Sequence-specific Amplification Polymorphism Research Articles

Hybridization and polyploidization effects on LTR-retrotransposon activation in potato genome.

Hybridization and polyploidization are major forces in plant evolution and potatoes are not an exception. It is proposed that the proliferation of Long Terminal Repeat-retrotransposons (LTR-RT) is related to genome reorganization caused by hybridization and/or polyploidization. The main purpose of the present work was to evaluate the effect of interspecific hybridization and polyploidization on the activation of LTR-RT. We evaluated the proliferation of putative active LTR-RT in a diploid hybrid between the cultivated potato Solanum tuberosum and the wild diploid potato species S. kurtzianum, allotetraploid lines derived from this interspecific hybrid and S. kurtzianum autotetraploid lines (ktz-autotetraploid) using the S-SAP (sequence-specific amplified polymorphism) technique and normalized copy number determination by qPCR. Twenty-nine LTR-RT copies were activated in the hybrid and present in the allotetraploid lines. Major LTR-RT activity was detected in Copia-27, Copia-12, Copia-14 and, Gypsy-22. According to our results, LTR-RT copies were activated principally in the hybrid, there was no activation in allotetraploid lines and only one copy was activated in the autotetraploid.

Sequence-Specific Amplified Polymorphism (SSAP) and Sequence Characterized Amplified Region (SCAR) Markers in Zea mays.

Transposable elements (TEs) are mobile, recurring DNA sequences scattered throughout genome and have a large impact on genome structure and function. Several genetic marker techniques were developed to exploit their ubiquitous nature. Sequence-specific amplified polymorphism (SSAP) is a TE-based genetic marker system that has been used in various purposes such as measuring genetic relatedness between species, deciphering the population structures, molecular tagging for agronomic development in marker-assisted breeding (MAS). In addition to SSAP, sequence characterized amplified region (SCAR) from the SSAP markers provides an added advantage in identifying qualitative traits. Once developed SCAR markers are efficient, fast, and reliable method for genetic evaluations. These methods can be useful especially for the crops which have no genetic sequence information. With improved discriminatory ability they offer access to dynamic and polymorphic regions of genome. These techniques can be useful in breeding programs to improve or develop high yielding crops.

Verification of taxonomic relationships within the genus Secale (Poaceae: Pooideae: Triticeae) based on multiple molecular methods

This study aimed to verify the taxonomic relationships within the genus Secale. The plant material included 16 wild rye accessions from four species. Two approaches were applied: 1) whole genome scanning using three molecular marker systems: diversity arrays technology sequencing, simple sequence repeats and sequence-specific amplification polymorphism; and 2) characterisation based on polymorphisms within the sequences of two genes involved in benzoxazinoid biosynthesis: ScBx1 and ScBx5. Bayesian and neighbour-joining clustering and principal coordinate analysis were applied to illustrate relationships among species and accessions of Secale based on genetic distance (GD) matrices. Pearson’s correlation analysis between GD matrices was conducted. Clustering of Secale accessions revealed that S. sylvestre samples were the most divergent. The remaining accessions formed two clusters. One of them comprised S. strictum accessions while the second cluster consisted of subspecies of S. cereale, the species S. vavilovii and S. strictum subsp. ciliatoglume.

Parental transposable element loads influence their dynamics in young Nicotiana hybrids and allotetraploids.

The genomic shock hypothesis suggests that allopolyploidy is associated with genome changes driven by transposable elements, as a response to imbalances between parental insertion loads. To explore this hypothesis, we compared three allotetraploids, Nicotiana arentsii, N.rustica and N.tabacum, which arose over comparable time frames from hybridisation between increasingly divergent diploid species. We used sequence-specific amplification polymorphism (SSAP) to compare the dynamics of six transposable elements in these allopolyploids, their diploid progenitors and in corresponding synthetic hybrids. We show that element-specific dynamics in young Nicotiana allopolyploids reflect their dynamics in diploid progenitors. Transposable element mobilisation is not concomitant with immediate genome merger, but occurs within the first generations of allopolyploid formation. In natural allopolyploids, such mobilisations correlate with imbalances in the repeat profile of the parental species, which increases with their genetic divergence. Other restructuring leading to locus loss is immediate, nonrandom and targeted at specific subgenomes, independently of cross orientation. The correlation between transposable element mobilisation in allopolyploids and quantitative imbalances in parental transposable element loads supports the genome shock hypothesis proposed by McClintock.

