Simple Sequence Repeat Molecular Markers Research Articles

Limited gene flow in adjacent valleys and genetic introgression in a mountain hybrid zone between <i>Aconitum</i> (Ranunculaceae) species

The number of polyploid plants in angiosperms is estimated at about 30-80%, depending on the study, but hybridization between diploids and tetraploids is not a common phenomenon. In this study, our aim was to determine the extent of hybridization and introgression between <i>Aconitum</i> species in a mountainous hybrid zone in the Tatra Mountains (Western Carpathians). We hypothesized that topography would affect their populations’ genetic structure. For this purpose, inter simple sequence repeat (ISSR) molecular markers and chloroplast DNA (cpDNA, region <i>trn</i>L(UAG)–<i>ndh</i>F) sequencing were used. The results revealed that diploid and tetraploid <i>Aconitum</i> species had specific cpDNA haplotypes, while the spontaneous hybrid species harbored a cpDNA haplotype typical for tetraploids. Bayesian STRUCTURE analysis showed that introgression occurred between diploid and tetraploid species above the tree line, where a spontaneous hybrid, a putative triploid <i>A. ×berdaui</i>, originated, probably via the triploid bridge mechanism. Molecular analysis of variance, principal coordinate analysis, and neighbor-net clustering revealed different genetic profiles of the diploids between neighboring mountain valleys. Hence, mountain topography, ridges, and valleys can form an effective geographical barrier, preventing gene flow over longer distances. Introgression happens near or above the tree line, where the hybridization zone is formed. In the introgressants, however, no morphological alterations were discernible.

Genetic Diversity Analysis of Bitter Gourd (Momordica charantia L.) Germplasm using Molecular Markers

Bitter gourd [Momordica charantia L.] is an important cucurbitaceous crop of medicinal and nutraceutical value. The present study was conducted to compare the efficacy of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) molecular markers for genetic diversity analysis of bitter gourd. Twenty-one genotypes of bitter gourd, collected from different parts of India, were used for diversity analysis using two different types of markers. RAPD and ISSR markers yielded an average of 7.16 and 5.73 amplicon per primer, respectively. In the present study, RAPD and ISSR showed 89 and 63% polymorphic bands, respectively. Combined data analysis of RAPD and ISSR markers provided 73.66% polymorphism with 5.33 polymorphic amplicons per primer. The better discriminatory power of ISSR markers over RAPD markers may be due to comparatively higher values of average polymorphic information content (0.175) as well as the diverse nature of the genotypes. The information generated in this study would be helpful in designing breeding strategies for bitter gourd improvement.

SSR-marker Assisted Evaluation of Genetic Diversity in Greengram [Vigna radiata (L.) Wilcezk

Background: Assessing genetic diversity in greengram is a prerequisite for its genetic improvement for yield and quality. DNA markers such as simple sequence repeats (SSRs) have been preferred in this crop for the analysis of genetic diversity because SSR markers are locus-specific, widely dispersed throughout the genome, highly polymorphic due to variation in repeat units, highly informative because of co-dominant nature, high reproducibility and ease of assay by PCR (Polymerase chain reaction). The current study aimed to study the genetic diversity among the mutants of greengram at the molecular level using SSR markers. Methods: In this study, twenty-five SSR markers were used to analyze the genetic diversity amongst twenty mutant genotypes of greengram along with their parents. Genomic DNA was isolated from the leaves by using the standard CTAB DNA extraction method. Then DNA purification and PCR amplifications were carried out. The genetic variability and diversity among genotypes was examined by assessing the scoring of the amplified bands by SSR -PCR amplification. Result: Seventeen SSR primers generated 102 polymorphic bands with an average of the six polymorphic bands per primer. The number of alleles per locus ranged from four to nine. The size of amplification product varied for each primer and the range found to be 100bp to 2000bp. The mean polymorphic information content (PIC) value for SSR markers was found to be 0.6335. The value of Jaccard’s similarity coefficient had ranged from 0.07-0.70 with an average value of 0.38. The dendrogram constructed on SSR molecular markers data through the unweighted pair group method with arithmetic averages (UPGMA) method had enabled grouping of the genotypes into thirteen clusters. The results indicate the usefulness of SSR markers in the assessment of genetic variability and diversity among the mutant genotypes of greengram.

