Polymorphic Simple Sequence Repeat Markers Research Articles

Molecular marker-assisted genetic diversity analysis in soybean cultivars from Himalayan region of Uttarakhand, India

‘Bhat’ is the soybean variety highly valued in the traditional food and therapeutic system of Uttarakhand state in the Indian Himalayan region. It is available in diverse colors, shapes, and sizes, often linked to their nutritional parameters. The present investigation was conducted to assess the diversity and genetic relatedness in ‘bhat’ genotype indigenous to the Garhwal division of Uttarakhand. 25 polymorphic simple sequence repeats (SSR) markers were used to determine the genetic diversity and varietal identification among the 45 genotypes collected from the different villages of Uttarakhand. Out of the different markers assessed only 13 primers showed amplification with the maximum of 2–4 alleles obtained with each primer with the molecular weight ranging between 80 and 400 kb. The highest band amplification was observed in Satt 257 and Satt 197. The allelic frequencies of amplified primers ranged from 0.5 to 0.833 with a mean value of 0.645 and the mean gene diversity and PIC value was found to be 0.43 and 0.33. Satt 183, Satt 288, and Satt 389 showed the highest polymorphism, while Satt257 and Satt245 exhibited the presence of unique alleles in some samples. The phylogenetic analysis grouped the genotypes into 4 major clusters having visually distinct phenotypes in each group, indicating the mixing of population and loss of authenticity also confirming that phenotypic attributes are not indicative of genetic relatedness among the genotype under study. The results indicate the need for the development of novel ‘bhat’ specific markers for more accurate genetic identification of the nutritionally rich indigenous soybean variety.

Population genetic variation and geographic distribution of suitable areas of Coptis species in China.

The rhizomes of Coptis plants have been used in traditional Chinese medicine over 2000 years. Due to increasing market demand, the overexploitation of wild populations, habitat degradation and indiscriminate artificial cultivation of Coptis species have severely damaged the native germplasms of species in China. Genome-wide simple-sequence repeat (SSR) markers were developed using the genomic data of C. chinensis. Population genetic diversity and structure of 345 Coptis accessions collected from 32 different populations were performed based on these SSRs. The distribution of suitable areas for three taxa in China was predicted and the effects of environmental variables on genetic diversity in relation to different population distributions were further analyzed. 22 primer pairs were selected as clear, stable, and polymorphic SSR markers. These had an average of 16.41 alleles and an average polymorphism information content (PIC) value of 0.664. In the neighbor-joining (N-J) clustering analysis, the 345 individuals clustered into three groups, with C. chinensis, C. chinensis var. brevisepala and C. teeta being clearly separated. All C. chinensis accessions were further divided into four subgroups in the population structure analysis. The predicted distributions of suitable areas and the environmental variables shaping these distributions varied considerably among the three species. Overall, the amount of solar radiation, precipitation and altitude were the most important environmental variables influencing the distribution and genetic variation of three species. The findings will provide key information to guide the conservation of genetic resources and construction of a core reserve for species.

Genetic Diversity and Population Structure of Maize (Zea mays L.) Inbred Lines in Association with Phenotypic and Grain Qualitative Traits Using SSR Genotyping

