Microsatellite Research Articles

Utilizing SSR-based core collection development to improve conservation and utilization of Corylus L. genetic resources.

Hazelnuts are traditional woody oilseed plants. Corylus L. resources are rich in variety and widely distributed in China. However, the identification of germplasm varieties and the selection of superior varieties remain quite limited. This study aimed to analyze the genetic diversity of 331 Corylus L. germplasms using 16 simple sequence repeat (SSR) markers. Based on this, 11 pairs of core primers were selected, a fingerprint database of germplasm resources was constructed, and a primary core collection was screened. The results indicated that these tested Corylus L. germplasms exhibited a high level of genetic diversity, with an average number of alleles (Na) per locus of 14.5 and a polymorphic information content of 0.777. The phylogenetic relationships among various hazelnut cultivars were characterized by complexity, and they were delineated into four distinct groups facilitated by genetic distance analyses. An SSR fingerprint database for 331 Corylus L. germplasms was successfully constructed using the 11 obtained core SSR markers to increase the discrimination efficiency. Ultimately, 127 primary core accessions of Corylus L. were selected. The retention rate for the observed Na and MAF (the minor allele frequency) in the primary core germplasm constructed based on a sampling proportion of 38.36% was 100% and 94.7%, respectively. Shannon's information index (I) was highly consistent between the core and original germplasms, indicating that the core germplasm could fully represent the genetic diversity of the original germplasm. Additionally, the principal coordinate analysis of the selected primary core germplasm was essentially consistent with that of the entire original germplasm, further supporting the broad representativeness of the core germplasm. This study provided a basis for precisely identifying and efficiently utilizing Corylus L. accession.

Pan-Chloroplast Genomes Reveal the Accession-Specific Marker for Gastrodia elata f. glauca.

Gastrodia elata rhizomes have been applied as traditional medicinal materials for thousands of years. In China, G. elata f. elata (red flower and stem, Ge), G. elata f. viridis (green, Gv), and G. elata f. glauca (black, Gg) represent the primary cultivars in artificial cultivation. Although the annual output of G. elata amounts to 150,000 tons, only 20% is Gg. The long production period, low yield, and high quality of Gg led to its extremely high market prices. However, an effective method to identify this crude drug based solely on its morphological or chemical characteristics is lacking. In this study, the complete chloroplast genomes of three G. elata variants were sequenced using the Illumina HiSeq 2500 platform. Another 21 chloroplast genomes from Gastrodia species, which have published in previous reports, were combined and analyzed together. Our results showed that larger genomic sizes, fewer long tandem repeats, and more simple sequence repeats were the major features of the Gg chloroplast genomes. Phylogenetic analysis showed that the Gg samples were separately clustered in a subclade. Moreover, an accession-specific marker was successfully developed and validated for distinguishing additional rhizome samples. Our study provides investigations of the taxonomic relationships of Gastrodia species. The molecular marker will be useful for differentiating Gastrodia products on the market.

Genetic Relationships and Population Structure among Nigerian Ethnic Groups (Ibibio, Igbo, Hausa, Tiv and Yoruba) Based on Nine DNA Loci

Abstract Context: Population-specific attributes typically determine the behavior of human genotypes and traits in individuals. Examining the genetic relationships between populations presents an opportunity to observe patterns of genetic changes over time. By comparing populations, it is possible to see factors that may have sparked the evolution of a trait or genotype as well as the genetic diversity of the populations. Genetic diversity in the groups in Nigeria is still largely undefined. This can be achieved with the use of genetic markers. Aims: To determine the genetic relationships between Igbo, Ibibio, Yoruba, Tiv, and Hausa ethnic groups using nine short tandem repeat DNA markers. Settings and Design: Observational studies of 250 consenting participants. Methods and Material: Individuals of Igbo, Yoruba, Hausa, Ibibio, and Tiv were randomly sampled from their ethnic homes in Nigeria at participating institutions. DNA was extracted from blood samples using commercial DNA kits, and tested for nine autosomal short tandem repeat loci alleles using PCR and electrophoresis. The allele types and sizes were read and recorded for each locus for every individual. Statistical Analysis Used: Allelic frequencies, population pairwise genetic distances (FST and RST, analysis of molecular variance (AMOVA), and principal component analysis (PCA) were calculated using GenAlEx v6.502. Results: The fixation index (FST) was between 0.001 and 0.500 in the five populations. The estimated variation was 99.98% within individuals and 0.10% among populations. Four heterogeneous clusters were observed with three axes accounting for 32.86% variation. Conclusions: The genetic relationships closely mirrored linguistic groupings showing that language may have shaped early interaction and ultimately gene transfer between these groups.