Development of KASP fingerprinting panel for clonal identification in red raspberry (Rubus idaeus L.)

AbstractRed raspberry is an economically important horticultural crop that is known for its fruit's sweet flavour and nutritional value. A reliable and economic genotyping platform is needed to facilitate clonal/variety identification. Previous attempts for clonal identification utilized morphological traits or low‐throughput, difficult to score dinucleotide‐containing simple sequence repeat molecular markers. Single nucleotide polymorphisms (SNPs), despite having lower allelic diversity, are numerous across the genome and more easily converted to high‐throughput assays restoring differential power. In this study, we use the kompetitive allele‐specific PCR (KASP™) chemistry, an affordable and high‐throughput platform, to develop a panel of SNPs to distinguish a diverse collection of red raspberry accessions for clonal identification. The panel consists of 48 KASP assays that show high concordance with whole genome sequencing, allelic balance, and recovery rate and a minimal set of 24 assays that distinguished the same accessions differentiated by the larger panel.

The Genetic Diversity and Genetic Structure of the Germplasm Resources of the Medicinal Orchid Plant Habenaria dentata.

Habenaria dentata has medicinal and ornamental value, but the number of wild populations is decreasing dramatically. Thus, conducting research on its genetic diversity and structure is necessary to provide a basis for its conservation. This study aimed to explore the genetic diversity of the wild plant H. dentata and protect and optimize its wild resources. The genetic diversity of 133 samples from six wild populations of H. dentata was analyzed using Inter Simple Sequence Repeat molecular markers to provide a scientific basis for the screening of improved germplasm resources. The results showed that the average number of alleles was 1.765, the average number of effective alleles was 1.424, the average Nei's gene diversity index was 0.252, the average Shannon diversity index was 0.381, and the average percentage of polymorphic loci was 76.499%. The variation within the populations was 77.34%, and the variation between the populations was 22.66%. The gene flow was 1.705, which was greater than 1. The results of the cluster analysis showed that the six populations were mainly divided into four clusters and were not classified according to their geographical location. There was no significant correlation between the geographical location and genetic distance between the populations (r = 0.557, p > 0.05). The genetic diversity of H. dentata is high. Among the six wild populations, the genetic diversity of the Mulun population was the highest and this population can be used as a key protection unit. The study on the genetic diversity of H. dentata can not only reveal the reasons for the decrease in the number of individuals in the population to a certain extent, and put forward the protection strategy, but also provide a scientific basis for the breeding of excellent seed resources.

Relationship between molecular markers and important fruit-related traits in almond (Prunus dulcis [Mill.] D.A. Webb syn. P. amygdalus Batsch) as revealed using multiple regression analysis (MRA).

Almond (Prunus dulcis [Mill.] D.A. Webb syn. P. amygdalus Batsch) is one of the most important nut crops, and its kernel is the edible part that has a high nutritional value and is used in the confectionery and cosmetics industries. The present research aimed to identify random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers associated with important fruit traits in late-blooming almond genotypes through multiple regression analysis (MRA). The studied genotypes showed significant differences from each other in terms of the measured fruit-related traits. The ISSR primers used produced a total of 125 bands in the studied germplasm, of which 112 showed polymorphic bands. The RAPD primers produced a total of 190 DNA fragments, of which 172 fragments showed polymorphism among genotypes. Some polymorphic fragments of ISSR and RAPD showed significant correlations with the fruit traits measured. Some of these informative markers were associated with more than one trait, which could be caused by the pleiotropic effects of quantitative trait loci related to each other in different traits. For instance, some of the markers showed significant correlations with both nut weight and kernel weight, which indicates a positive correlation between these two traits. Informative markers identified in this study can be used to select suitable parents for population generation for mapping. It is also useful for selecting superior genotypes, especially when information about their genetic basis, such as a linkage map, is not available.