Development of S-SAP Markers and Assessment of Their Potential in Genetic Studies of Representatives of the Genus Prunus L.

S-SAP (Sequence-Specific Amplified Polymorphism) markers developed on the basis of Cassandra retrotransposon LTR sequences were first used to study the genetic diversity and phylogeny of 65 representatives of seven species of the genus Prunus L. selected from the collection of the Krymskaya Experimental Breeding Station (KEBS). The use of six S-SAP markers developed within the framework of this study made it possible to identify from 29 to 185 polymorphic fragments per marker, and the mean polymorphism level was 92.75% per marker. This type of markers showed high efficiency for assessing intra- and interspecific genetic diversity in members of the genus Prunus. Cluster analysis of the results of S-SAP marking made it possible to distinguish six main groups corresponding to the investigated Prunus species, with the Prunus domestica genotypes occupying the isolated position. The reasons for the genetic isolation of Prunus domestica are discussed.

LTR retrotransposons from the Citrus x clementina genome: characterization and application

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis.

Analysis of genetic diversity in pigeon pea germplasm using retrotransposon-based molecular markers.

Pigeon pea (Cajanus cajan), an important legume crop is predominantly cultivated in tropical and subtropical regions of Asia and Africa. It is normally considered to have a low degree of genetic diversity, an impediment in undertaking crop improvement programmes.We have analysed genetic polymorphism of domesticated pigeon pea germplasm (47 accessions) across the world using earlier characterized panzee retrotransposon-based molecularmarkers. Itwas conjectured that since retrotransposons are interspersed throughout the genome, retroelements-based markers would be able to uncover polymorphism possibly inherent in the diversity of retroelement sequences. Two PCR-based techniques, sequence-specific amplified polymorphism (SSAP) and retrotransposon microsatellite amplified polymorphism (REMAP) were utilized for the analyses.We show that a considerable degree of polymorphism could be detected using these techniques. Three primer combinations in SSAP generated 297 amplified products across 47 accessions with an average of 99 amplicons per assay. Degree of polymorphism varied from 84-95%. In the REMAP assays, the number of amplicons was much less but up to 73% polymorphism could be detected. On the basis of similarity coefficients, dendrograms were constructed. The results demonstrate that the retrotransposon-based markers could serve as a better alternative for the assessment of genetic diversity in crops with apparent low genetic base.

English

Molecular markers have proven to be powerful tools in research related with diversity, variability, and improvement of economically important tropical crops. This study analyzed eight physiological and morphological fruit characters of economic interest in the cultivated Mexican guava (Psidium guajava L.), and assessed the suitability of two sequence specific amplified polymorphism (SSAP) and simple sequence repeat (SSR) markers developed for their use in early selection of individual plants with given fruit characteristics. Principal component analysis (PCA) explained 79% of the morphological variability observed among accessions. S-SAP was more informative than AFLP for studies of variability and diversity in guava, the former marker showing higher percentage of polymorphism (90%) and more intraspecific variability (0.58). It was analyzed by cluster analysis using the unweighted pair group with arithmetic means (UPGMA) method the relationships between accessions from nine guava varieties. S-SAP dendrograms clustered varieties in better agreement with pulp color and fruit shape, suggesting a possible association of the S-SAP marker with quantitative trait loci (QTL) related to fruit physiological and morphological fruit characters. According to results, the microsatellites mPgCIR131, mPgCIR136 and mPgCIR161 might also be linked to QTL related to internal and external pulp thickness, pulp color, and soluble solids, indicating that the SSR markers developed are appropriate for their use in early selection of guava individuals having specific fruit features, therefore being suitable for molecular marker assisted selection (MAS) of the crop. Key words: Microsatellites, quantitative trait loci (QTL), sequence specific amplified polymorphism (SSAP), simple sequence repeat (SSR), Psidium guajava L.

