Selected Simple Sequence Repeat Markers Research Articles

Morphological and Molecular Diversity of Traditional Upland Rice (Oryza sativa L.) Varieties for Drought Tolerance at Seedling Stage

Drought is considered the major threat and most damaging abiotic stress for rice production.However, some traditional upland rice varieties (TURVs) may have some forms of tolerance to various stresses compared to new elite varieties. Two experiments were conducted. Experiment to determine the morpho-physiological response, phenotypic diversity, and similarity of 13 TURVs and 2 check varieties to mild drought stress at the seedling stage (Experiment 1 (E1) and to determine the molecular diversity and genetic similarity of the genotypes using selected simple sequence repeat (SSR) markers ((Experiment 2 (E2). In E1, all TURVs were highly tolerant under mild drought stress for a physiological response. Standardized Shannon-Weaver Diversity Index(H’) showed zero to low phenotypic diversity for qualitative traits. However, the phenotypic diversity of quantitative traits ranged from moderate to high for well-watered conditions, low to high for drought conditions, and moderate to high for re-watered conditions. Cluster analysis through Ward’s minimum variance based on pooled phenotypic data grouped the genotypes into three major clusters at 7 Euclidean distances. For E2, polymorphic information content values from 24 polymorphic SSR markers ranged from slightly informative (RM256) to highly informative (RM206), with a mean of 0.61. Nei’s gene diversity mean was 0.62 and values varied from 0.26 (RM256) to 0.86 (RM206). Based on Jaccard’s similarity coefficient, rice genotypes were grouped into nine minor clusters. The results indicate that TURVs harbor moderate to high allelic diversity and there are potential tolerance genes for mild drought stress that can be utilized in breeding programs for drought-tolerant rice varieties.

Genetic diversity of leafy kale genetic resources (Brassica oleracea var. acephala L.)

Leafy kale (Brassica oleracea var. acephala L.) is one of the most important Brassicaceae species with health-promoting properties and great diversity. To investigate variability within and among accessions, 12 accessions (each accession comprised four individuals/genotypes) from the Balkan region were genotyped at 12 simple sequence repeats (SSRs) loci. The selected SSR markers originated from the genomes of B. oleracea (eight), B. rapa (two) and B. napus (two). DNA was extracted from seeds, cotyledons, shoots, and young leaves, depending on the germination energy of each seed, using magnetic extraction method. Genetic analysis included eight accessions from Serbia (24 genotypes), two accessions from Montenegro (eight genotypes), and one accession from Bosnia and Herzegovina and Croatia (with four genotypes each). Accession-specific polymorphism was found at the Ol12-FO2 and Ol10-F11a loci, where genotypes were qualitatively distinguished by horizontal agarose gel electrophoresis (binary detection). Among 12 loci, the average polymorphic information content (PIC) was 0.71, with the marker Ol13-C12 proving to be the most informative (PIC = 0.89). However, the analysis of molecular variability (AMOVA) showed 11% diversity between accessions, 19% diversity between genotypes, and 70% diversity within genotypes, explaining the molecular diversity of native Balkan leafy kale germplasm. According to the genetic structure, four genetic groups were formed with an average expected heterozygosity of 0.70 between clusters. In order to exploit the genetic diversity, it would be advisable to evaluate these accessions at phenotypic level and use their potential in breeding programmes.

Petal-assisted artificial pollination method enhanced the fruit setting ratios in tea plant (<i>Camellia sinensis</i>)

<bold/> The pollinated method plays an important role in developing F1 population. Our study presents an improved method of petal-assisted artificial pollination in tea plants. The field operation using this method exhibited high efficiency in which more than 500 pistillate flowers per day can be pollinated per capita, and the pollen amount from only one staminate flower was sufficient to pollinate more than 5 pistillate flowers. Additionally, we surveyed the fruit-setting ratio in three F1 groups, which were established using this method and the influence of rainfall on the fruit-setting ratio. The results indicate that the fruit-setting ratio was superior among the tested F1 population with an average value of 12.90%, and a significant negative correlation was observed between rainfall and fruit-setting ratio. By analyzing the growth states of each F1 population, both a high germination rate and survival rate were observed. Furthermore, the purity for 184 individuals in the F1 population of ‘Jiaming 1’ × ‘Longjing 43’ (J×L) were analyzed with 32 selected simple sequence repeats (SSRs). The purposes of this paper are (1) to demonstrate that this method is beneficial to establish high purity F1 population and (2) to identify the number of SSR markers that are suitable for identification. The parentage analysis detected nine false-hybrid individuals, and the rate of false-hybrid was only 4.89% at 95% confidence, demonstrating that a low false-hybrid rate can be achieved by the method. Furthermore, one to two SSR markers randomly selected from each linkage group or chromosome were recommended as a sufficient quantity for SSR identification.

