Simple Sequence Repeat Fragment Research Articles

Morphological and Molecular Characterisation of Nineteen (19) Tomato Cultivars from Northern Nigeria

Tomato is an autogamous vegetable crop whose genetic variability has been reduced by selection and breeding processes. Characterisation of tomato germplasm, therefore, aids the documentation of the genetic variability existing in a population. To morphologically characterise tomato varieties from Northern Nigeria, and molecularly characterise the varieties using (Simple Sequence Repeat (SSR) markers, nineteen (19) tomato varieties were collected from major markets in seven states in Northern Nigeria using a purposive sampling method. Tomato seeds were extracted, prepared, sowed in screen houses and morphologically characterised using cross-sectional shapes, core, pericarp, matured fruit colour, overall fruit shape, size and firmness. Leaves of tomato seedlings four weeks after sowing were pooled and used for genomic DNA extraction. The polymerase chain reaction (PCR) was performed and PCR products were visualized through electrophoresis. SSR fragment analysis was scored as the presence or absence of a specific fragment size from the Electropherograms.
 Results showed that 44% of the colour of tomato core was white, 17% were red and pink, 5% were orange and yellow and 11% were variants of other colours, showing wide variations in the qualitative traits. Similarities among the nineteen tomato samples ranged from 14 (Dan India with Derica sweet, Kaduna Tangino, Nassarawa Roma 1 and 2, Niger Roma2, UTC01 and UTC 02) to 86% (between Mai mara and Derica small). SSR gave three unique SSR bands that identified and characterised the cultivars. 
 The result of this study revealed genetic diversity within a group of tomato genotypes of different origins cultivated in Northern Nigeria using molecular (SSR) analysis. This confirmed the efficacy of the SSR markers as highly variable markers that detect the codominant single locus and are suitable to distinguish between the genetically related cultivars.

Chloroplast genomic characterization and phylogenetic analysis of Castanopsis hystrix

The structure and size of the chloroplast genome of Castanopsis hystrix was determined by Illumina HiSeq 2500 sequencing platform to understand the difference between C. hystrix and the chloroplast genome of the same genus, and the evolutionary position of C. hystrix in the genus, so as to facilitate species identification, genetic diversity analysis and resource conservation of the genus. Bioinformatics analysis was used to perform sequence assembly, annotation and characteristic analysis. R, Python, MISA, CodonW and MEGA 6 bioinformatics software were used to analyze the genome structure and number, codon bias, sequence repeats, simple sequence repeat (SSR) loci and phylogeny. The genome size of C. hystrix chloroplast was 153 754 bp, showing tetrad structure. A total of 130 genes were identified, including 85 coding genes, 37 tRNA genes and 8 rRNA genes. According to codon bias analysis, the average number of effective codons was 55.5, indicating that the codons were highly random and low in bias. Forty-five repeats and 111 SSR loci were detected by SSR and long repeat fragment analysis. Compared with the related species, chloroplast genome sequences were highly conserved, especially the protein coding sequences. Phylogenetic analysis showed that C. hystrix is closely related to the Hainanese cone. In summary, we obtained the basic information and phylogenetic position of the chloroplast genome of red cone, which will provide a preliminary basis for species identification, genetic diversity of natural populations and functional genomics research of C. hystrix.

Investigation of Genetic Diversity in Afghan Bread Wheat Genotypes Using SSR and AFLP Markers

Genetic diversity assessment is the principle component for conservation and characterization of germplasm. Genetic diversity study of Afghan bread wheat genotypes is a first step to identify and to select high performance genotypes and distribute to wheat breeding programs. The main objective of this study is to investigate of genetic diversity in 35 Afghan bread wheat genotypes by using Simple Sequence Repeat (SSR) and Amplified Fragment Length Polymorphism (AFLP) markers. DNA extraction according to Cetyl Trimethyl Ammonium Bromide (CTAB) method was conducted and the total genomic DNA was isolated from each variety. Sixty-four SSR primer markers were used and eighteen EcoRI+(N)/MseI+(N) primer combinations with their primer sequences were used for selective polymerase chain reaction (PCR) amplification. Every SSR and AFLP fragment was scored as present (1) or absent (0) within all genotypes under study. Marker/ Value ratio of pairwise genetic distance between genotypes according to the SSRs data was from 0.508 to 0.691 with an average distance of 0.599. Relatively different grouping pattern in comparison to AFLP data observed through cluster analysis. Both types of molecular markers (AFLP and SSR) used in this research proved to be suitable for investigating genetic diversity in the genotypes of Afghan bread wheat, however, AFLP markers gave better view of genetically relationships among genotypes than the SSR markers. The grouping generated by AFLP data showed a special agreement with the origin regions of genotypes (Ariana-07 and Mazar-99 originating from the north of Afghanistan, Lalmi-03 and Kabul-02. Large number of DNA bands identified with AFLP markers might provide a better estimation of genetic similarity than those of SSR markers.