RETRACTED ARTICLE: Comparison of traditional and new generation DNA markers declares high genetic diversity and differentiated population structure of wild almond species

Wild almond species as sources of genetic variation may have crucial importance in breeding. A total of 389 accessions of 18 species have been analysed using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplification polymorphism (S-SAP), amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR) and simple sequence repeats (SSR). Retrotransposon markers indicated the presence and movement of some Ty3-gypsy and Ty1-copia-elements in almond genome. Since transposable elements are associated with large-scale genome alterations, REMAP produced more reliable phylogenetic inferences than AFLP where homoplasy may affect clustering. In addition, high resolution melting (HRM) analysis was developed to detect SNPs. HRM analysis revealed 1:189 bp frequency of SNPs in exon positions, and the transition-to-transversion proportion was 1.84:1. The low transition bias suggests low methylation levels in almond genome. The polymorphic information content (PIC) was the highest for SSR markers, while SNPs had an average PIC of 0.59, which is close to the values of the rest of the markers. Huge genetic diversity, fragmented population structure and footprints of human selection was confirmed by merging information from all marker strategies. Considering time, cost and performance HRM can be a marker of choice in future studies of Prunus diversity.

Retrotransposon-based sequence-specific amplification polymorphism markers reveal that cultivated Pyrus ussuriensis originated from an interspecific hybridization

Retrotransposon-based sequence-specific amplification polymorphism markers reveal that cultivated Pyrus ussuriensis originated from an interspecific hybridization

Retrotransposon-based sequence-specific amplified polymorphism markers for the analysis of genetic diversity and phylogeny in Malus Mill. (Rosaceae)

Sequence-Specific Amplified Polymorphism (S-SAP)-analysis of 131 accessions of different species from five sections of the genus Malus was carried out to study genetic diversity, and to clarify phylogenetic and taxonomic issues. S-SAP-markers, based on long terminal repeat (LTR)-retrotransposons TRIM2 and dem1, were developed, which identified 679 polymorphic fragments in the studied Malus species. S-SAP technique proved to be effective for taxonomic studies in Malus. The obtained results generally support the existing sectional taxonomy in the genus Malus and allowed to determine the taxonomic status of some Russian landraces. The genetic diversity and taxonomic status of the Russian apple Antonovka landraces, widely used in breeding programs for their increased adaptation to abiotic stress and scab resistance, were clarified. All of them belong to M. domestica, section Malus. However, Antonovka Olginskaya might have a hybrid origin with some contribution from Gymnomeles species according to PCO analysis data. The taxonomic status was resolved for another Russian landrace, Yakutskaya, which exhibits increased winter hardiness and drought resistance; it belongs to section Gymnomeles, with a high resemblance to M. baccata.

Genetic diversity analysis of maize lines using AFLP and TE-based molecular marker systems

Maize has been domesticated in diverse environments ranging from low latitudes in tropical countries to high latitudes in Canada. Because maize breeding programs primarily focus on hybrid vigor by selectively crossing inbred lines to maximize recombination, we collected a diverse array of commercial hybrid and inbred lines from southern Asia, China, and Canada and analyzed them by amplified length fragment polymorphism (AFLP), sequence-specific amplified polymorphism (SSAP), and CACTA-transposon display (TD) analyses. Cluster analyses using these molecular marker systems clearly differentiated these maize lines into three groups: southern Asian lines, northern Asian lines, and Canadian lines. However, principal coordinate analysis (PCoA) based on Nei’s distances grouped them into two groups: Asian and Canadian lines. Thus, groupings by cluster dendrograms and PCoA showed that geographic origin was a more dominant factor than growing seasonal differences resulting from different latitudes. The overall genetic diversity (Ht) was found to be high (more than 80 % molecular variations) among the maize lines by all three of the marker systems.

