Integrating morphology, nuclear SSR, and chloroplast genomes to investigate hybrid origin and species delimitation in the Lycium barbarum L. and its varieties in China

BackgroundLycium has economic and ecological significance, but its natural hybridization has received little attention. Lycium qingshuiheense and L. ningxiaense exhibit intermediate morphological traits between or blended with L. barbarum and L. ruthenicum, inferring their potential hybrid origin. To clarify this hypothesis, the present study investigated their hybridization using morphological traits, nuclear simple sequence repeats (SSRs) markers, and chloroplast genomic data.ResultsComparative morphological analysis clearly distinguished L. barbarum from L. ruthenicum based on petiole length, leaf morphology and fruit color. In contrast, most L. qingshuiheense and L. ningxiaense individuals exhibited intermediate traits between L. barbarum and L. ruthenicum, although some L. ningxiaense individuals overlapped morphologically with L. ruthenicum. The genetic composition of all L. qingshuiheense and L. ningxiaense individuals represented a genetic mix of the two parent species, all of which are hybrids. The maximum likelihood (ML) and bayesian inference (BI) analyses based on the chloroplast genome revealed nearly congruent topologies, providing a strongly supported (BS = 100%; PP = 1) for a shared clade comprising L. qingshuiheense, L. ningxiaense, and L. ruthenicum.ConclusionBased on analyses of morphological traits, SSR markers, and cp. genome data, L. qingshuiheense and L. ningxiaense are likely hybrids resulting from out-crossing between L. ruthenicum and L. barbarum. However, genetic composition of L. qingshuiheense is derived from approximately 85% L. ruthenicum and 15% L. barbarum, Similarly, the variant of L. ningxiaense shows a genetic composition consisting of 40% derived from L. ruthenicum and 60% from Lycium barbarum. The present study elucidated the origin of two Lycium species, which enhances our understanding of the evolutionary mechanisms underlying interspecific hybridization in Lycium. These findings clarify the evolutionary history of these taxa, as well as provide molecular tools and genetic insights for developing improved Lycium varieties with desirable agronomic traits.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12150-3.

Cassava, family Euphorbiaceae, is the fifth most important staple food crop. The study of variability and diversity present in diverse cassava accessions maintained at the field genebank of TCRS, TNAU is essential to design the breeding program of cassava in TNAU. Hence, a study was carried out to assess the genetic diversity based on morphological traits and Simple Sequence Repeat (SSR) markers, identifying the SSR markers linked to a cassava mosaic disease-resistant QTL region. Genetic diversity among cassava germplasm accessions was estimated using 28 morphological traits and three SSR markers. The polymorphic information content (PIC) result gives high information for all the SSR markers. Morphological traits revealed 10 clusters and the SSR markers revealed 13 clusters at a similarity coefficient of 2.514 and 0.75, respectively. The SSR marker diversity revealed that the most promising clusters (II and XII) possessing accessions with yield attributing traits and cluster IX were grouped together due to low yield and CMD susceptibility. Morphological descriptors revealed variance for yield contributing traits. The first three principle components accounted for 10.8, 9.1, and 8.4%, respectively. Correlation studies showed significant correlation among yield and yield-attributing traits, which in turn influences yield. The morphological traits diversity reveals that cluster VI was the most promising cluster in which genotypes ME209 and ME460 possess higher yield and starch content with cassava mosaic disease (CMD) resistance. Most of the accessions grouped under cluster X belong to selection from International Centre for Tropical Agriculture (CIAT) collections and the cultivars under clusters I and II were true cassava seed (TCS) selections of India. The CMD-linked SSR marker which is reported in the previous studies have generated eight alleles and each were regressed with CMD resistance and resulted in non-significant linkage to CMD resistance.

Registration of crop varieties under Indian PPV & FR Act requires establishment of their Distinctiveness, Uniformity and Stability (DUS). At present, DUS of crop varieties are being established using morphological traits. DNA marker assay-based DUS testing would complement that based on morphological traits. Four markers, from a total of 220 tested SSR markers were polymorphic among two advanced breeding lines and two elite germplasm accessions (used as candidate varieties), and two released varieties (used as reference varieties) of dolichos bean. The four polymorphic simple sequence repeat (SSR) markers were used to establish distinctness of candidate varieties from reference varieties based on alleles unique to candidate varieties. Uniformity of candidate varieties were established using two criteria, (1) per cent homozygous plants (> 95%) and (2) SSR-homozygous loci ratio (HLR) (> 95%).The candidates which were found uniform at tested SSR loci were deemed stable. The candidate varieties were distinct from reference varieties at two of the four tested polymorphic SSR markers, as evident by the presence of alleles unique to candidate varieties. One hundred per cent homozygous plants and SSR-HLR within all the candidate varieties at all the four tested SSR marker loci suggested uniformity and deemed-stability of candidate varieties. The study demonstrated the utility of SSR markers to establish DUS of ABL/elite germplasm accessions in dolichos bean.