Characterisation of identity-informative genetic markers in the Australian population with European ancestry

Identity-informative single nucleotide polymorphisms (iiSNPs) are valuable genetic markers for human identification and kinship testing in forensic casework, especially when the quality and quantity of DNA evidence is not suitable for routine short tandem repeat (STR) profiling. This study analysed 105 buccal samples representing the Australian population with European ancestry in order to assign allele frequencies and conduct population genetic analyses for 94 iiSNPs and 20 STRs. The markers were assessed by calculating relevant forensic statistics and testing for deviations from Hardy-Weinberg and linkage equilibrium. No linkage of statistical significance was observed between any of the pair-wise combinations of the combined 114 identity-informative markers and only one STR exhibited deviation from Hardy-Weinberg equilibrium (D8S1179). The probability of matching genotypes being observed within this population was of the order of 10−23 for STRs, 10−38 for iiSNPs and 10−60 for the combined identity-informative marker panel, improving the ability to discriminate between individuals when calculating likelihood ratios in direct or indirect matching scenarios. Further, the addition of iiSNPs will facilitate identifications when suboptimal STR profiles are recovered from compromised or challenging samples and aid comparisons to genetic relatives for familial or kinship testing.

Leveraging genetics to support forensic toxicology analysis: Demonstrating concordance among marijuana samples

Cannabis sativa is an important plant for industrial purposes. Indeed, it is legal to cultivate and supply authorized low level Δ9-tetrahydrocannabinol (THC) cannabis plants for fiber and seeds (i.e., 0.2 % according to the European Union regulation on drugs). Today, chromatography/mass spectrometry-based procedures are successfully applied to determine THC concentration. Unfortunately, when cannabis samples have been degraded because wrongfully stored, toxicological results were affected, altering the final status of legal sample or illegal sample. Rather, genetic identification could give more information about the identity of these plants. For this reason, a degradation study was run to support how genetics can help to detect concordance in cannabis highly-degraded samples. Forty cannabis sub-samples were stored (for one week, two weeks, one month and two months) in auto-sealing bag to assess the effects of a wrong cannabis storage on weight reduction, genomic DNA changes, and corresponding STR profiles. Once respective time-period elapsed, each sub-sample’s weight was measured, the percentage weight loss calculated, and the genomic DNA was extracted and quantified, obtaining a complete STR profile for all samples. Furthermore, the aim of this study was to assess the same 13-loci short tandem repeat (STR) multiplex system used for the degradation experiment and evaluate the power of such markers in the identification and discrimination of highly degraded cannabis samples coming from real cases. The results of this study demonstrated that the 13-loci STR multiplex system successfully achieved the objective both for industry and forensic purposes. For each sample, all 13 loci were amplified, and degraded samples were correctly identified, suggesting that genetic typification could be a useful tool. The proposed procedure could be parallelly applied to toxicology analysis to detect if vegetable sample become from authorized plant, to help courts track back illegal samples or to achieve illegal cannabis genetic profiles for further comparison.

Genetic Diversity and Forensic Utility of X-STR Loci in Punjabi and Kashmiri Populations: Insights into Population Structure and Ancestry