Maize (Zea mays L.) is an important cereal and is affected by climate change. Therefore, the production of climate-smart maize is urgently needed by preserving diverse genetic backgrounds through the exploration of their genetic diversity. To achieve this, 96 maize inbred lines were used to screen for phenotypic yield-associated traits and grain quality parameters. These traits were studied across two different environments (Anand and Godhra) and polymorphic simple sequence repeat (SSR) markers were employed to investigate the genetic diversity, population structure, and trait-linked association. Genotype–environment interaction (GEI) reveals that most of the phenotypic traits were governed by the genotype itself across the environments, except for plant and ear height, which largely interact with the environment. The genotypic correlation was found to be positive and significant among protein, lysine and tryptophan content. Similarly, yield-attributing traits like ear girth, kernel rows ear−1, kernels row−1 and number of kernels ear−1 were strongly correlated to each other. Pair-wise genetic distance ranged from 0.0983 (1820194/T1 and 1820192/4-20) to 0.7377 (IGI-1101 and 1820168/T1). The SSRs can discriminate the maize population into three distinct groups and shortlisted two genotypes (IGI-1101 and 1820168/T1) as highly diverse lines. Out of the studied 136 SSRs, 61 were polymorphic to amplify a total of 131 alleles (2–3 per loci) with 0.46 average gene diversity. The Polymorphism Information Content (PIC) ranged from 0.24 (umc1578) to 0.58 (umc2252). Similarly, population structure analysis revealed three distinct groups with 19.79% admixture among the genotypes. Genome-wide scanning through a mixed linear model identifies the stable association of the markers umc2038, umc2050 and umc2296 with protein, umc2296 and umc2252 with tryptophan, and umc1535 and umc1303 with total soluble sugar. The obtained maize lines and SSRs can be utilized in future maize breeding programs in relation to other trait characterizations, developments, and subsequent molecular breeding performances for trait introgression into elite genotypes.

Does the geographic distance affect the genetic differentiation among bilberry populations sampled in Bosnia and Herzegovina?

Isolation-by-distance (IBD) pattern among bilberry (Vaccinium myrtillus L.) populations has previously been reported for this species in northern Europe. However, the number of molecular studies conducted on bilberry, using everything from isoenzymes, RAPDs to microsatellite markers, are very few and far between. Considering that Bosnia and Herzegovina (B&H) is a country rich with diverse fruit genetic resources, conducting a genetic characterization of the naturally occurring V. myrtillus populations could yield valuable data for the conservation and utilization of this resource. This study entailed genotyping samples collected from three bilberry populations located in Fojnica, Kladanj, and Srebrenica municipalities using seven polymorphic microsatellite or SSR (simple sequence repeats) markers. The obtained molecular data was used to calculate the correlation between the physical distance of the individual B&H populations and a parameter of the genetic differentiation (pairwise Fst). The results of the correlation analyses revealed an absence of a significant isolation-by-distance pattern among the three examined B&H bilberry populations. In addition, the most pronounced genetic differentiation was detected between the Srebrenica and each of the two remaining B&H populations. At the same time, the values for pFst were significant, albeit much lower, between the Fojnica and Kladanj populations. Bilberries from the sampled Srebrenica population appear to be distinct from the other B&H populations, possibly due to the different genetic origin of this population.

Inheritance pattern of tetraploids pummelo, mandarin, and their interspecific hybrid sour orange is highly influenced by their phylogenomic structure.

Citrus polyploidy is associated with a wide range of morphological, genetic, and physiological changes that are often advantageous for breeding. Citrus triploid hybrids are very interesting as new seedless varieties. However, tetraploid rootstocks promote adaptation to different abiotic stresses and promote resilience. Triploid and tetraploid hybrids can be obtained through sexual hybridizations using tetraploid parents (2x × 4x, 4x × 2x, or 4x × 4x), but more knowledge is needed about the inheritance pattern of tetraploid parents to optimize the efficiency of triploid varieties and tetraploid rootstock breeding strategies. In this work, we have analyzed the inheritance pattern of three tetraploid genotypes: 'Chandler' pummelo (Citrus maxima) and 'Cleopatra' mandarin (Citrus reticulata), which represent two clear examples of autotetraploid plants constituted by the genome of a single species, and the 'Sevillano' sour orange, which is an allotetraploid interspecific hybrid between C. maxima and C. reticulata. Polymorphic simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers were used to estimate parental heterozygosity restitution, and allele frequencies for centromeric loci were used to calculate the preferential pairing rate related to the proportion of disomic and tetrasomic segregation. The tetraploid pummelo and mandarin displayed tetrasomic segregation. Sour orange evidenced a clear intermediate inheritance for five of the nine chromosomes (1, 2, 5, 7, and 8), a slight tendency toward tetrasomic inheritance on chromosome 3, and intermediate inheritance with a tendency toward disomy for chromosomes 4, 6, and 9. These results indicate that the interspecific versus intraspecific phylogenomic origin affects preferential pairing and, therefore, the inheritance patterns. Despite its high level of heterozygosity, the important preferential chromosome pairing observed in sour orange results in a limited diversity of the genotypic variability of its diploid gametes, and consequently, a large part of the genetic value of the original diploid sour orange is transferred to the tetraploid progenies.