A genome-wide association study identified loci for yield component traits in sugarcane (Saccharum spp.).

Sugarcane (Saccharum spp.) has a complex genome with variable ploidy and frequent aneuploidy, which hampers the understanding of phenotype and genotype relations. Despite this complexity, genome-wide association studies (GWAS) may be used to identify favorable alleles for target traits in core collections and then assist breeders in better managing crosses and selecting superior genotypes in breeding populations. Therefore, in the present study, we used a diversity panel of sugarcane, called the Brazilian Panel of Sugarcane Genotypes (BPSG), with the following objectives: (i) estimate, through a mixed model, the adjusted means and genetic parameters of the five yield traits evaluated over two harvest years; (ii) detect population structure, linkage disequilibrium (LD) and genetic diversity using simple sequence repeat (SSR) markers; (iii) perform GWAS analysis to identify marker-trait associations (MTAs); and iv) annotate the sequences giving rise to SSR markers that had fragments associated with target traits to search for putative candidate genes. The phenotypic data analysis showed that the broad-sense heritability values were above 0.48 and 0.49 for the first and second harvests, respectively. The set of 100 SSR markers produced 1,483 fragments, of which 99.5% were polymorphic. These SSR fragments were useful to estimate the most likely number of subpopulations, found to be four, and the LD in BPSG, which was stronger in the first 15 cM and present to a large extension (65 cM). Genetic diversity analysis showed that, in general, the clustering of accessions within the subpopulations was in accordance with the pedigree information. GWAS performed through a multilocus mixed model revealed 23 MTAs, six, three, seven, four and three for soluble solid content, stalk height, stalk number, stalk weight and cane yield traits, respectively. These MTAs may be validated in other populations to support sugarcane breeding programs with introgression of favorable alleles and marker-assisted selection.

Development of SSR Marker Set to Identify Fourty Two Indonesian Soybean Varieties

<p>Profile of molecular marker can be used for variety identification, genetic purity monitoring of germplasm and additional<br />requirement in proposing intellectual property protection. DNA fingerprinting of soybean had been applied at the ICABIOGRADIAARD<br />since 2004 using simple sequence repeat (SSR) markers which were run automatically by CEQ 8000 Genetic Analyzer<br />platform based on capillary electrophoresis system. This method had produced unique DNA fingerprints of the varieties tested,<br />but the marker set to efficiently identify the varieties had not yet been developed. This study aimed to develop a set of SSR<br />markers as a tool to identify the Indonesian soybean varieties. Fourty two soybean varieties were analyzed using 14 random SSR<br />markersA total of 168 alleles that were obtained from the polymorphism analysis. The average of polymorphic information<br />content (PIC) value observed was 0.7337 per SSR locus. Based on marker reproducibility rate, PIC value, number of rare alleles,<br />frequency of dominant alleles, and percentage of SSR fragment detected by genetic analyzer, we identified five SSR markers i.e.<br />Satt414, Satt147, Satt308, Satt009, and Satt516 as a SSR marker set to be used for soybean variety identification purposes. This<br />marker set was used to develop the identity (ID) of the 42 Indonesian soybean varieties.</p>

Optimization of Polymerase Chain Reaction for Inter Simple Sequence Repeat Technique for Four Species of Plants

Polymerase chain reaction optimization for inter simple sequence repeat primers is a key factor to obtain accurate and reproducible results for gene mapping, studying the genetic structure of populations, plant passporting, phylogenetic analysis. Changing temperature conditions, the amount of amplification cycles and concentration of reaction mixture components is allowed to vary the number of bands obtained by this method. This article is result of preliminary research of method selection for molecular analysis. It is aimed to show how to adjust the profile of inter simple sequence repeat fragments by polymerase chain reaction for four model species Stipa lessingiana, Poa intricata, Equisetum fluviatile and Pteridium aquilinum. The working concentrations of magnesium chloride for primer ((СТС)3GC) and ((АС)8YG) were 2.5 mM for 0.63 units of Taq DNA polymerase and for primer ((СА)6GG) it was 4.5 mM for 1.25 units. Sharply defined banding was observed from the minimal amount of DNA 5 ng per reaction, with primer concentration from 10 to 80 pmol and dNTPs concentration 0.2 mM. Optimal hybridization temperatures were 51.9 °C for primers ((АС)8YG), ((СА)6GG) and 50.0 °C for ((СТС)3GC). The best imaging results were obtained when setting up electrophoresis in 1.9% agarose gel