Development of SSR Markers Based on Transcriptome Sequencing and Verification of Their Conservation across Species of Ornamental Pennisetum Rich. (Poaceae)

Pennisetum species have importance in foraging, agriculture, energy-production, the environment, and landscaping. To promote the preservation and utilization of ornamental Pennisetum resources, we developed simple sequence repeat (SSR) markers from the Pennisetum setaceum cv. ‘Rubrum’ transcriptome and verified their conservation in 38 sources. Our transcriptome sequencing efforts generated 58.91 Gb of clean data containing 55,627 unigenes. We functionally annotated 30,930 unigenes, with functions enriched in translation and ribosomal structure and biogenesis. Database comparisons indicated that the closest relative of P. setaceum cv. ‘Rubrum’ is Setaria italica. Over five thousand SSR markers were detected in the transcriptomic data. We selected 38 pairs of highly polymorphic SSR markers from 50 randomly selected SSR markers. Based on genetic diversity analysis of 38 ornamental Pennisetum sources, we obtained 312 polymorphic bands, with an average of 8.21 alleles per primer. Principal coordinate analyses and generation of a, which proved that Pennisetum has moderate genetic diversity. In addition, fingerprint maps were constructed to improve Pennisetum identification. The transcriptome data generated by our study enhances the transcriptional information available for P. setaceum. This study lays the foundation for the collection and utilization of ornamental Pennisetum resources and provides a basis for future breeding projects using this species.

Characterization of genetic diversity of citron watermelon landrace accessions of South Africa through simple sequence repeat markers

ABSTRACT Profiling the genetic diversity of germplasm collections is fundamental for selection programs and strategic conservation. Citron watermelon (Citrullus lanatus var. citroides [L.H. Bailey] Mansf. ex Greb.) is an under-researched cucurbit crop despite providing food and nutritional security in sub-Saharan Africa. There is limited information on genetic relationships of South African citron watermelon accessions for effective breeding. The objective of this study was to determine the extent of genetic diversity among 48 South African citron watermelon landrace accessions using selected simple sequence repeat (SSR) markers to identify genetically divergent accessions for trait integration and variety development. The SSR markers amplified a total of 24 alleles, with a mean expected heterozygosity value of 0.38, indicating moderate genetic diversity among the studied accessions. The mean polymorphic information content value of 0.21 indicated low discriminatory power of the SSR markers. Analysis of molecular variance attributed 8%, 75%, and 17% of the molecular variation between populations, among accessions and within accessions, respectively. Three distinctive genetic groups were identified based on cluster analysis, which aided the selection of genetically diverse citron watermelon accessions for breeding and cultivar development.

Analysis of genetic diversity and discrimination of Oil Palm DxP populations based on the origins of pisifera elite parents.

A total of 251 Dura cross Pisifera (DxP) hybrid palms from six populations descending from six parental African Pisifera origins and involving 12 progenies were analyzed with 19 selected Simple Sequence Repeats (SSR) markers. A total of 110 alleles were produced, ranging from three to eight per SSR, with a mean of 5.8 alleles per SSR locus. Of these, 68.5% were considered shared alleles by more than one population and the remaining 31.5% were population specific alleles. They generated between six and 21 haplotypes in all populations, and depending on the SSR marker, between one and 10 haplotypes within populations. Various parameters for analyzing genetic variability, differentiation and genetic structure were computed using GenAlEx, Structure and Darwin software. The obtained results confirmed microsatellites as a robust, feasible and trustful method for obtaining DNA fingerprints, tracing the source of oil palm samples. With respect to the authenticity of materials or for solving legitimacy issues, accession belonging to each population by SSR markers could be distinguished, but additional SSR should be screened for improving this process.