Construction of a genome-wide SSR marker library in Gerbera hybrida: Insights into genetic variation and germplasm resources

Gerbera hybrida is a highly sought-after cut flower globally, renowned for its diverse flower color and pattern resulting from its complex inflorescence meristem architecture. However, the scarcity of genomic information and molecular markers has impeded efforts to comprehend the genetic diversity of G. hybrida. To address this, our study aimed to construct genome-wide simple sequence repeat (SSR) markers in G. hybrida, utilizing genomic data from an inbred line. We employed 170 gerbera cultivars exhibiting distinct flower patterns and colors to analyze genetic variation and diversity. Our analyses revealed highly polymorphic SSR markers, a total of 69 alleles (Na) were amplified by 14 pairs of primers, in which polymorphic information content (PIC) ranging from 0.380 to 0.700 with an average value of 0.583. The polymorphic results of the observed heterozygosity (Ho) and Shannon's information index (I) were 0.499 and 1.195, respectively, showing a high level of genetic diversity within the enriched sequences of the G. hybrida genome. Leveraging these markers with strong differentiation, we successfully constructed a unique fingerprint map encompassing 170 gerbera germplasm lines. This study involved the construction of an SSR fingerprint library for G. hybrida germplasm resources, employing genetic analysis for screening and molecular-level identification. The findings provide valuable insights into the identification of gerbera germplasm resources and kinship studies among various groups. Moreover, this study lays a solid foundation for future advancements in molecular design breeding improvement and germplasm resource development in G. hybrida.

Development of polymorphic simple sequence repeat markers in Agastache rugosa and their application in genetic evaluation and cross-taxon transferability of Agastache species

Agastache is a genus of perennial herbaceous plants belonging to the mint family, Lamiaceae. Several Agastache species are commercially cultivated and used as medicinal, culinary, and ornamental plants. However, information on the genetic diversity and population structure of the species remains unclear. In the present study, genetic diversity within Agastache species was analyzed using simple sequence repeat (SSR) markers. In this study, 249,746 SSRs were identified in the A. rugosa genome and primer pairs were designed for 56,675 SSRs. The majority of SSR repeat types were dinucleotides (60.65%), followed by trinucleotides (12.38%), and pentanucleotides (12.10%). PCR conditions were established for 250 primer pairs, 111 of which were found to be polymorphic in A. rugosa germplasm. The number of alleles (NA) ranged from 2 to 19, major allele frequency (MAF) ranged from 0.11 to 0.95, observed heterozygosity (HO) ranged from 0 to 0.89, and polymorphic information content (PIC) ranged from 0.09 to 0.92. Cross-species amplification of SSRs markers in other Agastache species showed amplification rates of 82.6% for A. foeniculum and 78.1% in A. urticifolia, with an average of 80.37%. Cluster analysis of the 19 A. rugosa accessions using SSRs markers revealed four major clusters, and population STRUCTURE analysis using 79 SSRs markers revealed three groups and three subgroups among the A. rugosa populations. The SSRs markers developed can contribute to applications such as varietal identification, genetic diversity analysis, and population structure analysis of A. rugosa germplasm.

Simple Sequence Repeat Fingerprint Identification of Essential-Oil-Bearing Rosa rugosa via High-Resolution Melting (HRM) Analysis.

