR loci in coding sequences were associated with potential biological functions. Additionally, we used 30 pairs of primers to amplify SSR markers to gain a better understanding of the genetic variation in yellowhorn germplasms. The average values of observed heterozygosity and polymorphism information content were 0.625 and 0.517, respectively. Population structure, phylogeny and principal component analyses identified two distinct subclusters. Furthermore, yellowhorn germplasms with the same geographical distribution tended to group together. Moreover, a total of 26 yellowhorn core collections, which accounted for approximately 14.94% of the total yellowhorn germplasms, effectively represented the genetic diversity of all original germplasms. Our findings not only unveiled the genetic diversity and population structure of yellowhorn germplasms but also investigated the yellowhorn core collection, which will serve as a strong basis for yellowhorn management and genetic improvement.

9194 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C<A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

7151 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C<A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

Analysis of genetic diversity and environmental associations of wild citron (Citrus medica L.) in northeast India

Citron (Citrus medica L.) is a major citrus fruit crop known for its medicinal, nutritional, and aromatic uses. Native to northeast India, Myanmar, and south China, citron is cultivated mostly in the Mediterranean and South Asian countries. The truly wild populations of citron are found in the hills and valleys of northeast India. The extent of genetic diversity in wild citron in relation to environmental variables has not been documented. In the present study, we analyzed genetic diversity and environmental association of wild populations of C. medica in northeast India using 15 polymorphic nuclear SSR (simple sequence repeat) markers and 21 environmental variables. SSR profiling revealed a moderate genetic diversity (Ho = 0.40; He = 0.41; I = 0.70). Neighbor-joining (NJ) and principal coordinate analysis (PCoA) grouped 16 natural populations into two genetic clusters. Bayesian structure results showed two genetic populations with a high admixture of individuals. Analysis of molecular variance (AMOVA) revealed low genetic differentiation (Fst = 0.14; P < 0.001), indicative of high gene flow (Nm = 1.53) in citron. The isolation by distance (IBD) analysis showed a weak correlation, indicating that geographic distance was not a determining factor for genetic distance in C. medica populations. The higher metrics of outcrossing rate (tm > 0.9) revealed outbreeding nature of the species. The genetic diversity of citron showed a positive correlation with precipitation of driest month and precipitation of coldest quarter, but negative with mean diurnal range and isothermality. Overall, the results of the present study could benefit the conservation and management of citron resources and their natural habitat in northeast India. Genetically highly diverse populations identified in the present study can be used for conservation and utilization of citron genetic resources.

Complete chloroplast genome sequence of Artemisia littoricola (Asteraceae) from Dokdo Island Korea: genome structure, phylogenetic analysis, and biogeography study.

The Asteraceae family, particularly the Artemisia genus, presents taxonomic challenges due to limited morphological characteristics and frequent natural hybridization. Molecular tools, such as chloroplast genome analysis, offer solutions for accurate species identification. In this study, we sequenced and annotated the chloroplast genome of Artemisia littoricola sourced from Dokdo Island, employing comparative analyses across six diverse Artemisia species. Our findings reveal conserved genome structures with variations in repeat sequences and junction boundaries. Notably, the chloroplast genome of A. littoricola spans 150,985bp, consistent with other Artemisia species, and comprises 131 genes, including 86 protein-coding, 37 tRNA, and 8 rRNA genes. Among these genes, 16 possess a single intron, while clpP and ycf3 exhibit two introns each. Furthermore, 18 genes display duplicated copies within the IR regions. Moreover, the genome possesses 42 Simple Sequence Repeats (SSRs), predominantly abundant in A/T content and located within intergenic spacer regions. The analysis of codon usage revealed that the codons for leucine were the most frequent, with a preference for ending with A/U. While the chloroplast genome exhibited conservation overall, non-coding regions showed lower conservation compared to coding regions, with the Inverted Repeat (IR) region displaying higher conservation than single-copy regions. Phylogenetic analyses position A. littoricola within subgenus Dracunculus, indicating a close relationship with A. scoparia and A. desertorum. Additionally, biogeographic reconstructions suggest ancestral origins in East Asia, emphasizing Mongolia, China (North East and North Central and South Central China), and Korea. This study underscores the importance of chloroplast genomics in understanding Artemisia diversity and evolution, offering valuable insights into taxonomy, evolutionary patterns, and biogeographic history. These findings not only enhance our understanding of Artemisia's intricate biology but also contribute to conservation efforts and facilitate the development of molecular markers for further research and applications in medicine and agriculture.