Background: X-chromosomal short tandem repeats (X-STRs) are crucial in forensic applications, particularly in complex kinship cases, and play an important role in population genetics. However, there is limited data on X-STR variation in Pakistani populations, especially among ethnic groups like Kashmiri and Punjabi. Methodology: This study investigates the forensic and genetic properties of 12 X-STRs from the Investigator Argus X-12 Kit (QIAGEN, Hilden, Germany) in 125 families (75 Kashmiri, 50 Punjabi) from Azad Jammu and Kashmir and Punjab, Pakistan. Results: In both populations, a total of 222 alleles were identified across the 12 X-STR loci (Punjabi 171 alleles, Kashmiri 161 alleles), with allele frequencies ranging from 0.0056 to 0.3033. DXS10148 was the most polymorphic locus with 28 alleles, while DXS7132 was the least polymorphic with 9 alleles. Most loci were in linkage equilibrium, except for the DXS10135/DXS10148 pair in males, with no loci exhibiting significant linkage disequilibrium in females. The combined power of discrimination was 0.999 999 9977 for Kashmiri males, 0.999 999 999 999 9746 for Kashmiri females, and 0.999 999 999 999 9781 for Punjabi females. In Kashmiri males, 34, 31, 28, and 32 haplotypes were observed across the four linkage groups (LG1, LG2, LG3, and LG4), though these groups did not form stable haplotypes, as indicated by Linkage Equilibrium within and significant Linkage Disequilibrium between groups. Conclusions: Genetic structure analysis using Principal Component Analysis and STRUCTURE revealed distinct clustering patterns for the Kashmiri and Punjabi populations, indicating unique genetic backgrounds and ancestry influences, particularly distinguishing them from East Asian populations. This study provides a comprehensive analysis of X-STR variation in Punjabi and Kashmiri populations, offering valuable insights for forensic and population genetic studies.

Microsatellites explorer: A database of short tandem repeats across genomes

Short tandem repeats (STRs) are widespread, repetitive elements, with a number of biological functions and are among the most rapidly mutating regions in the genome. Their distribution varies significantly between taxonomic groups in the tree of life and are highly polymorphic within the human population. Advances in sequencing technologies coupled with decreasing costs have enabled the generation of an ever-growing number of complete genomes. Additionally, the arrival of accurate long reads has facilitated the generation of Telomere-to-Telomere (T2T) assemblies of complete genomes. Nevertheless, there is no comprehensive database that encompasses the STRs found per genome across different organisms and for different human genomes across diverse ancestries. Here we introduce Microsatellites Explorer, a database of STRs found in the genomes of 117,253 organisms across all major taxonomic groups, 15 T2T genome assemblies of different organisms, and 94 human haplotypes from the human pangenome. The database currently hosts 406,758,798 STR sequences, serving as a centralized user-friendly repository to perform searches, interactive visualizations, and download existing STR data for independent analysis. Microsatellites Explorer is implemented as a web-portal for browsing, analyzing and downloading STR data. Microsatellites Explorer is publicly available at https://www.microsatellitesexplorer.com.

“CLADE-FINDER”: Candida auris Lineage Analysis Determination by Fourier Transform Infrared Spectroscopy and Artificial Neural Networks

In 2019, Candida auris became the first fungal pathogen included in the list of the urgent antimicrobial threats by the Centers for Disease Control (CDC). Short tandem repeat (STR) analysis and whole-genome sequencing (WGS) are considered the gold standard, and can be complemented by other molecular methods, for the genomic surveillance and clade classification of this multidrug-resistant yeast. However, these methods can be expensive and require time and expertise that are not always available. The long turnaround time is especially not compatible with the speed needed to manage clonal transmission in healthcare settings. Fourier transform infrared (FTIR) spectroscopy, a biochemical fingerprint approach, has been applied in this study to a set of 74 C. auris isolates belonging to the five clades of C. auris (I-V) in combination with an artificial neural network (ANN) algorithm to create and validate “CLADE-FINDER”, a tool for C. auris clade determination. The CLADE-FINDER classifier allowed us to discriminate the four primary C. auris clades (I-IV) with a correct classification for 96% of the samples in the validation set. This newly developed genotyping scheme can be reasonably applied for the effective epidemiological monitoring and management of C. auris cases in real time.

Genetic Diversity and Association Analysis of Traits Related to Water-Use Efficiency and Nitrogen-Use Efficiency of Populus deltoides Based on SSR Markers.