Oil-bearing Rosa rugosa are popular in the essential oil and perfume markets. The similar botanical characteristics between high-oil-yield or low-oil-yield cultivars are confusing and it is hard for farmers or breeders to identify the high-oil-yield cultivar by phenotype difference. High-resolution melting (HRM) analysis of simple sequence repeats (SSRs) can construct accurate DNA fingerprints quickly, which was shown to be effective for identification of closely related cultivars of R. rugosa. Optimization of HRM-SSR indicated that the 10 µL HRM reaction mixture containing 20 ng of genomic DNA of R. rugosa and 0.75 µL of 10 µmol/L of each primer with an annealing temperature of 64 °C was a robust SSR genotyping protocol. Using this protocol, 9 polymorphic SSR markers with 3-9 genotypes among the 19 R. rugosa cultivars were identified. The top three polymorphic makers SSR9, SSR12 and SSR19 constructed a fingerprint of all cultivars, and the rare insertion in the flanking sequences of the repeat motif of SSR19 generated three characteristic genotypes of three high-oil-yield cultivars. These results may be economical and practical for the identification of high-oil-yield R. rugosa and be helpful for the selection and breeding of oil-bearing roses.

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.

Genome-wide survey and characterization of microsatellites in cashew and design of a web-based microsatellite database: CMDB.

The cashew is an edible tree nut crop having a wide range of food and industrial applications. Despite great economic importance, the genome-wide characterization of microsatellites [simple sequence repeats (SSRs)] in cashew is lacking. In this study, we carried out the first comprehensive genome-wide microsatellites/SSRs characterization in cashew and developed polymorphic markers and a web-based microsatellite database. A total of 54526 SSRs were discovered in the cashew genome, with a mean frequency of 153 SSRs/Mb. Among the mined genome-wide SSRs (2-6 bp size motifs), the dinucleotide repeat motifs were dominant (68.98%) followed by the trinucleotides (24.56%). The Class I type of SSRs (≥20 bp) were 45.10%, while Class II repeat motifs (≥12-<20 bp) were 54.89% of the total genomic SSRs discovered here. Further, the AT-rich SSRs occurred more frequently in the cashew genome (84%) compared to the GC-rich SSRs. The validation of the in silico-mined genome-wide SSRs by PCR screening in cashew genotypes resulted in the development of 59 polymorphic SSR markers, and the polymorphism information content (PIC) of the polymorphic SSR markers ranged from 0.19 to 0.84. Further, a web-based database, "Cashew Microsatellite Database (CMDB)," was constructed to provide access to the genome-wide SSRs mined in this study as well as transcriptome-based SSRs from our previous study to the research community through a user-friendly searchable interface. Besides, CMDB provides information on experimentally validated SSRs. CMDB permits the retrieval of SSR markers information with the customized search options. Altogether, the genome-wide SSRs characterization, the polymorphic markers and CMDB database developed in this study would serve as valuable marker resources for DNA fingerprinting, germplasm characterization, genetic studies, and molecular breeding in cashew and related Anacardium species.

Genetic Diversity and Population Structure of Juniperus seravschanica Kom. Collected in Central Asia.

Juniperus seravschanica Kom. is a species that grows widely in the mountain ranges from Central Asia to Oman. It is an important tree for the formation of shrub-forest massifs in mountainous areas and for draining and fixing soils from middle to high altitudes. A comprehensive study of the species' genetic diversity and population structure is a basic approach to understanding the current status of J. seravschanica resources for the development of future conservation strategies. Samples from 15 populations of J. seravschanica were collected from the mountain ranges of Uzbekistan, Kyrgyzstan, and Kazakhstan. The genetic diversity and population structure of 15 Central Asian populations of J. seravschanica were assessed using 11 polymorphic simple sequence repeat (SSR) markers. Genetic diversity parameters, including the number of alleles (na), the effective number of alleles (ne), Shannon's information index (I), the percentage of polymorphic loci (PPL), Nei's genetic diversity index (Nei), principal coordinate analysis (PCoA), etc., were evaluated. The analysis of 15 J. seravschanica populations based on 11 polymorphic SSRs detected 35 alleles. The average PIC value was 0.432, and the highest value (0.662) was found in the JT_40 marker. Nei's genetic diversity index for the J. seravschanica populations was 0.450, ranging from 0.407 (population 14) to 0.566 (population 4). The analysis of molecular variance (AMOVA) showed that 90.3% of total genetic variation is distributed within the population. Using the alleles of all the populations, the gene flow (Nm) was found to be 4.654. Population structure analysis revealed poor clustering in the studied populations and confirmed our AMOVA results. The output of this work can be efficiently used for the maintenance of the species across the Central Asian region.

