Optimization of sequence alignment for simple sequence repeat regions

Abstract Background Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs). SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. Findings To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type. When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. Conclusions The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship.</p

Genomic DNA extraction from sapwood of Pinus roxburghii for polymerase chain reaction studies

A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged from 6.98 to 19.668 μg / 100 mg tissue and A260 / A280 ratio ranged from 1.70 to 1.87. The polymerase chain reaction (PCR) amplification of the DNA extracted from sapwood tissue using random amplification of polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers was similar to that of DNA extracted from the needle tissues.Keywords: Pinus roxburghii, DNA extraction, sapwood, random amplification of polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR)African Journal of Biotechnology Vol. 12(15), pp. 1732-173

Genetic diversity analysis and subspecies classification of Thailand rice landraces using DNA markers

Genetic diversity among 126 rice accessions, including 110 Thai landraces and 16 varieties used as subspecies reference, were evaluated by three types of DNA markers, InDel (Insertion/Deletion), inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. Twelve InDel primer pairs, based on DNA sequence polymorphism between ‘93-11’ (indica) and ‘Nipponbare’ (japonica), were used to identify subspecies of landraces. Most of the local rice samples had either ‘93-11’ alleles or ‘Nipponbare’ alleles. The scatter plotting of the principal component analysis (PCA) and dendrogram results based on InDel data could clearly classify landraces into two groups, indica and japonica. InDel and SSR markers showed the average polymorphic information content (PIC) values of 0.3707 and 0.6367, respectively. The dendrogram, based on combining InDel, ISSR and SSR data, could classify rice samples into five clusters at a cut-off genetic similarity value of about 0.70. The genetic similarity within landraces was low, indicating that Thai local rice samples have a great genetic diversity. The results of this experiment provide helpful data for rice germplasm management in breeding program.Key word: Rice, genetic diversity, DNA markers, simple sequence repeat (SSR), inter-simple sequence repeat (ISSR), insertion/deletion (InDel)

Molecular marker analysis of ‘Shatangju’ and ‘Wuzishatangju’ mandarin (Citrus reticulata Blanco)

‘Wuzishatangju’(Citrus reticulata Blanco) is an excellent cultivar derived from a bud sport of a seedy ‘Shatangju’ cultivar found in Guangdong Province in the 1980s. In this study, six molecular markers including random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-related amplified polymorphism (SRAP), inter-retrotransposn amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) were used to study the genetic variations between ‘Shatangju’ and ‘Wuzishatangju’. 1196 RAPD, seven SSR, 28 IRAP and 56 REMAP primers were used to detect the genetic variations between ‘Shatangju’ and ‘Wuzishatangju’. However, no difference was observed between the two cultivars. These results indicate that there was a very close genetic relationship between ‘Shatangju’ and ‘Wuzishatangju’ and RAPD, SSR, IRAP and REMAP markers could not distinguish them. Two and 21 specific bands were obtained using 100 ISSR and 153 SRAP primers, respectively. The present research could be a valuable tool for identification of Citrus bud sport clones, which laid the foundations for the further study of the mechanisms of Citrus bud sports.Key words: Citrus reticulata Blanco, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-related amplified polymorphism (SRAP), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), identification

Simple sequence repeat (SSR) and inter simple sequence repeat (ISSR) analyses of genetic diversity in tissue culture regenerated plants of cotton

Cotton is one of the main economic crop plants of Iran cultivated under continuous artificial selection and cultivation which may lead to genetic erosion and possible loss of useful genetic loci resulting in vulnerability to pests and diseases. For this reason increasing and improving the amount of genetic diversity in cotton germplasm through tissue culture is important. The present report considers genetic diversity induced in tissue culture regenerated plants of three cotton cultivars namely Mehr, Sindose and their hybrid Mehr X Sindose. Surface of seeds were disinfected with 70% ethanol for 2 min and then treated with 5% hypochlorite solution for 20 min. Finally, they were washed 3 to 4 times with sterile distilled water and inoculated aseptically on Murashige and Skoog (MS) basal medium free hormones. Single nodes resulted from seedlings cultured as explants. Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) primers used produced different number of bands in the genotypes studied showing different levels of molecular polymorphisms in each cultivar. Some common and few specific ISSR/SSR loci were indentified while some bands were present in all the genotypes except one indicating genetic changes in them. Analysis of molecular variance (AMOVA) test showed significant difference (p &lt; 0.05) for ISSR markers but not for SSR markers. Molecular trees obtained showed genetic variations among the regenerated plants of each cultivar due to tissue culture.Keywords: Cotton, genetic diversity, ISSR, RAP