Identifying SSR markers associated with seed characteristics in Perilla (Perilla frutescens L.).

The online version of this article (10.1007/s12298-021-00933-3) contains supplementary material, which is available to authorized users.

Genetic diversity and population structure of groundnut (Arachis hypogaea L.) accessions using phenotypic traits and SSR markers: implications for rust resistance breeding

Groundnut (Arachis hypogaea L.) is a multi-purpose legume serving millions of farmers and their value chain actors globally. Use of old poor-performing cultivars contributes to low yields (< 1 t/ha) of groundnut in sub-Saharan Africa including Tanzania. The objectives of this study were to determine the extent of genetic variation among diverse groundnut collections using phenotypic traits and simple sequence repeat (SSR) markers to select distinct and complementary genotypes for breeding. One hundred and nineteen genotypes were evaluated under field conditions for agronomic traits and susceptibility to rust and leaf spot diseases. The study was conducted in two locations across two seasons. In addition, the 119 accessions were profiled with 13 selected SSR markers. Genotype and genotype by environment interaction effects were significant (p < 0.05) for days to flowering (DTF), late leaf spot score at 85 and 100 days after planting, pod yield (PDY), kernel yield (KY), hundred seed weight (HSW) and shelling percentage (SP). Principal components analysis revealed that plant stand, KY, SP, NPP (number of pods per plant), late leaf spot and rust disease scores accounted for the largest proportion of the total variation (71.9%) among the tested genotypes. Genotypes ICGV-SM 08587 and ICGV-SM 16579 had the most stable yields across the test environments. Moderate genetic variation was recorded with mean polymorphic information content of 0.34 and gene diversity of 0.63 using the SSR markers. The majority (74%) of genotypes showed high membership coefficients to their respective sub-populations, while 26% were admixtures after structure analysis. Much of the variation (69%) was found within populations due to genotypic differences. The present study identified genotypes ICGV-SM 06737, ICGV-SM 16575, ICG 12725 and ICGV-SM 16608 to be used for development of mapping population, which will be useful for groundnut improvement. This study provided a baseline information on characterization and selection of a large sample of groundnut genotypes in Tanzania for effective breeding and systematic conservation.

Population structure and diversity assessment of barley (Hordeum vulgare L.) introduction from ICARDA

This study was undertaken to measure the genetic diversity and population structure of 48 barley accessions introduced from ICARDA using 51 polymorphic simple sequence repeat (SSR) markers to select unique parents for breeding. The mean polymorphic information content was 0.491, suggesting high polymorphism for the selected SSR markers among the barley accessions. The population structure indicated a fine genetic base only with two major clusters. All accessions had 100% membership probability in their respective clusters. Analysis of molecular variance revealed that most (78%) of the variation was attributed between populations, while 22% was due to variation among individuals within populations. Neighbour-joining (NJ) tree was constructed using this distance matrix and two major clusters were observed in it. Cluster 1 had all hulled barley accessions and cluster 2 had all hulless barley accessions. Cluster 2 could be further divided into three subclusters. Principal coordinates analysis results were similar to the NJ tree, where the hulled and hulless barley accessions were grouped into separate clusters. This study established the existence of considerable genetic diversity among the 48 tested accessions. The selected genetic resources will be useful for barley breeding in India and other countries.

Simple sequence repeat markers revealed genetic divergence and population structure of okra [Abelmoschus esculentus] collections of diverse geographic origin

The study was carried out to assess the genetic diversity and population structure of okra collections from diverse geographic origin using selected polymorphic simple sequence repeat (SSR) markers. Thirty-two okra accessions collected from three geographic regions were genotyped using 16 selected SSR markers. The SSR markers generated a total of 71 loci of which 67% were polymorphic. Genetic distances among accessions ranged from 2.2 to 7.1, with a mean of 4.65. Gene diversity ranged from 0.10 to 0.78 with a mean of 0.52. Genetic clustering classified the accessions into three major clusters and four sub-clusters. Each cluster and sub-cluster consisted of accessions derived from different sources. Analysis of molecular variance revealed that 67%, 81% and 83% of the total genetic variation detected was found within populations of geographic origin, altitude and collection district, respectively. The observed moderate to high population differentiation could partly be attributed to limited germplasm exchange, agro-ecological differences, and partly by selection pressure. The present study revealed the presence of high genetic diversity and population divergence among okra collections from Ethiopia. The study demonstrated that a collection strategy for conservation of okra genetic resources should focus on sampling of representative genotypes covering wide geographic regions and altitudinal ranges of target agro-ecologies.