Populus deltoides is one of the primary tree species for bioenergy production in temperate regions. In arid/semi-arid northern China, the scarcity of water and nitrogen significantly limits the productivity of poplar plantations. The identification of relevant molecular markers can promote the breeding of resource-efficient varieties. In this study, 188 genotypes of P. deltoides from six provenances served as experimental material. Genetic differentiation analysis, analysis of molecular variance (AMOVA), principal coordinate analysis (PCoA), unweighted pair group method with arithmetic mean (UPGMA) clustering, and genetic structure analysis were performed using selected simple sequence repeat (SSR) markers. Based on these analyses, the association analysis of water-use efficiency (WUE) and nitrogen-use efficiency (NUE) were conducted using general linear model (GLM) and mixed linear model (MLM) approaches. The results showed that 15 pairs of SSR primers successfully amplified across all 188 individuals, with an average of 7.33 alleles (Na) observed per primer pair. The polymorphism information content (PIC) ranged from 0.060 to 0.897, with an average of 0.544, indicating high genetic diversity in the selected markers. The average inbreeding coefficient intra-population (Fis), inbreeding coefficient inter-population (Fit), and inter-population genetic fraction coefficient (Fst) values were 0.005, 0.135, and 0.132, respectively, indicating high heterozygosity, substantial inbreeding within populations, and moderate genetic differentiation, with an average gene flow (Nm) of 1.964, suggesting substantial gene flow between populations. Additionally, molecular variance was primarily within individuals (84.12%). Genetic structure analysis revealed four subgroups, with some degree of genetic admixture among the provenances. In the GLM model, 11 markers were significantly associated with five traits (p < 0.05), with an average contribution rate of 15.82%. Notably, SSR132 and SSR143 were significantly associated with multiple traits (p < 0.05). The MLM model identified two markers (SSR47 and SSR85) significantly associated with ground diameter (p < 0.05) and one marker (SSR80) significantly associated with NUE (p < 0.05). This study identifies loci associated with WUE and NUE, laying a foundation for future genetic improvement and marker-assisted breeding strategies in poplar.

Genetic diversity and fingerprinting of Elaeagnus angustifolia based on SSR molecular markers

DNA fingerprinting can reveal the genetic diversity of Elaeagnus angustifolia germplasm resources and clarify the source and genetic background of E. angustifolia germplasm, which are the preconditions for the breeding of new varieties, the identification and protection of germplasm resources, and the comprehensive development of the E. angustifolia industry considering both ecological and economic benefits. We employed 11 pairs of primers with high polymorphism, clear bands, and high reproducibility to analyze the genetic diversity of 150 E. angustifolia germplasm accessions from Gansu and Beijing by the simple sequence repeat (SSR) molecular markers. We then employed the unweighted pair-group method with arithmetic means (UPGMA) to perform the cluster analysis based on genetic distance and analyzed the genetic structure of the 150 germplasm accessions based on a Bayesian model in Structure v2.3.3. The genetic diversity analysis revealed the mean number of alleles (Na) of 7.636 4, the mean number of effective alleles (Ne) of 2.832 6, the mean Shannon genetic diversity index (I) of 1.178 1, the mean Nei's gene diversity index (H) of 0.582 1, the mean observed heterozygosity (Ho) of 0.489 9, the mean expected heterozygosity (He) of 0.584 0, the mean polymorphism information content (PIC) of 0.535 4, and the mean genetic similarity (GS) of 0.831 5. These results suggested that the E. angustifolia germplasm resources we studied exhibited significant genetic differences and rich genetic diversity. The cluster analysis revealed that the tested materials can be classified into 3 groups, with the main genetic distance (GD) of 0.422 9. The clustering results were not completely consistent with the geographic origin. The population structure analysis classified the germplasm accessions into 2 populations. We used 8 pairs of primers with high PIC to construct the fingerprints of 150 E. angustifolia germplasm accessions. The present study successfully constructs the DNA fingerprints and clarified the genetic relationship of the E. angustifolia germplasm resources in Gansu and Beijing, providing a theoretical basis for germplasm resource identification, breeding of elite varieties, application in gardening, and molecular-assisted breeding of E. angustifolia.

Comprehensive genome analysis and variant detection at scale using DRAGEN.

Research and medical genomics require comprehensive, scalable methods for the discovery of novel disease targets, evolutionary drivers and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size or location. Here we present DRAGEN, which uses multigenome mapping with pangenome references, hardware acceleration and machine learning-based variant detection to provide insights into individual genomes, with ~30 min of computation time from raw reads to variant detection. DRAGEN outperforms current state-of-the-art methods in speed and accuracy across all variant types (single-nucleotide variations, insertions or deletions, short tandem repeats, structural variations and copy number variations) and incorporates specialized methods for analysis of medically relevant genes. We demonstrate the performance of DRAGEN across 3,202 whole-genome sequencing datasets by generating fully genotyped multisample variant call format files and demonstrate its scalability, accuracy and innovation to further advance the integration of comprehensive genomics. Overall, DRAGEN marks a major milestone in sequencing data analysis and will provide insights across various diseases, including Mendelian and rare diseases, with a highly comprehensive and scalable platform.