A set of morphological traits and SSR markers were used to determine the genetic relationship among 12 elite thermosensitive genic male sterile (TGMS) lines developed at three different research institutions of India. Agro-morphological data recorded on 20 morphological traits revealed a wide base of genetic variation and a set of four morphological traits could distinguish most of the TGMS lines. Analysis with 30 SSR markers (20 EST-SSRs and 10 genomic SSRs) revealed 27 markers to be polymorphic, amplifying a total of 83 alleles. Each SSR marker amplified 2-6 alleles with an average of 2.76 alleles per marker and a PIC value varying from 0.54 to 0.96. Cluster analysis based on SSR and morphological data clearly differentiated the lines according to their source of origin. Correlation analysis between morphological and molecular data revealed a very poor association (r = 0.06), which could be attributed to selection pressure, genetic drift, sampling error and unknown relationship among related lines. The SSR markers discriminated the genotypes distinctly and quantified the genetic diversity precisely among the TGMS lines. Data on the yield per plant indicated that the genotypes grouping under a similar cluster showed same heterotic behaviour as compared to the genotypes from different clusters when crossed to similar pollinators.

BackgroundDespite the rapid advancement of high-throughput sequencing, simple sequence repeats (SSRs) remain indispensable molecular markers for various applied and research tasks owing to their cost-effectiveness and ease of use. However, existing SSR markers cannot meet the growing demand for research on lotus (Nelumbo Adans.) given their scarcity and weak connections to the lotus genome.MethodsUsing whole-genome resequencing, active SSR loci were identified throughout the genomes of eight typical Asian lotus. After that, high polymorphism SSR molecular markers were mined from each 2n + 0.5 Mb site on each chromosome (e.g., Chr.1-2.5, 4.5, 6.5 Mb) through four steps: online primer design, primer pair evaluation, agarose gel electrophoresis testing using six Asian lotus, one American lotus, and two their hybrids, and DNA sequence alignment. Finally, the polymerase chain reaction (PCR) efficiency of several SSR markers was validated in 20 Asian temperate lotus, eight Asian tropical lotus, and one American lotus.ResultsA total of 463 SSR markers were developed based on each 2n + 0.5 Mb site of the eight lotus chromosomes (totaling 821.29 Mb). These markers were evenly distributed throughout the lotus genome at a density of 1 SSR per 1.76 Mb. The chromosomal locations of the SSR markers were determined precisely, and the specificity of the primer pairs for each site was verified by sequencing the PCR products. We further provided a set of genome-wide SSR loci, covering 129 per Mb, identified from eight representative Asian lotus, allowing other researchers to independently discover specific SSR markers for particular experiments.ConclusionThese SSR markers, which have a density of 1 SSR marker per 1.76 Mb in this study, will act as a bridge connecting lotus phenotypes with the genome. This work reveals a novel and convenient strategy for developing highly polymorphic SSR markers at any location throughout the lotus genome, and it sheds light on the development of SSR molecular markers in other plant species.

Narbon vetch (Vicia narbonensis L.) is a promising forage legume with good resistance to cold and drought. The assessment of genetic diversity of Narbon vetch is an essential component in germplasm management. In this study, we analysed the genetic diversity of 13 local and introduced Narbon vetch accessions from three continents using 27 morphological traits and 13 simple sequence repeat (SSR) markers. Significant differences among accessions for morphological and phenological traits were observed. The SSR markers showed a total of 126 alleles with a mean number of two alleles per locus. Polymorphic information content values were in the range of 0.772–0.915 with an average of 0.858. A high level of diversity (Nei’s genetic differentiation index of 59) was observed among accessions. Analysis of genetic distances separated the studied accessions into three groups based on both morphological and SSR markers. Cluster analysis of the SSR markers separated the accessions into three groups according to geographical origin. The Tunisian populations shared the same morphological traits but differed genetically from each other and were similar to those from Lebanon. A significant correlation was detected between morphological traits and SSR markers. The results suggested that SSR markers can be used to efficiently distinguish Narbon vetch accessions and estimate their genetic diversity.