Construction of a Tomato (Solanum lycopersicum L.) Introgression Line Population and Mapping of Major Agronomic Quantitative Trait Loci

Tomato as a fresh fruit has a large market share in China, but few new materials have been developed for such cultivar breeding in recent years. This study aims to create innovative breeding materials for fresh fruit tomatoes with consistent genetic backgrounds and take advantage of beneficial genes from wild germplasm resources. An introgression line (IL) population was constructed using freshly cultivated tomato S. lycopersicum 1052 and wild tomato S. pennellii LA0716 through hybridization and five consecutive backcrossings, with molecular marker-assisted selection techniques during seedling stages. A total of 447 cleaved amplified polymorphic sequences (CAPS) and 525 simple sequence repeat (SSR) markers were used to screen polymorphic markers among the two parental lines, resulting in 216 polymorphic CAPS and 236 polymorphic SSR markers, with 46.5% parental polymorphism. Then, 200 molecular markers uniformly distributed over the entire genome were further selected, and 107 ILs were finally obtained from 541 BC5 candidate plants. The physical distance between adjacent markers was 6.3~10.0 cm, with an average interval of 7.29 cm, and the IL population constructed covered the whole genome of S. pennillii LA0716, with an average introgression segment of 31.5 cm. Moreover, phenotype data of major agronomic traits in BC5 progeny after selfing two times, were analyzed for quantitative trait locus (QTL) mapping, and a total of 11 QTLs distributed on 6 chromosomes were identified, including 3 QTLs regulating plant height, 1 QTL regulating leaf size, 1 QTL regulating fruit color, 4 QTLs regulating fruit weight, and 2 QTLs regulating soluble solids content in ripening fruits. The IL population constructed in this study provided good materials for fresh fruit tomato breeding with improved yield and quality in the future.

Unravelling the genetic diversity and population structure of common walnut in the Iranian Plateau

BackgroundCommon walnut (Juglans regia L.) has a long cultivation history, given its highly valuable wood and rich nutritious nuts. The Iranian Plateau has been considered as one of the last glaciation refugia and a centre of origin and domestication for the common walnut. However, a prerequisite to conserve or utilize the genetic resources of J. regia in the plateau is a comprehensive evaluation of the genetic diversity that is conspicuously lacking. In this regard, we used 31 polymorphic simple sequence repeat (SSR) markers to delineate the genetic variation and population structure of 508 J. regia individuals among 27 populations from the Iranian Plateau.ResultsThe SSR markers expressed a high level of genetic diversity (HO = 0.438, and HE = 0.437). Genetic differentiation among the populations was moderate (FST = 0.124), and genetic variation within the populations (79%) significantly surpassed among populations (21%). The gene flow (Nm = 1.840) may have remarkably influenced the population genetic structure of J. regia, which can be attributed to anthropological activities and wind dispersal of pollen. The STRUCTURE analysis divided the 27 populations into two main clusters. Comparing the neighbor-joining and principal coordinate analysis dendrograms and the Bayesian STRUCTURE analysis revealed the general agreement between the population subdivisions and the genetic relationships among the populations. However, a few geographically close populations dispersed into different clusters. Further, the low genetic diversity of the Sulaymaniyah (SMR) population of Iraq necessitates urgent conservation by propagation and seedling management or tissue culture methods; additionally, we recommend the indispensable preservation of the Gonabad (RGR) and Arak (AKR) populations in Iran.ConclusionsThese results reflected consistent high geographical affinity of the accession across the plateau. Our findings suggest that gene flow is a driving factor influencing the genetic structure of J. regia populations, whereas ecological and geological variables did not act as strong barriers. Moreover, the data reported herein provide new insights into the population structure of J. regia germplasm, which will help conserve genetic resources for the future, hence improving walnut breeding programs’ efficiency.