Morphological and molecular characterization of some Chrysanthemum (Dendranthema grandiflora) cultivars

The diversity and genetic relationships among seven commercial chrysanthemum cultivars were analyzed using morphological and molecular markers. Vegetative growth, flowering, flower yield, and flower quality parameters were evaluated to assess genetic variability across the cultivars. Cultivars Crystal Red, Kodiack, and Crystal White exhibited superior vegetative growth, while Abrun, Crystal Red, and Kodiack displayed better flowering characteristics, particularly in terms of the number of inflorescences per plant and mass of colored flowering. Crystal White, Coca Bleach, and Crystal Red cultivars demonstrated the highest inflorescence stalk length, while cvs. Crystal Red, Crystal Yellow, Crystal Pink, and Kodiack Yellow recorded the maximum number of ray floret inflorescences. Other quality parameters such as inflorescence diameter and ray floret length were found to be optimal in Kodiack, Crystal White, Crystal Pink, Coca Bleach, and Crystal Yellow cultivars. Simple Sequence Repeat (SSR) markers were employed to distinguish and identify standard-type chrysanthemum cultivars, utilizing twelve SSR markers from the chrysanthemum SSR database. The results suggest that these SSR markers hold promise for identifying additional chrysanthemum cultivar types and assessing genetic relationships among them. Association studies combining morphological and molecular data offer a valuable approach to identifying informative markers for plant breeding purposes.

Diversity of Improved Diploids and Commercial Triploids from Musa spp. via Molecular Markers

Banana breeding consists of obtaining diploid, triploid, and tetraploid intra- and interspecific hybrids by conventional breeding methods with the objective of aggregating characteristics of agronomic and commercial interest. Given the narrow genetic base of bananas, Embrapa’s Banana Genetic Breeding Program (BGBP) aims at crosses between improved diploids (ID) (ID × ID) and between improved diploids (ID) and commercial triploids (ID × CTP) and tetraploids (ID × CTT), in order to increase the genetic base and variability in bananas regarding agronomic traits of interest and resistance to main biotic and abiotic factors. These improved diploids are resistant to main fungal diseases such as yellow (YSD) and black Sigatoka (BSD) disease and Fusarium wilt (race 1 and subtropical race 4), the latter being one of the most devastating diseases in bananas. The genetic diversity between 22 improved diploids and seven commercial banana triploids was analyzed using DNA molecular markers. Five IRAP (Inter-Retrotransposon Amplified Polymorphism, 7 ISSR (Inter-Simple Sequence Repeats) and 12 SSR (Simple Sequence Repeat) markers were used. The genetic dissimilarity matrix was based on the Jaccard dissimilarity index; clusters were separated using the UPGMA (Unweighted Pair Group Method With Arithmetic Mean) method and cophenetic correlation of 0.8755. This study of the genetic diversity between improved diploids and commercial triploids, based on the genetic dissimilarity matrix, revealed that the most dissimilar diploids were DM23 and DM15 (74%) and DM16 and DM15 (74%). The smallest genetic distances between the improved diploids and commercial triploids were between TCGN25 and DM17 (50%) and TCN26 and DM17 (50%). The genetic distance matrix also revealed important genotypes to be used in crosses in order to maintain good characteristics in commercial triploids when crossed with improved diploids. The results of our study provide better breeding strategies for one of the largest banana-breeding programs worldwide focused on the development of banana varieties resistant to main biotic and abiotic factors.