Molecular diversity of the entomopathogenic fungal Metarhizium community within an agroecosystem

The entomopathogenic fungal Metarhizium anisopliae lineage harbors cryptic diversity and was recently split into several species. Metarhizium spp. are frequently isolated from soil environments, but the abundance and distribution of the separate species in local communities is still largely unknown. Entomopathogenic isolates of Metarhizium spp. were obtained from 32 bulked soil samples of a single agroecosystem in Denmark using Tenebrio molitor as bait insect. To assess the Metarhizium community in soil from the agricultural field and surrounding hedgerow, 123 isolates were identified by sequence analysis of 5′ end of elongation factor 1-α and their genotypic diversity characterized by multilocus simple sequence repeat (SSR) typing. Metarhizium brunneum was most frequent (78.8%) followed by M. robertsii (14.6%), while M. majus and M. flavoviride were infrequent (3.3% each) revealing co-occurrence of at least four Metarhizium species in the soil of the same agroecosystem. Based on SSR fragment length analysis five genotypes of M. brunneum and six genotypes of M. robertsii were identified among the isolates. A single genotype within M. brunneum predominated (72.3% of all genotypes) while the remaining genotypes of M. brunneum and M. robertsii were found at low frequencies throughout the investigated area indicating a diverse Metarhizium community. The results may indicate potentially favorable adaptations of the predominant M. brunneum genotype to the agricultural soil environment.

Molecular Variation and Application from Aerospace Mutagenesis in Upland Rice Huhan 3 and Huhan 7

To further improve upland rice varieties Huhan 3 and Huhan 7, seed samples were sent to outer space with two recoverable spaceships for approximately 1 and 5 d and were propagated for 7 and 5 generations, respectively. Phenotypic analysis revealed that the morphological traits and the protein and amylose contents of grains changed. Characterization of genomic mutations by the gene-associated simple sequence repeat (SSR) and insertion-delete (InDel) markers indicated that the mutation pattern was very complex. Most of the mutations occurred at the 3′- or 5′-end of the fragments in the simple sequence repeat fragment. Reverse transcription-polymerase chain reaction (RT-PCR) assay showed that mutations in those parts of the SSR affected their gene expression, indicating that gene associated markers would be helpful to isolate functional genes. Field survey for breeding also revealed that more lines with high yield, high quality and drought-tolerance could be selected through aerospace breeding. The results indicate that aerospace mutagenesis resulted in molecular variation, as well as physiological and morphological changes for rice breeding.

Efficient multiplex simple sequence repeat genotyping of the oomycete plant pathogen Phytophthora infestans

Genotyping is fundamental to population analysis. To accommodate fast, accurate and cost-effective genotyping, a one-step multiplex PCR method employing twelve simple sequence repeat (SSR) markers was developed for high-throughput screening of Phytophthora infestans populations worldwide. The SSR markers reported for this species were evaluated and the twelve most informative and easily scored were selected. To accomplish a single step genotyping procedure, we optimized primers, fluorescent labels and PCR conditions to genotype using a capillary electrophoresis system with four fluorescent labels (FAM, NED, PET and VIC) and a labeled LIZ standard for sizing of the SSR fragments. The results obtained using commercially available multiplex PCR kits on a set of reference isolates were in agreement with that obtained using primer pairs in simplex reactions. In testing on many thousands of isolates, we have found the markers appropriate for resolving distinct multilocus genotypes (MLGs) of isolates of European and wider populations. Here we demonstrate the utility of the assay on a set of 19 reference isolates plus 77 others sampled from The Netherlands and Great Britain. In most isolates one to two alleles were observed at each locus but the presence of three alleles at a single locus in some isolates was consistent with increased ploidy. Methods are presented that are appropriate for the analysis of datasets comprising isolates of mixed ploidy levels. We also report on the direct P. infestans genotyping from infected field material to collect, store and extract pathogen DNA. A critical step in this multiplex method was the standardization of the protocol between two laboratories in The Netherlands and Great Britain. Reference isolates were exchanged and an allele nomenclature and scoring system agreed. Such co-operation is facilitating the genotyping of international collections of P. infestans isolates in wider networks of laboratories and providing the data required to expand an existing international database of pathogen diversity.