Due to its beneficial effects on river ecosystems, black alder (Alnus glutinosa) is one of the tree species selected for planting on riverbanks in the cross-border area encompassing Wallonia in Belgium, Lorraine in France, and Luxembourg. The preservation of this species, however, is threatened by an invasive pathogen that particularly targets and kills young alder individuals. The objectives of this study were to characterize the genetic diversity and the genetic structure of A. glutinosa at this local level with the aim of assisting the conservation and replanting strategies and to determine if a germplasm collection comprising individuals from the same cross-border area captures the diversity present in the region. Nuclear simple sequence repeat (SSR) and chloroplastic DNA (cpDNA) markers were used to analyze four local wild populations and the germplasm collection which is representative of two river catchments and six legal provenance regions. Three populations distant from the studied area were also included. A panel of 14 nuclear SSR loci revealed high allelic diversity and very low differentiation among wild populations (mean F ST = 0.014). The germplasm collection displayed a range of alleles that were representative of the different populations, and no significant differentiation between the germplasm collection and the local wild populations was observed, making this collection, as far as allelic diversity is concerned, suitable for providing trees for riverbank replanting programs. Using SSR markers, various statistical approaches consistently indicated the lack of a significant geographical structure at the level of the river catchments or provenance regions. In contrast, two cpDNA haplotypes were detected and displayed a cross-border geographically structured distribution that could be taken into account in defining new cross-border provenance regions.

BackgroundThis study aimed to develop a set of perfect simple sequence repeat (SSR) markers with a single copy in the cotton genome, to construct a DNA fingerprint database suitable for authentication of cotton cultivars. We optimized the polymerase chain reaction (PCR) system for multi-platform compatibility and improving detection efficiency. Based on the reference genome of upland cotton and 10× resequencing data of 48 basic cotton germplasm lines, single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers. The SSR markers were detected by denaturing polyacrylamide gel electrophoresis (PAGE) for initial screening, then fluorescence capillary electrophoresis for secondary screening. The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.ResultsUsing bioinformatics techniques, 1 246 SSR markers were designed from 26 626 single-copy SSR loci. Adopting a stepwise (primary and secondary) screening strategy, a set of 60 perfect SSR markers was selected with high amplification efficiency and stability, easy interpretation of peak type, multiple allelic variations, high polymorphism information content (PIC) value, uniform chromosome distribution, and single-copy characteristics. A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.ConclusionsA set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established. This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

Comparative Analysis of Genetic Diversity and Structure in Rice Using ILP and SSR Markers

The characterisation of traditional barley varieties from North Africa is promising with respect to detect traits linked with resilience to drought. Nine Algerian barley varieties comprising the economically relevant local landraces Saïda183 and Tichedrett were genotyped by random amplifying polymorphic DNA (RAPD) and by Simple Sequence Repeat (SSR) markers to get insight into their phylogenetic relationship and to compare the efficiency of the two approaches. The SSR markers were chosen such that all chromosomes of barley were represented by at least one marker. In addition, a principal component analysis (PCA) of morpho-agronomical traits was conducted. We found that the phylogeny based on the RAPD markers reflected the geographical distribution of the tested varieties, while the phylogeny based on the SSR markers reflected the pedigree. Among the RAPD markers, the maximal polymorphism information content was obtained for BY14 with a value of 0.82, for the SSR markers, Bmg13 was most informative with a value of 0.60. The similarity matrix used to the construct the phylogenetic tree, inferred a close relationship (Jaccard Index 0.8 based on RAPD markers) between the accessions Fouara and Oued-Athmania occurring in close vicinity. The SSR markers group the autochthonous varieties Saïda183 and Tichedrett together with a Jaccard Index of 0.8. The close phylogenetic relationship between Saïda183 and Tichedrett is also supported by the PCA of morphological and agronomic traits. Our data underpin the diversity of Algerian barley varieties and lend support to the autochthonous origin of these two Algerian landraces.