The genetic diversity and population structure of wild and cultivated Avena species in Ethiopia using a SSR markers

Oats are grains that can be consumed by both animals and humans. They have thrived in Ethiopia, where certain oat species are considered native to the region. This work represents the first investigation of the population structure and genetic diversity of Ethiopian and other country oats. This led the scientists to explore the genetic diversity and population structure of wild and cultivated Ethiopian oats (Avena) species as well as oat cultivated in USA, the Netherlands and Austria. This study's main objective looks to be to investigate the variation in genetic makeup of cultivated and wild oat species. Studying the population structure of the oat species in the germplasm of Ethiopia, USA, the Netherlands and Austria. We used nineteen fluorescent SSR (simple sequence repeat) markers since previous research had indicated that these markers had high PIC (polymorphism information content) values. Five species of Avena were studied among the 176 oat accessions: A. sativa (cultivated oats) and four wild oats, such as A. abyssinica, A. vaviloviana, A. fatua, and A. sterilis. The AMOVA investigation revealed significant genetic distinctions among populations, individuals, and within individuals, explaining 18 % of the variance within populations, 4 % among populations, and 78 % within individuals. The AMOVA analysis of Avena species demonstrated extensive variance, with 33 % variation among species and 67 % within each species, underscoring robust species differentiation. The study also discovered gene interchange between wild oat and cultivated populations, defining two Avena species: domesticated oats and wild oats. Using the STRUCTURE software at K = 2, PCoA, and UPGMA, a distinct genetic structure was displayed in the dataset. Despite variations in ploidy levels and genomes, A. sterilis and A. vaviloviana were determined to be more closely linked, whereas A. abyssinica and A. fatua demonstrated a close association. This research delivers valuable insights for scientists and can be employed in oat breeding programs to improve future oat yield and productivity.

Assessment of Genetic Diversity and Population Structure of Exotic Sugar Beet (Beta vulgaris L.) Varieties Using Three Molecular Markers.

Sugar beet (Beta vulgaris L.) is a biennial herb belonging to the Amaranthaceae family. It contributes to approximately 30% of the world's total sucrose production and is an economically important crop. In this study, we analyzed the genetic diversity and population structure of 132 exotic sugar beet varieties using three molecular makers: four pairs of simple sequence repeat (SSR) primers, three pairs of insertion-deletion sequence (InDel) primers, and 20 cis-element amplification polymorphism (CEAP) primers. The results indicated that the number of alleles (Na) was 298, among which the number of effective alleles (Ne) was 182.426 (accounting for approximately 61.2%). The mean value of the genetic diversity index was 0.836. The polymorphic information content (PIC) was 0.639-0.907 (mean = 0.819), indicating a high level of polymorphism. These sugar beet varieties were classified into six clusters using the UPGMA method of cluster analysis. Population structure analysis revealed that the most ideal K value was 6. This indicated that the test materials could be divided into six categories, consistent with the clustering results. The clustering results indicated that most sugar beet varieties from the same breeding company clustered together, and the genetic distance between them was small, indicating that they may share the same male and/or female parent. Some varieties from different companies clustered together, indicating a narrow genetic base and potential exchange of germplasm resources between breeding companies. This study revealed the genetic differences among exotic sugar beet varieties and characteristics of the population structure. It provided a scientific basis for the identification of sugar beet varieties and markers-assisted breeding in China in the future.

Comparative and phylogenetic analysis of the chloroplast genomes of four commonly used medicinal cultivars of Chrysanthemums morifolium

‘Boju’ and ‘Huaiju’ are cultivars of the Chrysanthemum (Chrysanthemum morifolium Ramat.) in the family Asteraceae, valued for their medicinal, tea, and ornamental properties, and valued by individuals. However, the yield and quality of medicinal chrysanthemums are limited by the characteristics of the germplasm resources, including the identification at the varieties and cultivation levels. Currently, research characterizing the chloroplast genomes of medicinal Chrysanthemum flowers is relatively limited. This study conducted chloroplast whole-genome sequencing on two cultivars of Chrysanthemum, ‘Boju’ and ‘Huaiju’, and compared them with the previously published chloroplast genomes of ‘Hangbaiju’ and ‘Gongju’. The study analyzed the chloroplast genome structures of these four medicinal Chrysanthemums, identifying mutation hotspots and clarifying their phylogenetic relationships. The chloroplast genome sizes of four medicinal Chrysanthemum cultivation products ranged from 151,057 to 151,109 bp, with GC content ranging from 37.45% to 37.76%. A total of 134 genes were identified, including 89 protein-coding genes, 37 ribosomal RNA genes, and 8 transfer RNA genes. Comparative genomic analysis revealed 159 large repeat sequences, 276 simple sequence repeats, 1 gene, and 8 intergenic regions identified as highly variable regions. Nucleotide diversity (Pi) values were high (≥ 0.004) for the petN-psbM, trnR-UCU-trnT-GGU, trnT-GGU-psbD, ndhC-trnV-UCA, ycf1, ndhI-ndhG, trnL-UGA-rpl32, rpl32-ndhF, and ndhF-ycf1 fragments, aiding in variety identification. Phylogenetic analysis revealed consistent results between maximum likelihood and Bayesian inference trees, showing that the four medicinal Chrysanthemum cultivars, along with their wild counterparts and related species, evolved as a monophyletic group, forming a sister clade to Artemisia and Ajania. Among the six Chrysanthemum species, the wild Chrysanthemum diverged first (Posterior probability = 1, bootstrap = 1,000), followed by Ajania, while C. indicum and C. morifolium clustered together (Bootstrap = 100), indicating their closest genetic relationship. The chloroplast whole-genome data and characteristic information provided in this study can be used for variety identification, genetic conservation, and phylogenetic analysis within the family Asteraceae.