Identification and characterization of SSR markers of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and transferability to other bamboo species.

The aim of this study was to develop simple sequence repeat (SSR) markers for genetic studies on G. chacoensis, as well as to evaluate their transferability to other bamboo species. Genomic DNA was isolated from G. chacoensis and its partial sequencing was used to find SSR loci. The obtained sequencing data were de novo assembled using the software CLC Genomics Workbench® 8.0v. The SSR loci primers were identified and designed with the software SSRLocator. The selected markers were validated using 56 plants sampled in seven populations from southern Brazil. The markers with potential polymorphism were selected and fluorescently labeled for characterization by capillary electrophoresis. In total, 92 SSR loci were found in G. chacoensis contigs. Suitable primers were designed for 70 SSR loci, and the remaining 22 SSR loci did not have sequences for primer development. Out of 35 selected SSR markers, after PCR optimization, 10 with high polymorphism potential were characterized. These loci can be used in genetic analyses of G. chacoensis and all of them were successfully transferred to other bamboo species. Non-polymorphic loci require further tests with additional plants, from different populations, to identify possibilities of their use.

Assessment of the genetic diversity and population structure of rice genotypes using SSR markers

ABSTRACTGenetic diversity is a pre-requisite for rice (Oryza sativa L.) breeding and population development. Hence, the objective of this study was to assess the genetic diversity and population structure of 54 rice accessions using 14 polymorphic simple sequence repeats (SSR) markers to select unique parents for breeding. Data analysis was based on marker and population genetic parameters. The mean polymorphic information content (PIC) was 0.61 suggesting high polymorphisms for the selected SSR markers among the rice accessions. The population structure revealed a narrow genetic base with only two major sub-populations. Analysis of molecular variance revealed that only 30% of the variation was attributed to population differences while 47% and 23% were due to variation among individuals within populations and within individual variation, respectively. The genetic distance and identity among genotypes varied from 0.083 to 1.834 and 0.159 to 0.921, respectively. Dendrogram grouped the genotypes into three clusters with wide variation among the accessions. The study established the existence of considerable genetic diversity among the tested 54 accessions. The selected genetic resources will be useful resources for rice breeding in Tanzania or other African countries.

Molecular breeding of ameliorating commercial pearl millet hybrid for downy mildew resistance

Downy mildew (DM) caused by Sclerospora graminicola is the most calamitous disease of pearl millet. Therefore, for introgression of DM resistance (DMR) in HHB 197 (MH-1302), an elite pearl millet hybrid, a marker-assisted breeding was undertaken by targeting three DMR loci on linkage groups (LGs) 1, 2 and 4. Breeding programme was initiated by crossing HBL 11 (DM susceptible), male parent of HHB 197 hybrid with ICMP 451 (DM-resistant) to produce true F1 plants. By conducting three rounds of backcrossing and selection, BC3F1 lines were generated. Foreground selection was employed using six polymorphic simple sequence repeat (SSR) markers of the 18 total selected markers. Four of these markers were linked to LG 1, five to LG 2 and nine to LG 4. Background selection was performed in BC3F1 generation using 33 polymorphic SSR markers of a total of 56 evenly spread SSR markers in the pearl millet genome to check recovery of recurrent parent genome. On the basis of genotypic selection (foreground as well as background) using selected SSR markers, agronomic performance in field and DM screening in greenhouse; 10 improved HBL 11 lines were selected and crossed with ICMA 97111 to produce DM-resistant HHB 197 hybrid versions. Six putatively improved HHB 197 hybrids were successfully tested in first year trials at Hisar and Bawal locations of Haryana and two selected versions with higher yield and zero DM incidence will be further tested in multilocation trials.