Genetic diversity and population structure of Polygonatum cyrtonema Hua in China using SSR markers.

Polygonatum cyrtonema Hua is a perennial herbaceous plant of the Polygonatum genus, belonging to the Liliaceae family, with significant medicinal and nutritional value. In China, this species is a traditional medicinal and edible herb with a long history of application and is widely appreciated by the people. However, as the demand for medicinal herbs continues to grow, excessive harvesting has led to the depletion of wild resources and the risk of genetic erosion. In addition, the chaotic cultivation of varieties and the lack of high quality germplasm resources have led to inconsistent quality of medical materials. Therefore, it is urgent to conduct genetic diversity evaluation of this species and establish a sound conservation plan. This study assessed the genetic diversity and population structure of 96 samples collected from seven regions in China using the simple sequence repeat (SSR) molecular marker technology. In this study, a total of 60 alleles (Na) were detected across the 10 polymorphic SSR markers used, with an average of 6.0 alleles generated per locus. The values of polymorphic information content (PIC) values ranged from 0.3396 to 0.8794, with an average value of 0.6430. The average value of the effective number of alleles (Ne) was 2.761, and the average value of the Shannon's information index (I) was 1.196. The population structure analysis indicates that the Polygonatum cyrtonema Hua germplasm can be classified into three subpopulations (JZ, QY, JD) at the molecular level, which corresponds to the previous subgroups identified based on individual plant phenotypic traits. Analysis of Molecular Variance (AMOVA) showed that 74% of the genetic variation was between individuals within populations in different regions. The phylogenetic analysis of the 96 germplasm samples divided them into three main populations. The QY and JD subpopulations are largely clustered together, which could be attributed to their mountainous distribution and the local climate environment. The genetic differentiation coefficient (Fst) value was low at 0.065, indicating relatively low population differentiation. The ratio of the genetic differentiation coefficient (Fst) between the JZ population and the other two populations (QY and JD) is much higher than the ratio between the QY and JD populations. Based on the clustering results and the ratio of the genetic differentiation coefficient (Fst), it can be inferred that the genetic relationship between the QY and JD subpopulations is closer, with a certain degree of genetic differentiation from the JZ subpopulation. This study supports the conservation of germplasm resources of Polygonatum cyrtonema Hua in China and provides new parental material for germplasm genetic improvement and breeding programs.

Development, Identification and Validation of a Novel SSR Molecular Marker for Heat Resistance of Grapes Based on miRNA

The adverse effects of high temperatures on fruit quality and yield have been reported in many studies of grapevines. MiRNAs play crucial roles in plant growth and development and also fulfill functions in regulating the high-temperature response. In this research, miRNA-based SSR (simple sequence repeat) polymorphisms were analyzed according to the grape genome sequence and then combined with polymerase chain reaction (PCR) amplification and Sanger sequencing methods to analyze the miRNA-SSR diversity of different heat-resistant grape varieties. A statistical analysis of SSR sequences showed that 391 and 12 SSRs were specific to pri-miRNA and pre-miRNA, respectively. In pri-miRNA containing SSR, hexa-nucleotide repeats were the most abundant (52.69%), followed by tetra-nucleotide (13.04%) and minimum penta-nucleotide (4.09%), which were also observed in pre-miRNA sequences. On the other hand, differences in heat resistance among grape varieties were observed. Based on the results of leaf Fv/Fm images, values and phenotypic changes under high-temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 heat-sensitive (e.g., Shine Muscat and Jumeigui) grape varieties were identified. Further, PCR-Sanger sequencing was used to screen SSR differences in four thermos-tolerant and four thermos-sensitive grape varieties, and finally, eight SSR differential primers were found to be able to distinguish these varieties. Of these, four pairs of primers were selected for validation in 40 grape germplasm resources (20 thermo-tolerance and 20 thermo-sensitivity). The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties was 90%. In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and heat-sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and will be beneficial to the breeding of heat-resistant varieties in the future.