High-Throughput Microsatellite Markers Development for Genetic Characterization of Emerging Sporothrix Species.

Sporotrichosis is the main subcutaneous mycosis worldwide transmitted by animal or plant vectors and often escalates to outbreaks or epidemics. The current cat-transmitted sporotrichosis driven by Sporothrix brasiliensis has become a significant public health issue in South America. Transmission dynamics remain enigmatic due to the lack of development of polymorphic markers for molecular epidemiological analysis. This study used a high-throughput mining strategy to characterize simple sequence repeat (SSR) markers from Sporothrix genomes. A total of 118,140-143,912 SSR loci were identified (82,841-98,369 unique markers), with a 3651.55-3804.65 SSR/Mb density and a majority of dinucleotides motifs (GC/CG). We developed a panel of 15 highly polymorphic SSR markers suitable for genotyping S. brasiliensis, S. schenckii, and S. globosa. PCR amplification revealed 240 alleles in 180 Sporothrix isolates with excellent polymorphic information content (PIC = 0.9101), expected heterozygosity (H = 0.9159), and discriminating power (D = 0.7127), supporting the effectiveness of SSR markers in uncovering cryptic genetic diversity. A systematic population genetic study estimated three clusters, corresponding to S. brasiliensis (population 1, n = 97), S. schenckii (population 2, n = 49), and S. globosa (population 3, n = 34), with a weak signature of mixed ancestry between populations 1 and 2 or 3 and 2. Partitioning of genetic variation via AMOVA revealed highly structured populations (ΦPT = 0.539; Nm = 0.213; p < 0.0001), with approximately equivalent genetic variability within (46%) and between (54%) populations. Analysis of SSR diversity supports Rio de Janeiro (RJ) as the center of origin for contemporary S. brasiliensis infections. The recent emergence of cat-transmitted sporotrichosis in northeastern Brazil indicates an RJ-Northeast migration resulting in founder effects during the introduction of diseased animals into sporotrichosis-free areas. Our results demonstrated high cross-species transferability, reproducibility, and informativeness of SSR genetic markers, helping dissect deep and fine-scale genetic structures and guiding decision making to mitigate the harmful effects of the expansion of cat-transmitted sporotrichosis.

Microsatellite Genome-Wide Database Development for the Commercial Blackhead Seabream (Acanthopagrus schlegelii)

Simple sequence repeats (SSRs), the markers with the highest polymorphism and co-dominance degrees, offer a crucial genetic research resource. Limited SSR markers in blackhead seabream have been reported. The availability of the blackhead seabream genome assembly provided the opportunity to carry out genome-wide identification for all microsatellite markers, and bioinformatic analyses open the way for developing a microsatellite genome-wide database in blackhead seabream. In this study, a total of 412,381 SSRs were identified in the 688.08 Mb genome by Krait software. Whole-genome sequences (10×) of 42 samples were aligned against the reference genome and genotyped using the HipSTR tools by comparing and counting repeat number variation across the SSR loci. A total of 156,086 SSRs with a 2-4 bp repeat were genotyped by HipSTR tools, which accounted for 55.78% of the 2-4 bp SSRs in the reference genome. High accuracy of genotyping was observed by comparing HipSTR tools and PCR amplification. A set of 109,131 loci with a number of alleles ≥ 3 and with a number of genotyped individuals ≥ 6 were reserved to constitute the polymorphic SSR database. Fifty-one polymorphic SSR loci were identified through PCR amplification. This strategy to develop polymorphic SSR markers not only obtained a large set of polymorphic SSRs but also eliminated the need for laborious experimental screening. SSR markers developed in this study may facilitate blackhead seabream research, which lays a certain foundation for further gene tagging and genetic linkage analysis, such as marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Association mapping of major economic traits and exploration of elite alleles in Prunus sibirica