Genetic interrelationship of sweet stem sorghum genotypes assessed with simple sequence repeat markers

Knowledge of genetic diversity within and among populations provides essential information in employing appropriate management strategies for germplasm conservation and for establishing a successful breeding program. The objective of this study was to evaluate the genetic interrelationships among phenotypically selected sweet stem sorghum genotypes using polymorphic simple sequence repeat (SSR) markers. Eighteen sweet sorghum cultivars were genotyped using 25 selected SSR markers. The number of alleles detected per marker ranged from 1 to 10 with a mean of 4.64 per locus. The number of effective alleles varied from 1 to 6.75 with a mean of 2.9. The observed heterozygosity varied from 0.00 to 0.39 with a mean of 0.16. The inbreeding coefficient varied from 0.11 to 1.00 with a mean of 0.7. The polymorphism information content (PIC) values for all markers ranged from 0.00 to 0.85 with a mean value of 0.56. Sixty-eight percent of the markers used had a PIC value > 0.50. Analysis of molecular variance revealed highly significant differences (p < 0.001) among the test population. Among and within individual variances contributed to 78% and 21% of total genetic variance, respectively. Based on SSR analysis and agronomic traits, genotypes AS391, SS27, AS204 and AS244 were selected to breed for biofuel production.

Phenotypic characterization of elite quality protein maize (QPM) inbred lines adapted to tropical-highlands and the association studies using SSR markers

A well-characterized germplasm is a requisite to develop productive and better adapted crop cultivars. Morphological and molecular markers and pedigree analyses are widely used in germplasm characterization, and to establish genetic diversity and relationship in maize. The objectives of this study were to characterise newly developed quality protein maize (QPM) inbred lines adapted to tropical-highlands using phenotypic traits and to determine the association with simple sequence repeat (SSR) markers. Thirty-six maize inbred lines (30 QPM and six non-QPM) adapted to tropical-highlands of Ethiopia were evaluated using 18 phenotypic traits and 26 selected SSR markers. Significant phenotypic variations were observed among inbred lines for all measured traits. Grain yield showed moderate and high genotypic and phenotypic coefficients of variation, respectively. Ear height and thousand kernel weight had high heritability (≥ 0.70) which could be a source of high genetic advance through selection. Principal component and cluster analysis using unweighted paired group method using arithmetic averages (UPGMA) revealed the presence of two to four distinct genetic groups, while population structure analysis based on phenotypic data showed the presence of four to six genetic groups. Seven QPM inbred lines (KIT32Q, 142-1eQ, SRSYN20Q, FS67(BC2), FS170Q, FS60, and F7215) with complementary phenotypic traits and relatively better yield performance were selected using the phenotypic traits and SSR based genotyping for cultivar development and/or conservation.

Newly developed SSR markers reveal genetic diversity and geographical clustering in Paeonia suffruticosa based on flower colour

ABSTRACTTree peony (Paeonia suffruticosa Andr.), considered by many to be the national flower of China, is famous for its ornamental, medicinal and culinary attributes. In this study, a total of 8,663 Simple Sequence Repeats (SSRs) were detected by means of a microsatellite search of unigene sequences identified from the de novo assembly of sequence data from different genotypes of tree peony from the Zhongyuan group. Among 100 randomly selected SSR markers, 25 were successfully amplified and showed polymorphism in 31 tree peony accessions. The number of polymorphic alleles ranged from 3 to 11 for each locus and the polymorphism information content value (PIC) ranged from 0.58 to 0.85, with a mean of 0.73, indicating a high level of discriminative capability. To analyse the genetic diversity, a phylogenetic tree was constructed, which demonstrated that tree peonies of similar flower colour were clustered together. The large number of tree peony SSR markers identified in this study will be valuable in studies in genetic diversity and linkage map construction, in gene localisation and cloning and in molecular-marker-assisted breeding in tree peony.