Characterization of a gene conferring resistance to US Russian wheat aphid biotypes in the Iranian wheat landrace PI 625139

AbstractRussian wheat aphid (RWA, Diuraphis noxia Kurdjumov) is a highly invasive and destructive wheat pest evolving rapidly to overcome host resistance. Novel genes conferring resistance to multiple RWA biotypes are needed to sustain wheat production. The Iranian landrace PI 625139 is resistant to all five US RWA biotypes. To map the RWA resistance gene in PI 625139, both F2 and F2:3 populations were developed from cross PI 625139 × Smith's Gold, and the F2:3 population was evaluated for responses to five RWA biotypes, RWA1, RWA2, RWA3/7, RWA6, and RWA8. RWA assays identified a single gene in PI 625139, designated Dn625139, conditioning resistance to all five US RWA biotypes. Selective genotyping of a subset of F2 plants using genotyping‐by‐sequencing (GBS) revealed a set of single‐nucleotide polymorphism (SNP) markers on the short arm of chromosome 7D that are closely associated with RWA resistance. Kompetitive allele‐specific PCR (KASP) markers were developed from selected GBS‐SNPs in 7DS. Genetic analysis using the new KASP and simple sequence repeat (SSR) markers placed Dn625139 to a 2.6 cM interval. Dn625139 was 1.2 cM proximal to the KASP marker Stars‐KASP1007 (169.57 Mb) and 1.4 cM distal to the SSR marker Stars1039 (195.27 Mb) in the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v2.1 reference sequence, and co‐segregated with the SSR marker Stars1029 (180.82 Mb). Dn625139 is a valuable gene conferring resistance to multiple RWA biotypes, and the molecular markers developed in this study can facilitate its rapid introgression into locally adapted cultivars to enhance wheat RWA resistance.

Characterization of Bread Wheat Genotypes Using SSR Markers for Terminal Heat Tolerance

Wheat is a major global food crop, but its productivity is increasingly threatened by terminal heat stress due to climate change. This study aimed to characterize the genetic diversity and heat tolerance of widely grown bread wheat genotypes using SSR markers to identify heat-tolerant cultivars adaptable to various regions in Bangladesh. A total of 15 genotypes were screened, and 13 polymorphic SSR (Simple Sequence Repeats) markers were used to determine the genetic similarity and categorize genotypes based on their heat tolerance. The molecular analysis revealed 13 polymorphic SSR markers that produced distinct PCR (Polymerase Chain Reaction) bands across the different genotypes. By the conclusion of the study, bread wheat genotypes were categorized as either tolerant or sensitive to heat, and the genetic similarity among the varieties was assessed using molecular markers. The genetic similarity coefficients obtained from the SSR primer screening ranged from 0.00 to 0.925. The lowest genetic distance (0.000) was found in Nadi 2 vs BAW1147 variety pair indicating that they are genetically similar to each other. Comparatively higher genetic distance (0. 925) was observed between BAW 1290 vs BARI Gom 28. The dendrogram divided the fifteen genotypes into two main groups, A and B, both formed at a similar coefficient of 0.05. Group A included seven genotypes and was further divided into two clusters, while Group B comprised eight genotypes, which were also divided into two clusters. The categorization of the genotypes was based on the average Heat Susceptibility Index (HSI), Thousand Grain Weight (TGW), grain yield, and the relative reduction in TGW and grain yield under stress conditions compared to timely sown conditions, aligning with the molecular data. Among the fifteen genotypes, BARI Gom 25, BARI Gom 26, BARI Gom 27, BARI Gom 28, BARI Gom 29, BARI Gom 30, and BARI Gom 31 demonstrated their adaptability to late sown conditions. The heat tolerance observed in these genotypes, as indicated by their SSR marker scores, is anticipated to provide valuable insights for molecular breeding studies focused on heat resistance.