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, Ficus carica L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.

Comparison of SSR and AFLP analysis for genetic diversity assessment of Ethiopian arabica coffee genotypes

Knowledge of genetic diversity within and among genotypes of any crop is fundamental for estimation of the potential genetic gain in a breeding programme and effective conservation of available genetic resources. Currently, different molecular marker techniques are being developed for measuring genetic diversity. Comparison among molecular marker techniques is important for effective marker selection. However such types of efforts are rare for arabica coffee. This study was conducted to compare the efficiency of simple sequence repeat (SSR) analysis for determining genetic relationships of 28 Coffea arabica L. genotypes collected from different parts of Ethiopia with work previously done using amplified fragment length polymorphism (AFLP) analysis. A total of 22 SSR fragments were amplified and compared with 712 previously amplified AFLP fragments. AFLP and SSR markers were positively and significantly correlated (0.217) in estimating genetic similarity among genotypes. The average genetic similarity coefficient calculated using SSR markers was much lower (0.560 with a range of 0.286–1.000) compared to AFLP markers (0.915 with a range of 0.860–0.982) indicating the higher information content of SSR markers. AFLP markers distinguished all genotypes, while SSR markers distinguished 64.3% of the genotypes. Results indicated that AFLP markers were more efficient compared to SSR markers for characterization of the evaluated coffee genotypes.

Genotyping of Peach and Nectarine Cultivars with SSR and SRAP Molecular Markers

Simple sequence repeat (SSR) and sequence related amplified polymorphism (SRAP) molecular markers were evaluated for detecting intraspecific variation in 38 commercially important peach and nectarine (Prunus persica) cultivars. Out of the 20 SSR primer pairs 17 were previously developed in sweet cherry and three in peach. The number of putative alleles revealed by SSR primer pairs ranged from one to five showing a low level of genetic variability among these cultivars. The average number of alleles per locus was 2.2. About 76% of cherry primers produced amplification products in peach and nectarine, showing a congeneric relationship within Prunus species. Only nine cultivars out of the 38 cultivars could be uniquely identified by the SSR markers. For SRAP, the number of fragments produced was highly variable, ranging from 10 to 33 with an average of 21.8 per primer combination. Ten primer combinations resulted in 49 polymorphic fragments in this closely related set of peaches and nectarines. Thirty out of the 38 peach and nectarine cultivars were identified by unique SRAP fingerprints. UPGMA Cluster analysis based on the SSR and SRAP polymorphic fragments was performed; the relationships inferred are discussed with reference to the pomological characteristics and pedigree of these cultivars. The results indicated that SSR and SRAP markers can be used to distinguish the genetically very close peach and nectarine cultivars as a complement to traditional pomological studies. However, for fingerprinting, SRAP markers appear to be much more effective, quicker and less expensive to develop than are SSR markers.

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties.

Genetic distances (GDs) based on molecular markers are important parameters for identifying essentially derived varieties (EDVs). In this context information about the variability of molecular markers within maize inbred lines is essential. Our objectives were to (1) determine the variation in the size of simple sequence repeat (SSR) fragments among different accessions of maize inbreds and doubled haploid (DH) lines, (2) attribute the observed variation to genetic and marker system-specific sources, and (3) investigate the effect of SSR fragment size differences within maize lines on the GD between maize lines and their consequences for the identification of essentially derived varieties. Two to five accessions from nine inbred lines and five DH lines were taken from different sources or drawn as independent samples from the same seed lot. Each accession was genotyped with 100 SSR markers that evenly covered the whole maize genome. In total, 437 SSR fragments were identified, with a mean of 4.4 alleles per locus. The average polymorphic information content (PIC) was 0.58. GD estimates between two accessions of the same genotype ranged from 0.00 to 0.12 with an average of 0.029 for inbred lines and 0.001 for DH lines. An average of 11.1 SSRs was polymorphic between accessions of the same inbred line due to non-amplification (8.1 SSRs), heterogeneity (4.0 SSRs) or unknown alleles (2.6 SSRs). In contrast to lab errors, heterogeneity contributed considerably to the observed variation for GD. In order to decrease the probability to be suited for infringing an EDV threshold by chance, we recommend to increase the level of homogeneity of inbred lines before applying for plant variety protection.