Development of Expressed Sequence Tag–Simple Sequence Repeat Markers Related to the Salt-Stress Response of Kenaf (Hibiscus cannabinus)

Kenaf is one of the most important natural cannabis plants. Molecular marker-assisted breeding is vital for accelerating the breeding process of kenaf. However, the number of kenaf markers is insufficient for molecular marker-assisted breeding. Using transcriptome sequencing data for salt-stressed kenaf plants, the number and distribution of simple sequence repeats (SSRs) and single nucleotide variations (SNVs) in the expressed sequences were determined. The objectives of this study were to elucidate the sequence variations in kenaf genes expressed in response to salt stress and to identify stable and dependable molecular markers. Primers were designed for SSR loci and then EST-SSR molecular markers were generated. The subsequent analyses revealed that 30.50% of the unigenes contained SSR motifs, most of which were single nucleotides followed by trinucleotides and dinucleotides. The unigenes containing SSRs were mostly associated with kenaf salt tolerance. Additionally, 10,483 SNVs were detected in contig sequences. Of the 3995 differentially expressed genes encoding interacting proteins, 1297 contained SSRs. Most of these genes were associated with metabolic pathways (e.g., 03000 transcription factors, B09132 signal transduction, and 04122 sulfur relay system). We designed 20 pairs of EST-SSR primers to genotype 30 kenaf varieties (lines), of which 9 primer pairs were ideal for genotyping (e.g., 1 highly polymorphic marker and 2 moderately polymorphic markers). The primer pairs designed for the EST-SSR markers in the kenaf genome may be useful SSR molecular markers for future research on kenaf. The verified polymorphic markers may be applicable to the molecular marker-assisted breeding of salt-tolerant kenaf varieties.

Genetic fingerprint construction and genetic diversity analysis of sweet potato (Ipomoea batatas) germplasm resources

BackgroundChina is the largest producer of sweet potato in the world, accounting for 57.0% of the global output. Germplasm resources are the basis for promoting innovations in the seed industry and ensuring food security. Individual and accurate identification of sweet potato germplasm is an important part of conservation and efficient utilization.ResultsIn this study, nine pairs of simple sequence repeat molecular markers and 16 morphological markers were used to construct genetic fingerprints for sweet potato individual identification. Combined with basic information, typical phenotypic photographs, genotype peak graphs, and a two-dimensional code for detection and identification were generated. Finally, a genetic fingerprint database containing 1021 sweet potato germplasm resources in the “National Germplasm Guangzhou Sweet Potato Nursery Genebank in China” was constructed. Genetic diversity analysis of the 1021 sweet potato genotypes using the nine pairs of simple sequence repeat markers revealed a narrow genetic variation range of Chinese native sweet potato germplasm resources, and Chinese germplasm was close to that from Japan and the United States, far from that from the Philippines and Thailand, and the furthest from that from Peru. Sweet potato germplasm resources from Peru had the richest genetic diversity, supporting the view that Peru is the center of origin and domestication of sweet potato varieties.ConclusionsOverall, this study provides scientific guidance for the conservation, identification, and utilization of sweet potato germplasm resources and offers a reference to facilitate the discovery of important genes to boost sweet potato breeding.

Identification of SSR Molecular Markers for Jassid Resistance in Cotton

Background: This study has focussed on identifying the Simple Sequence Repeat (SSR) markers associated with the sucking pests (more precisely on jassid) tolerance/resistance loci. The study was initiated with 4348 mapped SSR markers to differentiate between resistant (R-1) and susceptible (BS-1) genotypes. Out of 4348 markers (retrieved from cotton microsatellite database and cottongen.org) used,17 markers were showing polymorphism between the parental lines. Methods: The F1 population was generated by crossing Resistant line (R-1) and the elite Susceptible line (Bs-1). F1 progeny were selfed to obtain F2 population. Following the screening, to identify the SSR markers associated with trait, Single Marker Analysis was performed. Result: The results indicated that two of the seventeen polymorphic markers - BL1646 and DOW047 had significant LoD of 4.5 and 7.2 respectively and PVE% of 6.6 and 10.3 respectively. Hence, these were identified as the two markers associated with jassid resistance trait having the potential for their use in marker assisted selection (MAS).