Exploring elite alleles based on association analysis of economic traits will promote the breeding, development, and utilization of Prunus sibirica resources. This study used 113 P. sibirica clones with diverse provenances to perform association mapping of 20 economic traits based on 149 pairs of highly polymorphic simple sequence repeat (SSR) markers. Population structure analysis split the clones into three subgroups. General linear model (GLM) and mixed linear model (MLM) identified 117 SSR loci associated with 20 P. sibirica economic traits, specifically fruit (62 loci), nuclear (69), and kernel (46) traits. Phenotypic variation in these three sets of traits ranged 8.93–82.12% (GLM) or 0.23–84.82% (MLM), 10.96–56.65% (GLM) or 0.17–52.54% (MLM), and 5.17–59.41% (GLM) or 4.86–59.89% (MLM), respectively. Of the 117 marker SSR loci, 35 were simultaneously associated with two or more economic traits. In addition, phenotypic effects analysis identified 1842 elite alleles and seven typical materials related to key economic traits. We then designed elite parental combinations through crossing the typical materials. In conclusion, our study detected alleles and plant materials that allowed the identification of clones expressing desired traits of interest. These findings can be used for improved, targeted breeding of P. sibirica through marker-assisted selection.

Development and validation of SSR markers related to flower color based on full-length transcriptome sequencing in Chrysanthemum

Chrysanthemum (Chrysanthemum moriforlium Ramat.) is one of the most popular flowers worldwide, with very high ornamental and economic values. However, the limitations of available DNA molecular markers and the lack of full genomic sequences hinder the study of genetic diversity and the molecular breeding of chrysanthemum. Here, we developed simple sequence repeat (SSR) from the full-length transcriptome sequences of chrysanthemum cultivar ‘Hechengxinghuo’. A total of 11,699 SSRs with mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats were identified, of which eight out of eighteen SSR loci identified based on sixteen transcripts participated in carotenoid metabolism or anthocyanin synthesis were validated as polymorphic SSR markers. These SSRs were used to classify 117 chrysanthemum accessions with different flower colors at the DNA and cDNA levels. The results showed that four SSR markers of carotenoid metabolic pathway divided 117 chrysanthemum accessions into five groups at cDNA level and all purple chrysanthemum accessions were in the group III. Furthermore, the SSR marker CHS-3, LCYE-1 and 3MaT may be related to green color and the PSY-1b marker may be related to yellow color. Overall, our work may be provide a novel method for mining SSR markers associated with specific traits.

Distribution of Genetic Diversity in Beta patula Aiton Populations from Madeira Archipelago, Portugal

Beta patula Aiton is a crop wild relative (CWR) which belongs to the Gene Pool 1b and is considered a Critically Endangered species, and is present in very specific environments, such as the Desembarcadouro islet (DI) in Ponta de São Lourenço or Chão islet (CI) in the Desertas Islands. The ISOPlexis Center (University of Madeira) has been providing continuous support for its in situ conservation by keeping a management plan of wild populations and an ex situ conservation strategy through the storage of accessions in the ISOPlexis GeneBank. The present work intends to present the spatial distribution of genetic variability and diversity in these B. patula populations using eight polymorphic Simple Sequence Repeat (SSR) markers. The overall results lead to the identification of three spots with a high genetic diversity. CI with a small cluster of individuals shows a genetic footprint different from DI, having unique alleles present in its population. DI has two distinct areas: the western area, with a higher individual density but with a lower genetic diversity and higher allele fixation; and the central area, with a lower individual count but with a higher genetic diversity and with the presence of unique alleles. Despite some genetic differences, the comparison of the two islets’ DI and CI populations shows that they have more similarities than differences. Analysis of the Molecular Variance, based on the hierarchical cluster, showed a 9% diversity between populations, 68% among individuals, and 23% within individuals. This data will be used for the establishment of a protocol to monitor and manage B. patula genetic diversity under a genetic reserve, subsequently contributing to the European Genetic Reserve network implementation and the protection of this important CWR.