Development of genomic simple sequence repeat markers for yam

Yam ( Dioscorea spp.) is a major staple crop widely cultivated for its starchy tubers. To date, very few marker resources are publicly avail - able as tools for genetic and genomic studies of this economically important crop. In this study, 90 simple sequence repeat (SSR) markers were developed from an enriched genomic library of yellow Guinea yam ( D. cayenensis Lam.). Cross- amplification revealed that 85 (94.4%) and 51 (56.7%) of these SSRs could be successfully transferred to the two major cultivated species of D. rotundata Poir. and D. alata L., respec - tively. Polymorphisms in 30 markers selected on the basis of reliability and reproducibility of DNA bands were evaluated using a panel of 12 D. cayenensis , 48 D. rotundata , and 48 D. alata accessions. Accordingly, number of alleles per locus ranged from 2 to 8 in D. cayenensis (mean = 3.9), 3 to 30 in D. rotundata (mean = 13.9), and 2 to 22 in D. alata (mean = 12.1). The average observed and expected heterozygosi - ties were 0.156 and 0.634 ( D. cayenensis ), 0.326 and 0.853 ( D. rotundata ), and 0.247 and 0.836 ( D. alata ), respectively. Clustering based on six SSRs that were polymorphic in at least four of the five cultivated Dioscorea species studied, including D. cayenensis , D. rotundata , D. alata , D. dumetorum (Kunth) Pax., and D. bulbifera L., detected groups consistent with the phyloge - netic relationships of the species except for D. dumetorum . These new SSR markers are invalu - able resources for applications such as genetic diversity analysis and marker-assisted breedingYam ( Dioscorea spp.) is a major staple crop widely cultivated for its starchy tubers. To date, very few marker resources are publicly avail - able as tools for genetic and genomic studies of this economically important crop. In this study, 90 simple sequence repeat (SSR) markers were developed from an enriched genomic library of yellow Guinea yam ( D. cayenensis Lam.). Cross- amplification revealed that 85 (94.4%) and 51 (56.7%) of these SSRs could be successfully transferred to the two major cultivated species of D. rotundata Poir. and D. alata L., respec - tively. Polymorphisms in 30 markers selected on the basis of reliability and reproducibility of DNA bands were evaluated using a panel of 12 D. cayenensis , 48 D. rotundata , and 48 D. alata accessions. Accordingly, number of alleles per locus ranged from 2 to 8 in D. cayenensis (mean = 3.9), 3 to 30 in D. rotundata (mean = 13.9), and 2 to 22 in D. alata (mean = 12.1). The average observed and expected heterozygosi - ties were 0.156 and 0.634 ( D. cayenensis ), 0.326 and 0.853 ( D. rotundata ), and 0.247 and 0.836 ( D. alata ), respectively. Clustering based on six SSRs that were polymorphic in at least four of the five cultivated Dioscorea species studied, including D. cayenensis , D. rotundata , D. alata , D. dumetorum (Kunth) Pax., and D. bulbifera L., detected groups consistent with the phyloge - netic relationships of the species except for D. dumetorum . These new SSR markers are invalu - able resources for applications such as genetic diversity analysis and marker-assisted breedin