Genetic diversity and origin of Kazakh Tobet Dogs

The Kazakh Tobet is an indigenous Kazakh dog breed that has been used to guard livestock since ancient times. To understand the genetic structure and phylogenetic relationship of the Kazakh Tobet breed with other herding and livestock guarding dog breeds, we analysed short tandem repeat data of 107 Kazakh Tobet dogs from different regions of Kazakhstan and Mongolia, as well as whole genome sequencing data from two Kazakh Tobet dogs and 43 dogs from 24 working breeds. Our results indicate a high genetic diversity of the Kazakh Tobet, with the average number of alleles per locus ranging from 6.00 to 10.22 and observed heterozygosity ranging from 76 to 78%. The breed has a complex genetic structure characterised by seven different clusters. The neighbour-joining tree constructed based on 14,668,406 autosomal and the maximum likelihood tree based on mitochondrial D-loop sequences indicate a common genetic heritage between the Kazakh Tobet, the Central Asian Shepherd Dog and the Turkish Akbash. The presence of haplotype A18 in the Kazakh Tobets supports the hypothesis of the ancient origin of the breed, which was previously suggested by archaeological finds and written sources. These results provide an important genetic basis for the ongoing efforts to improve the Kazakh Tobet breed, to ensure its preservation as an independent genetic lineage and to recognise a breed on an international level.

Detection of putative loci affecting milk yield in Turkish Awassi sheep using microsatellite markers.

On the basis of comparisons between bovine and ovine genome mapping information, the aim of the study was to analyze the genetic diversity of selected DNA microsatellites from the bovine genome and to investigate their correlation with the average daily milk yield in Awassi sheep. 18 informative microsatellite markers were selected from the significant QTL regions affecting milk yield identified in the bovine genome in previous studies. The selected microsatellite markers were then amplified by PCR as reciprocal amplifications on the genomic DNA of Awassi sheep, with standard daily milk yield records. Thus, in this study, 18 microsatellite markers associated with milk yield in the bovine genome were examined for both determination of genetic polymorphism within the flock and the effects of marker loci on average daily milk yield in Awassi sheep. Allele frequencies of markers were determined based on the results of fragment analysis. The analysis of variance showed that the 123bp allele at the marker locus BMS1341 on BTA2 significantly influenced the average daily milk yield of Ivesi sheep (P < 0.01). On the other hand, the BMS381 locus with a 115bp allele on BTA2, the MCM140 locus with a 185bp allele on BTA6, the BMS2721 locus with a 155bp allele, the BM1237 locus with 174 and 180bp alleles on BTA7, and finally, the BMS1967 locus with a 117bp allele, the BM4208 locus with 176 and 182bp alleles, and the INRA locus with a185 bp allele on BTA8 showed moderately significant effects on the average daily milk yield of Ivesi ewes (P < 0.05).

Genetic Diversity of Local Chili (Capsicum annum L.) from West Sumatra Indonesia Based on Inter Simple Sequences Repeat (ISSR) Markers

Capsicum annum L. is the most widely cultivated species due to its economic importance and health benefits. West Sumatra is one of the largest chili pepper producers in Indonesia, so it has a lot of local chili genetic resources. There is inadequate information on the genetic diversity of local chilies in West Sumatra. The purpose of this research was to analyze the genetic diversity of 23 genotypes of chili pepper from West Sumatra amplified with 10 markers of ISSR. The material used was leaves from 23 genotypes of West Sumatra’s local chili which are collections of the Agriculture Faculty, Andalas University, Padang, West Sumatra. The results showed that ISSR markers (ISSR primer 12, ISSR UBC, and ISSR 3M) had produced 84 polymorphic DNA bands with a total polymorphic percentage of 61.45%. The dendrogram based on 10 ISSR markers showed that the 23 chili genotypes were retrieved into three main groups (I, II, III, IV) with genetic similarity coefficient values ranging from 0.67 to 0.97 or a diversity of features of 0.03–0.33 (30%). Group I consisted of 1 genotype, group II consisted of 3 genotypes, and group III consisted of 14 genotypes. Kopay chili (G20) has a large genetic distance compared to the other genotypes, while Lolai and Gero chilies have a very high similarity. This genetic diversity information can be used as a basis for generating crosses between genotypes in a breeding program. The farther the genetic distance will produce a high population diversity, the greater the chance of getting superior hybrids. Keywords: Capsicum annum, ISSR, local chili, West Sumatra

Population structure analysis of Phlebotomus papatasi populations using transcriptome microsatellites: possible implications for leishmaniasis control and vaccine development

BackgroundPhlebotomus papatasi is considered the primary vector of Leishmania major parasites that cause zoonotic cutaneous leishmaniasis (ZCL) in the Middle East and North Africa. Phlebotomus papatasi populations have been studied extensively, revealing the existence of different genetic populations and subpopulations over its large distribution range. Genetic diversity and population structure analysis using transcriptome microsatellite markers is important to uncover the vector distribution dynamics, essential for controlling ZCL in endemic areas.MethodsIn this study, we investigated the level of genetic variation using expressed sequence tag-derived simple sequence repeats (EST-SSRs) among field and colony P. papatasi samples collected from 25 different locations in 11 countries. A total of 302 P. papatasi sand fly individuals were analyzed, including at least 10 flies from each region.ResultsThe analysis revealed a high-level population structure expressed by five distinct populations A through E, with moderate genetic differentiation among all populations. These genetic differences in expressed genes may enable P. papatasi to adapt to different environmental conditions along its distribution range and likely affect dispersal.ConclusionsElucidating the population structuring of P. papatasi is essential to L. major containment efforts in endemic countries. Moreover, the level of genetic variation among these populations may improve our understanding of Leishmania–sand fly interactions and contribute to the efforts of vaccine development based on P. papatasi salivary proteins.Graphical

Generation and Characterization of hiPS Lines from Three Patients Affected by Different Forms of HPDL-Related Neurological Disorders.