Assessment of the Genetic Relationship and Population Structure in Oil-Tea Camellia Species Using Simple Sequence Repeat (SSR) Markers.

Oil-tea camellia trees, the collective term for a class of economically valuable woody oil crops in China, have attracted extensive attention because of their rich nutritional and pharmaceutical value. This study aimed to analyze the genetic relationship and genetic diversity of oil-tea camellia species using polymorphic SSR markers. One-hundred and forty samples of five species were tested for genetic diversity using twenty-four SSR markers. In this study, a total of 385 alleles were identified using 24 SSR markers, and the average number of alleles per locus was 16.0417. The average Shannon's information index (I) was 0.1890, and the percentages of polymorphic loci (P) of oil-tea camellia trees were 7.79-79.48%, indicating that oil-tea camellia trees have low diversity. Analysis of molecular variance (AMOVA) showed that the majority of genetic variation (77%) was within populations, and a small fraction (23%) occurred among populations. Principal coordinate analysis (PCoA) results indicated that the first two principal axes explained 7.30% (PC1) and 6.68% (PC2) of the total variance, respectively. Both UPGMA and PCoA divided the 140 accessions into three groups. Camellia oleifera clustered into one class, Camellia vietnamensis and Camellia gauchowensis clustered into one class, and Camellia crapnelliana and Camellia chekiangoleosa clustered into another class. It could be speculated that the genetic relationship of C. vietnamensis and C. gauchowensis is quite close. SSR markers could reflect the genetic relationship among oil-tea camellia germplasm resources, and the results of this study could provide comprehensive information on the conservation, collection, and breeding of oil-tea camellia germplasms.

Evaluation of morphological and molecular characterization of rice (Oryza sativa L.) through SSR marker under saline stress condition

Aim: To assess 43 rice genotypes for their resistance to salinity and to assess the hereditary variety by 10 quantitative characteristics just as 40 SSR markers. Methodology: The seedlings were scored on the basis of 1-9 scale of SES following 10 days of 12 dS/m EC. The morphological traits (10 quantitative traits) were evaluated under natural saline soils (EC: 3.1; pH 7.8) to assess the diversity. At molecular level, 40 Simple Sequence Repeat (SSR) markers were also used to evaluate the diversity in different rice genotypes. Results: The cluster analysis of genotypes based on the morphological traits, identified the Amogh, Vasundhara, AC39040, PS336, and Paiyur-1 clustered in the Pokkali and Nona Bokra clusters. A total of 111 alleles were produced from 26 polymorphic SSR markers, with an average of 4.26 alleles per locus. The PIC value ranged from 0.314 (RM 13) to 0.824 (RM 6283) with an average of 0.633 and the expected heterozygosity varied from 0.498 to 0.844 with a mean of 0.690. Clustering of genotypes based on the molecular data clubbed Pokkali, AC35534, TRY-1, AC39014, Paiyur-1, JGL3844, Vasundhara, White Ponni, PS 349, AC39040, CR143202, and Nona Bokra in one cluster. Interpretation: The combination of screening at the seedling stage, morphological diversity, and molecular diversity clearly show the Vasundhara, AC39040, and Paiyur-1 as new sources of salinity. These genotypes may be further explored to identify novel genes/(QTLs) Quantitative Trait Loci, which could increase the production under salt-affected soils. Key words: Morphological diversity, Molecular diversity, Saline stress, Salinity screening