Development of SSR molecular markers and genetic diversity analysis of Clematis acerifolia from Taihang Mountains.

Investigating the genetic diversity and population structure is important in conserving narrowly distributed plants. In this study, 90 Clematis acerifolia (C. acerifolia) plants belonging to nine populations were collected from the Taihang Mountains in Beijing, Hebei, and Henan. Twenty-nine simple sequence repeats (SSR) markers developed based on RAD-seq data were used to analyze the genetic diversity and population structure of C. acerifolia. The mean PIC value for all markers was 0.2910, indicating all SSR markers showed a moderate degree of polymorphism. The expected heterozygosity of the whole populations was 0.3483, indicating the genetic diversity of both C. acerifolia var. elobata and C. acerifolia were low. The expected heterozygosity of C. acerifolia var. elobata (He = 0.2800) was higher than that of C. acerifolia (He = 0.2614). Genetic structure analysis and principal coordinate analysis demonstrated that C. acerifolia and C. acerifolia var. elobata showed great genetic differences. Molecular variance analysis (AMOVA) demonstrated that within-population genetic variation (68.31%) was the main contributor to the variation of the C. acerifolia populations. Conclusively, C. acerifolia var. elobata had higher genetic diversity than C. acerifolia, and there are significant genetic differences between C. acerifolia and C. acerifolia var. elobata, and small genetic variations within the C. acerifolia populations. Our results provide a scientific and rational basis for the conservation of C. acerifolia and provide a reference for the conservation of other cliff plants.

Genetic diversity analysis of Wamena Arabica Coffee (Coffea arabica L.) based on SSR markers

Wamena is notable for producing and exporting high-quality coffee. However, a molecular method based on its polymorphism and not affected by external factors is required since morphological observations alone are frequently insufficient for identifying the coffee. The objective of this study is to identify Wamena Arabica coffee in the Jayawijaya District using simple sequence repeats (SSRs) molecular markers. This research involved several stages, including DNA isolation and purification, PCR SSR amplification with 5 primers, polymorphism, and heterozygosity level analysis. The results analysis proved that 30 alleles of 32 coffee genotypes were successfully amplified, with fragment sizes ranging from 176 to 553bp. The Car M052 locus (0.29%) had the least polymorphism with only two alleles, while the CarM101 locus (0.82%) had the most with 12 alleles, that high and low polymorphism indicated a measure of PIC scores. Heterozygosity analysis showed that the coffee samples were highly heterozygous. And based on the results of the bootstrap analysis, the phenogram shows that the coffee samples are divided into six clusters, with a cophenetic correlation coefficient (r) of 0.948 (excellent fit). This study proved that all SSR loci succeeded in amplifying 30 alleles and could be identified molecularly based on the genetic variation of the Wamena Arabica coffee genotypes in Papua. It seems highly possible that there is a mix of traits through gene flow and exchange between coffee genotypes, and the heterozygosity in the Arabica population on plantations. As a result, it is very important further analysis to confirm the findings.

Microsporogenesis in the triploid hybrid ‘Beilinxiongzhu 1#’ and detection of primary trisomy in 2x × 3 × Populus hybrids