Detection of Salt Tolerant Rice Genotypes at the Seedling Stage Using Molecular Markers: A Genotypic Analysis

Selection of salinity tolerant genotypes of rice based on the phenotypic variation is not consistent and thereby, will hinder the advancement in breeding. However, the recent introduction of the use of molecular markers, microsatellites or simple sequence repeats (SSRs) to detect the salt tolerant rice genotypes is reliable. The objective of the present study was to screen the four indigenous rice genotypes collected at Coastal Saline Research Centre, Ramanathapuram, Tamil Nadu, India, under saline and non-saline conditions using the microsatellite markers for the identification of salt tolerant genotypes at the seedling and reproductive stages. Two selected SSR markers were used to determine salinity tolerance in rice genotypes. SSR based marker identified three genotypes as tolerant at least tolerant to one of the two SOS markers but one variety was susceptible for both the markers. The detected salt tolerant genotypes can be a probable germplasm resource for prospective breeding program.

Development of simple sequence repeat markers in the halophytic turf grass Sporobolus virginicus and transferable genotyping across multiple grass genera/species/genotypes

Simple sequence repeat (SSR) markers are highly informative and widely used in genetic studies and plant breeding. Sporobolus virginicus is a halophytic turf grass that shows a high tolerance to salinity of up to 1.5 M NaCl. In the present study, we developed 148,411 SSR markers on 48,512 transcriptome contigs derived from RNA sequencing data. Of 33 randomly selected SSR markers, 23 (69.7%) produced clean amplification products, and an average of 1.25 alleles per marker was detected in S. virginicus genotypes. These markers were also examined by detecting polymorphisms across 19 different genera/species/genotypes in Poaceae, resulting in a high percentage (40%) of transferability. The sequencing of amplified products from these genera/species/genotypes revealed a high level of sequence similarity; however, substitutions, deletions, and insertions were detected not only in the objective SSRs but also in their flanking sequences. This is the first study on SSR marker development from S. virginicus. These SSR markers are useful for mapping the genes in and the breeding of S. virginicus and also for comparative genomics across genera/species/genotypes in Poaceae.

Combining Ability and Molecular Genetic Distances in Tropical Maize Lines for Grain Yield and Agronomic Traits

Ascertaining the relationship of genetic diversity and combining ability would aid the design of a breeding strategy for maize hybrids. Twelve tropical maize inbreds were crossed in a half-diallel to study gene action for yield and other traits and also to determine the relationship between heterosis, combining ability for grain yield and molecular genetic distance in the set of lines. Sixty-six crosses and their 12 parental lines were evaluated in five Kenyan locations in 2011 and 2012. Genetic distances between lines was quantified using 28 SSR markers. General combining ability (GCA) and specific combining ability (SCA) mean squares were both important for grain yield and other traits. However, SCA was more important than GCA for grain yield. Line C92 had the best GCA effects (0.63; p < 0.001), whereas MUL71 × OSU23i (1.11; p < 0.01) had the highest SCA. The correlation coefficient between genetic distance (GD) and SCA was high, positive and significant (0.62; p < 0.001), as was that between GD and heterosis (0.68; p < 0.001), suggesting some predictive value. Cluster analysis based on simple sequence repeat (SSR) markers revealed three groups that were in conformity with SCA and heterosis clusters. This study supports the use of the selected SSR markers for further exploration as a complementary tool to field evaluations for heterotic grouping.

Phylogenetic Relationship ofEchinochloaSpecies Based on Simple Sequence Repeat and Phenotypic Marker Analyses

Echinochloaspecies are among the most troublesome weeds in rice cultivation, and grow in a broad habitat range in Korea. Although various ecotypes ofEchinochloahave been collected as germplasm for future studies, it has been difficult to classify them due to their high level of morphological similarity. This study was thus conducted to develop and investigate the phylogenetic relationships between 77Echinochloaaccessions with the use of 23 simple sequence repeat (SSR) markers and 24 morphological traits. Of 77Echinochloaaccessions, including 57 accessions from Korea and 5 reference species, late watergrass was clearly clustered as a distinctive group from barnyardgrass and otherEchinochloaspecies. In this analysis, we also identified core genetic and morphological markers that can be used for the future identification and classification ofEchinochloaspecies. Five out of 23 SSR makers produced distinctive bands that discriminate late watergrass from barnyardgrass and otherEchinochloaspecies. Four morphological traits of the reproductive organs were the most influential contributors for classifyingEchinochloaspecies. Although there was no clear consensus generated in this study between SSR markers and morphological trait analyses, our results support the potential use of the selected SSR markers and morphological traits in future studies ofEchinochloa.