Isolated Lateralized Overgrowth - Phenotypic Spectrum and Molecular Alterations.

To evaluate the molecular aberrations at 11p15.5 locus in thirty-two patients with isolated lateralized overgrowth (ILO). Among selected 32 cases of ILO, methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) was performed initially followed by short tandem repeats (STR) marker analysis to confirm uniparental disomy (UPD). In those patients with normal MLPA reports, cyclin dependent kinase inhibitor 1C (CDKN1C) gene and whole exome sequencing was performed. Molecular analysis by MS-MLPA showed methylation aberrations in 28% (9/32) of patients. Gain of methylation at IC1 imprinting center (H4, H7) and loss of methylation at IC2 (H6, H9) was observed in 2 patients each. Uniparental disomy was observed in 9% cases. Except one, all patients with methylation aberration had more than one limb hypertrophy. Two patients (H22/H29) also had loss of methylation at IC1. Though this molecular alteration is specifically associated with Silver Russel syndrome (SRS), but the affected children did not completely fulfill the diagnostic criteria for SRS. In a recent study, a discrepancy was reported between the diagnosis of Beckwith-Wiedemann syndrome (BWS)/SRS and the molecular findings in the patients. Many times, it is very difficult to differentiate between hemi hypertrophy/hemi hypotrophy. Patients, in whom no aberrations were detected on MS-MLPA, whole exome sequencing (WES) was performed and no pathogenic variant was identified. Thus, ILO may be considered as a mild presentation on the extreme edge of BWS spectrum with methylation aberration and UPDin one thirdof cases which has implications in follow up.

Abstract B058: Nous-209 off-the-shelf vaccine targets neoantigens mutations well represented both in primary and metachronous incident CRCs from Lynch syndrome patients

Abstract Lynch Syndrome (LS) is the most common hereditary cancer syndrome, caused by germline mutations in one of the four DNA mismatch repair (MMR) genes. LS patients have a high risk to develop microsatellite instable (MSI) colorectal cancers. Tumors developed by LS carriers display high levels of microsatellite instability, which leads to the accumulation of large numbers of mutations, among which frameshift insertion/deletions (indels) within microsatellite (MS) loci are very common and shared among different tumors. When indels occur in coding genes they give rise to frameshift peptides (FSPs) with a very high potential to act as safe and potent neoantigens for a vaccine. NOUS-209 is a genetic vaccine encoding 209 neoantigens, shared across sporadic and hereditary MSI tumors, developed for interception and treatment of MSI tumors. Phase 1b trial evaluating NOUS-209 monotherapy in healthy LS carriers showed the induction of a potent and broad immune response. Here, we characterize indels encoding FSPs in a cohort of 50 incident MSI CRC from LS patients detected during routine screening (38 primary tumors; 22 metachronous tumors) by whole Exomeseq NGS. Overall, primary and metachronous tumors have a similar number of indels in coding genes. The repertoire of detected indels is only partially shared (27%) between the two categories of tumors. The 209 indels selected for the NOUS-209 vaccine are instead highly shared (95%) between primary and metachronous tumors. Moreover, the analysis of 8 patients for which both the primary and the metachronous tumor were available confirmed the presence of the 209 mutations both in the first and the second tumor. Therefore, the NOUS 209 vaccine has the potential to prevent tumor occurrence both in previvors and survivors by inducing a broad T cell immune response against recurrent FSP. Citation Format: Lorenzo De Marco, Elisa Micarelli, Joni Panula, Anna Morena D'Alise, Guido Leoni, Kalle E. Hokkanen, Eija Hämäläinen, Jukka-Pekka Mecklin, Elisa Scarselli, Toni Seppälä. Nous-209 off-the-shelf vaccine targets neoantigens mutations well represented both in primary and metachronous incident CRCs from Lynch syndrome patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B058.