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene, leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear, though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here, we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations, using episomal reprogramming. The patients' clinical characteristics are carefully listed. The hiPS lines were meticulously characterized, demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4, TRA1-60, NANOG, and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition, HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation, similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83, offering insights into its molecular mechanisms and potential for developing targeted therapies.

Limit Detection of Short Tandem Repeats (STR) Analysis on Touch DNA Samples

Forensic short tandem repeats (STR) profiling on touch DNA samples has emerged as a primary method for human identification. The stability and uniqueness of STR combination from the targeted locus in each individual make it a precision marker for human identification. Touch DNA samples can be found in traces of biological material shed from a person. This work aimed to identify the lowest concentration limit required for generating an interpretable DNA profile and the sensitivity of the STR loci applied. Touch DNA samples were collected from donors who were asked to hold a rope for 5 min. A double swab technique was used to lift the touch samples from the rope. These samples are subjected to DNA extraction and quantification. Two STR amplification cycles, 29 and 34 cycles, were used. DNA concentration greatly influences the success of amplifying the target allele at each STR locus to be interpreted into a complete DNA profile, shown by its allele peak. Touch DNA concentration &gt;0.25 ng can produce a complete DNA profile. LCN method successfully amplified touch DNA with a concentration 0.0625–0.25 ng/µL. Limit detection of touch DNA analysis is 0.25 ng/µL. Low-copy DNA can still be analyzed within 0.0625–0.25 ng/µL.

Genotyping of Candida tropicalis isolates uncovers nosocomial transmission of two lineages in Italian tertiary-care hospital

Candida tropicalis is a medically important yeast with rising antifungal resistance, while nosocomial transmission is rarely reported. Here we genotyped C. tropicalis isolates from Italian hospitals to uncover potential nosocomial transmission and assess resistance. A total of 197 C. tropicalis isolates from 161 patients was collected from five centres from 2013 to 2023. Short tandem repeat (STR) genotyping was conducted on all isolates and a selection of 24 isolates was typed with whole genome sequencing (WGS) and the novel Fourier-transform infrared (FTIR) spectroscopy method. Antifungal resistance was investigated with microbroth dilution and WGS. STR genotyping revealed seven clusters with isolates from multiple patients. WGS single nucleotide polymorphism (SNP) analysis on five groups of isolates with related STR genotypes also separated these isolates into five groups, of which two groups contained a cluster of isolates from different patients distinguished by 59 or fewer SNPs. In comparison, sequential isolates within three patients were differentiated by 141 SNPs at most. The two C. tropicalis WGS clusters also clustered based on FTIR genotyping, although this method did not separate the isolates into five groups. None of the 24 isolates were resistant to common antifungals. WGS SNP analysis indicated nosocomial transmission of two lineages within the same hospital, highlighting the need for enforced infection prevention measures and the need for routine genotyping on this common yeast in clinical settings. While both STR and FTIR genotyping also clustered these lineages, WGS SNP analysis is required to determine whether isolates were clonally transmitted.

New simple sequence repeat markers reveal undetected diversity in Spanish and Californian Diplodia sapinea populations

Diplodia sapinea is the causal agent of Diplodia shoot blight, an emerging disease affecting pine forests worldwide. The range expansion of this pathogen in northern Europe has been suggested to be partially facilitated by recent warmer conditions. Although D. sapinea has been studied extensively, critical aspects of its infection biology and population structure remain unexplored. In this study, we developed nine simple sequence repeat (SSR) markers mined from D. sapinea genomes to assess the genetic diversity at higher resolution. Isolates from northern Spain, an area formerly regarded as having low genetic diversity and samples from a Californian population that was formerly regarded as clonal, were analysed in the study. In Spain, the nine SSR markers identified 56 genotypes in 285 samples. Isolates from symptomatic shoots, cones and asymptomatic tissues collected from different stands, suggested admixture between local populations. The same genotype tended to dominate within a single cone, and the same genotypes were usually found in both symptomatic and asymptomatic shoot tissues. The nine new SSR markers developed in this study revealed a high level of genetic diversity in both the northern Spanish and northern Californian populations than previous anticipated. Analyses using these nine SSR markers should contribute to a better understanding of the epidemiology, evolution and origin of D. sapinea, a pathogen that is gaining prominence in many parts of the world.

Mining of Novel Simple Sequence Repeat (SSR) Markers Derived from Chicken (Gallus gallus) Genome Assembly

Mining of Novel Simple Sequence Repeat (SSR) Markers Derived from Chicken (Gallus gallus) Genome Assembly