Kentucky bluegrass (Poa pratensis L.) is an important perennial forage and amenity turfgrass species used throughout temperate regions in North America and Europe. Kentucky bluegrass cultivars and accessions exhibit a wide range of ploidy levels, morphological traits, turfgrass quality characteristics, and tolerances to both biotic and abiotic stresses. As such, cultivar classification systems have been developed to categorize Kentucky bluegrass cultivars and accessions on the basis of pedigree, turf performance, morphological traits, and genetic relatedness (DNA markers). The objectives of the current study were to assess the genetic diversity among and within Kentucky bluegrass entries using simple sequence repeat (SSR) markers, and to categorize new germplasm in the Kentucky bluegrass classification system. In this study, 21 SSR markers were used to genotype eight individuals from each of 144 bluegrass cultivars and accessions (1152 total samples). The SSR markers successfully categorized all entries into distinct classification groups and provided justification for a partial revision to the previous DNA marker‐based classification system. The majority of cultivars and accessions were uniquely identified with the current set of SSR markers, and SSR marker‐based genetic relationships of individuals within classification types showed agreement with breeding history records. Over 60 new Kentucky bluegrass National Turfgrass Evaluation Program entries were placed into updated classification groups, and the SSR markers used in this study can be used in the future to genotype and assign new cultivars and accessions into Kentucky bluegrass classification types and assess the genetic relatedness among entries.

BackgroundLiriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm.ResultsWe performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79.ConclusionsThe genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

India is blessed with an abundance of diverse rice landraces in its traditional cultivated areas. Two marker systems (simple sequence repeats (SSR) and single nucleotide polymorphism (SNP)) were used to study a set of 298 rice landrace accessions collected from six different regions of India (Andaman and Nicobar Islands, Chhattisgarh, Jharkhand, Uttar Pradesh, Uttarakhand, and West Bengal). Thirty hyper-variable simple sequence repeats (HvSSRs) and 32,782 single nucleotide polymorphisms (SNPs) were used in inferring genetic structure and geographical isolation. Rice landraces from Uttar Pradesh were the most diverse, with a gene diversity value of 0.42 and 0.49 with SSR and SNP markers, respectively. Neighbor-joining trees classified the rice landraces into two major groups with SSR and SNP markers, and complete geographical isolation was observed with SSR markers. Fast STRUCTURE analysis revealed four populations for SSR markers and three populations for SNP markers. The population structure with SSR markers showed that few individuals from Uttarakhand and Andaman and Nicobar Islands were grouped in small clusters. Population structure analysis with SNP markers showed not very distinct region-wise clustering among the rice landraces. Discriminant analysis of principal components (DAPC) and minimum spanning network (MSN) using SSR markers showed region-wise grouping of landraces with some intermixing, but DAPC and MSN with SNP markers showed very clear region-wise clustering. Genetic differentiation of rice landraces between the regions was significant with both SSR (Fst 0.094–0.487) and SNP markers (Fst 0.047–0.285). A Mantel test revealed a positive correlation between the genetic and geographic distance of rice landraces. The present study concludes that rice landraces investigated in this study were very diverse, and unlinked SSR markers show better geographical isolation than a large set of SNP markers.

Only a limited number of simple sequence repeat (SSR) markers is available for the genome of garlic (Allium sativum L.) despite the fact that SSR markers have become one of the most preferred DNA marker systems. To develop new SSR markers for the garlic genome, garlic expressed sequence tags (ESTs) at the publicly available GarlicEST database were screened for SSR motifs and a total of 132 SSR motifs were identified. Primer pairs were designed for 50 SSR motifs and 24 of these primer pairs were selected as SSR markers based on their consistent amplification patterns and polymorphisms. In addition, two SSR markers were developed from the sequences of garlic cDNA-AFLP fragments. The use of 26 EST-SSR markers for the assessment of genetic relationship was tested using 31 garlic genotypes. Twenty six EST-SSR markers amplified 130 polymorphic DNA fragments and the number of polymorphic alleles per SSR marker ranged from 2 to 13 with an average of 5 alleles. Observed heterozygosity and polymorphism information content (PIC) of the SSR markers were between 0.23 and 0.88, and 0.20 and 0.87, respectively. Twenty one out of the 31 garlic genotypes were analyzed in a previous study using AFLP markers and the garlic genotypes clustered together with AFLP markers were also grouped together with EST-SSR markers demonstrating high concordance between AFLP and EST-SSR marker systems and possible immediate application of EST-SSR markers for fingerprinting of garlic clones. EST-SSR markers could be used in genetic studies such as genetic mapping, association mapping, genetic diversity and comparison of the genomes of Allium species.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.