BackgroundPrimary trisomy is a powerful genetic tool in plants. However, trisomy has not been detected in Populus as a model system for tree and woody perennial plant biology.ResultsIn the present study, a backcross between Populus alba × Populus glandulosa ‘YXY 7#’ (2n = 2x = 38) and the triploid hybrid ‘Beilinxiongzhu 1#’ (2n = 3x = 57) based on the observation of microsporogenesis and an evaluation of the variations in pollen was conducted to create primary trisomy. Many abnormalities, such as premature migration of chromosomes, lagging of chromosomes, chromosome bridges, asymmetric separation, micronuclei, and premature cytokinesis, have been detected during meiosis of the triploid hybrid clone ‘Beilinxiongzhu 1#’. However, these abnormal behaviors did not result in completely aborted pollen. The pollen diameter of the triploid hybrid clone ‘Beilinxiongzhu 1#’ is bimodally distributed, which was similar to the chromosomal number of the backcross progeny. A total of 393 progeny were generated. We provide a protocol for determining the number of chromosomes in aneuploid progeny, and 19 distinct simple sequence repeat (SSR) primer pairs covering the entire Populus genome were developed. Primary trisomy 11 and trisomy 17 were detected in the 2x × 3 x hybrid using the SSR molecular markers and counting of somatic chromosomes.ConclusionsNineteen distinct SSR primer pairs for determining chromosomal number in aneuploid individuals were developed, and two Populus trisomies were detected from 2x × 3 x hybrids by SSR markers and somatic chromosome counting. Our findings provide a powerful genetic tool to reveal the function of genes in Populus.

Development and Application of EST-SSR Markers Related to Lead Stress Responses in Kenaf Based on Transcriptome Sequencing Data

Kenaf is an important bast fiber crop. In order to diversify the available kenaf simple sequence repeat (SSR) molecular markers and generate markers potentially useful for kenaf breeding, we developed expression sequence tag simple sequence repeat (EST-SSR) molecular markers based on lead-stressed kenaf transcriptome sequencing data and spliced unigene sequences. Additionally, the distribution of the SSRs in the transcriptome and the potential functions of the SSR-containing genes were determined. Moreover, SSR markers in the differentially expressed genes (DEGs) of a protein–protein interaction (PPI) network were analyzed to screen for polymorphic markers, which were used to examine the genetic diversity and population structure of kenaf germplasm resources. The genetic diversity and population structure of 138 kenaf germplasm materials revealed that 22 EST-SSR markers could be used to distinguish the kenaf germplasms. The 22 EST-SSR markers enrich the kenaf molecular markers database and provide an important tool for future genetic improvement of kenaf resistance to lead stress.

Variabilidade genética em genótipos de Cártamo via marcador molecular SSR

ABSTRACT The safflower is an oleaginous plant belonging to the Asteraceae family. It is used as a raw material for various purposes. These plants are popular for the quality and quantity of oil produced and, and thus, studying their genetic variability using markers is necessary for determining genetic resources to conduct breeding programs. Therefore, we evaluated the genetic variability of safflower genotypes using Simple Sequence Repeat (SSR) molecular markers. The study was conducted at the State University of Mato Grosso “Carlos Alberto Reyes Maldonado”, in the Campus of Cáceres-MT. In total, 121 safflower genotypes from the Germplasm collection were evaluated using 21 SSR markers. The programs GenAlEx 6.5, GENES, and Structure were used to analyze the data. We identified 158 alleles at 21 loci among the genotypes. The expected heterozygosity (He) was high (0.551 - 0.804), but the observed heterozygosity (Ho) was low (0.000 - 0.502), and the indices of the endogamy coefficient (F) were positive in all loci and all populations, with an overall average of 0.958. The genetic differentiation (FST) values among populations were low, with an average of 0.010, which suggested a low population structure. The modified Tocher clustering and the UPGMA hierarchical clustering yielded 19 and 15 distinct groups, respectively. The genetic structure showed two populations, with few intermixes in the genome. The evaluated safflower genotypes showed genetic variability, and these genetically different variants might be used in breeding programs to obtain cultivars adapted to Brazil.