Simple sequence repeat variation in the Daphnia pulex genome

Background: Simple sequence repeats (SSRs) are highly variable features of all genomes. Their rapid evolution makes them useful for tracing the evolutionary history of populations and investigating patterns of selection and mutation across gnomes. The recently sequenced Daphnia pulex genome provides us with a valuable data set to study the mode and tempo of SSR evolution, without the inherent biases that accompany marker selection. Results: Here we catalogue SSR loci in the Daphnia pulex genome with repeated motif sizes of 1-100 nucleotides with a minimum of 3 perfect repeats. We then used whole genome shotgun reads to determine the average heterozygosity of each SSR type and the relationship that it has to repeat number, motif size, motif sequence, and distribution of SSR loci. We find that SSR heterozygosity is motif specific, and positively correlated with repeat number as well as motif size. For non-repeat unit polymorphisms, we identify a motif-dependent end-nucleotide polymorphism bias that may contribute to the patterns of abundance for specific homopolymers, dimers, and trimers. Our observations confirm the high frequency of multiple unit variation (multistep) at large microsatellite loci, and further show that the occurrence of multiple unit variation is dependent on both repeat number and motif size. Using the Daphnia pulex genetic map, we show a positive correlation between dimer and trimer frequency and recombination. Conclusions: This genome-wide analysis of SSR variation in Daphnia pulex indicates that several aspects of SSR variation are motif dependent and suggests that a combination of unit length variation and end repeat biased base substitution contribute to the unique spectrum of SSR repeat loci

In silico comparative analysis of EST-SSRs in three cotton genomes

In this study, expressed sequence tags- simple sequence repeat (EST-SSRs) were surveyed in three cotton genomes (Gossypium arboreum, Ga; Gossypium raimondii, Gr and Gossypium hirsutum, Gh). The frequency of EST-SSRs was highest in Gr, and motif type for hexanucleotide was obviously abundant in Gr. Trinucleotide repeats were the most abundant motif; AT and AG, AAG and ATC were the most frequent motifs for dinucleotide and trinucleotide, respectively. The repeat number was greatly diverse between the three genomes with the highest variation in Gh. AG and AAG had a high frequency both in homologue groups (HGs) with and without repeat number change between genomes. The range of repeat number change in each HG was wider in Gr-Gh. The annotation of the SSR-ESTs showed that more Gene Ontology (GO) items targeted by SSR-ESTs of Ga and Gr than those of Gh. This study gave us new insights into the difference between the three cotton genomes, which will be more helpful to understand the differentiation and evolution of the three genomes.Key words: Cotton, simple sequence repeat, expressed sequence tags, motif, gene ontology

Applicazione di marcatori molecolari per la caratterizzazione di genotipi di <i>Myrtus communis</i> l.

The application of molecular markers technology to evaluate the genetic diversity within the species Myrtus communis L. has been aimed to analyse 38 selected genotypes, collected from different natural populations growing in Sardinia and prospected as improved varieties for myrtle cultivation. The results obtained showed the high efficiency in evidencing polymorphisms of ISSR (Inter Simple Sequence Repeat) molecular marker, thus proving to be useful in the evaluation of genetic relationship among the selections. The application of cpSSR (Chloroplast Simple Sequence Repeat) marker gave less reliable results, since data obtained were not suitable for the genotypes fingerprinting

Identification of simple sequence repeat markers for sweetpotato weevil resistance

The development of sweetpotato [Ipomoea batatas (L.) Lam] germplasm with resistance to sweetpotato weevil (SPW) requires an understanding of the biochemical and genetic mechanisms of resistance to optimize crop resistance. The African sweetpotato landrace, ‘New Kawogo’, was reported to be moderately resistant to two species of SPW, Cylas puncticollis and Cylas brunneus. Resistance has been associated with the presence of hydroxycinnamic acids esters (HCAs), but the underlying genetic basis remains unknown. To determine the genetic basis of this resistance, a bi-parental sweetpotato population from a cross between the moderately resistant, white-fleshed ‘New Kawogo’ and the highly susceptible, orange-fleshed North American variety ‘Beauregard’ was evaluated for SPW resistance and genotyped with simple sequence repeat (SSR) markers to identify weevil resistance loci. SPW resistance was measured on the basis of field storage root SPW damage severity and total HCA ester concentrations. Moderate broad sense heritability (H2 = 0.49) was observed for weevil resistance in the population. Mean genotype SPW severity scores ranged from 1.0 to 9.0 and 25 progeny exhibited transgressive segregation for SPW resistance. Mean genotype total HCA ester concentrations were significantly different (P < 0.0001). A weak but significant correlation (r = 0.103, P = 0.015) was observed between total HCA ester concentration and SPW severity. A total of five and seven SSR markers were associated with field SPW severity and total HCA ester concentration, respectively. Markers IBS11, IbE5 and IbJ544b showed significant association with both field and HCA-based resistance, representing potential markers for the development of SPW resistant sweetpotato cultivars