Further fine mapping and candidate gene prediction for a new restoring fertility gene Rf(fa) in rice

Rf(fa), a new restoring fertility gene in rice, was previously located to a large region on Chromosome 10. The large number of genes within the region made cloning of Rf(fa) difficult. To perform the cloning and further elucidate the molecular mechanism, we reconstructed a mapping segregation population (BC<sub>1</sub>F<sub>1</sub>) of 12 000 plants. Using the population and polymorphism of simple sequence repeat (SSR) molecular markers, we finally mapped Rf(fa) between the two SSR molecular markers MM2000 and RM25658, within a 78.87 kb region. By de novo sequencing of a restoring line of CMS-FA hybrid rice, we obtained the genomic sequence of the mapping region, which provided the basis for the prediction of the candidate gene(s) of the target gene and for the comparison of genomic sequence differences between wild and cultivated rice. Within the mapping region, the genomic sequence of the wild rice was significantly different from that of cultivated rice. There were ten genes in the final mapping region. A pentatricopeptide repeat (PPR) protein gene was predicted as the candidate gene of Rf(fa). Our results laid a solid foundation for the final cloning and molecular mechanism analysis of the gene. The identified molecular markers tightly linked to Rf(fa) will facilitate the marker assisted selection in breeding of CMS-FA hybrid rice.

Genetic Diversity Analysis and Core Germplasm Collection Construction of Camellia oleifera Based on Fruit Phenotype and SSR Data.

Many Camellia oleifera germplasm resources were collected from Guizhou Province, but the fruit morphological variation and genetic diversity of C. oleifera germplasm resources remain unclear. The genetic diversity of C. oleifera germplasms resources in Guizhou was studied based on fruit traits and simple sequence repeat (SSR) molecular markers to build a core collection. This paper aims to provide a scientific basis for the collection, management, development, and utilization of C. oleifera resources in Guizhou province. The variation coefficients among and within varieties of seven fruit phenotypic traits of C. oleifera ranged from 11.79% to 61.76% and from 8.15% to 42.31%, respectively, showing rich phenotypic variation. Furthermore, 12 SSR markers were used to analyze the genetic diversity. These primers generated 214 polymorphic bands, and the average number was 17.833. The average number of effective alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC), and major allele frequency (MAF) were 8.999, 2.301, 0.965, 0.50, 0.836, and 0.238, respectively. The results showed that 12 SSR markers had high polymorphism, and the genetic diversity of 167 C. oleifera germplasm resources was high. Based on SSR molecular marker information and fruit traits clustering, 167 C. oleifera germplasm resources were divided into three groups. When constructing core collections based on fruit traits and molecular marker information, the PowerCore-25 of core collections greatly preserves fruit traits and improves genetic diversity. This paper can provide a reference for the genetic diversity and fruit traits variation of C. camellia germplasm resources in Guizhou Province. It is significant for establishing a core collection, thus promoting germplasm innovation and the development of the oil tea industry in Guizhou.

Genetic diversity and population structure of cowpea [Vigna unguiculata (L.) Walp.] accessions from Togo using SSR markers.

Cowpea [Vigna unguiculata (L.) Walp.] is a crop with significant agronomic and nutritional value. In Togo, the crop is very appreciated by local people. It is the third food habit in Togo after maize and rice. However, several accessions of cowpea cultivated in Togo are now prone to extinction, creating a risk of genetic erosion. It is therefore urgent to assess the genetic diversity of accessions in order to set up a good conservation program. To achieve this, genetic diversity and phylogenetic relationships among 70 accessions of cowpea collected in the five (5) administrative regions of Togo were assessed using Simple Sequence Repeat (SSR) molecular markers. The twenty-eight SSR primers used in this study generated a total of 164 alleles with an average of 5.82 alleles per locus. Polymorphic Information Content (PIC) values ranged from 0.20 to 0.89 with an average value of 0.58. Population structure analysis using model-based revealed that the cowpea germplasm was grouped into two subpopulations. The analysis of molecular variance (AMOVA) revealed that 98% of genetic variation existed among accessions within regions. The fixation index (Fst) value, which was 0.069 was low, indicating relatively low population differentiation. The phylogenetic analysis grouped the 70 accessions into two main groups that can be further divided into four groups independent of their origins. This study provides a foundation for a Togolese cowpea germplasm conservation program and can serve for the selection of parental material for further studies aimed at the genetic improvement of local germplasm.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.