Isolation, characterization, and marker utility of KCRE1, a transcriptionally active Ty1/copia retrotransposon from Kandelia candel.

Transposable elements are key players in eukaryotic genomic diversity. Due to their high abundance, great heterogeneity, and replicative transposition, long terminal repeat (LTR) retrotransposons are well suited as molecular markers for breeding and biodiversity studies, whereas their utilities in non-model organisms have been hindered by the lack of prior sequence knowledge. In this study, a putative complete (5362bp) LTR retrotransposon was isolated and characterized in the mangrove species Kandelia candel (named KCRE1), and its transcription, insertional polymorphisms and copy number variations were also investigated. KCRE1 has all the features of a typical copia retroelement, and its transcription initiation and termination sites were identified by 5' and 3' rapid amplification of cDNA ends (RACE), respectively. KCRE1 exhibits high sequence similarity with the tomato retroelement Rider and is constitutively expressed in the leaf, root, flower, and hypocotyl tissues of K. candel. Based on KCRE1, sequence-specific amplification polymorphism (SSAP) markers were developed to explore genetic diversity across 30 individuals from three natural populations of K. candel in China. Six primer combinations yielded a total of 204 SSAP bands with an averaged percentage of polymorphic loci of 55.37 and 41.35% at the species and population levels, respectively. Each individual had a distinct SSAP phenotype, and 14-23 unique bands were observed for each population. Accordingly, KCRE1 was highly abundant in the genome of K. candel and showed considerable copy number variation among the three populations. In conclusion, KCRE1 is the first transcriptionally active retrotransposon reported in K. candel, providing a useful tool for the elucidation of untapped genetic diversity in mangrove genomes.

Primitive Genepools of Asian Pears and Their Complex Hybrid Origins Inferred from Fluorescent Sequence-Specific Amplification Polymorphism (SSAP) Markers Based on LTR Retrotransposons

Recent evidence indicated that interspecific hybridization was the major mode of evolution in Pyrus. The genetic relationships and origins of the Asian pear are still unclear because of frequent hybrid events, fast radial evolution, and lack of informative data. Here, we developed fluorescent sequence-specific amplification polymorphism (SSAP) markers with lots of informative sites and high polymorphism to analyze the population structure among 93 pear accessions, including nearly all species native to Asia. Results of a population structure analysis indicated that nearly all Asian pear species experienced hybridization, and originated from five primitive genepools. Four genepools corresponded to four primary Asian species: P. betulaefolia, P. pashia, P. pyrifolia, and P. ussuriensis. However, cultivars of P. ussuriensis were not monophyletic and introgression occurred from P. pyrifolia. The specific genepool detected in putative hybrids between occidental and oriental pears might be from occidental pears. The remaining species, including P. calleryana, P. xerophila, P. sinkiangensis, P. phaeocarpa, P. hondoensis, and P. hopeiensis in Asia, were inferred to be of hybrid origins and their possible genepools were identified. This study will be of great help for understanding the origin and evolution of Asian pears.

Analysis of the genetic diversity of super sweet corn inbred lines using SSR and SSAP markers.

In this study, we compared the efficiency of simple sequence repeat (SSR) and sequence specific amplified polymorphism (SSAP) markers for analyzing genetic diversity, genetic relationships, and population structure of 87 super sweet corn inbred lines from different origins. SSR markers showed higher average gene diversity and Shannon's information index than SSAP markers. To assess genetic relationships and characterize inbred lines using SSR and SSAP markers, genetic similarity (GS) matrices were constructed. The dendrogram using SSR marker data showed a complex pattern with nine clusters and a GS of 53.0%. For SSAP markers, three clusters were observed with a GS of 50.8%. Results of combined marker data showed six clusters with 53.5% GS. To analyze the genetic population structure of SSR and SSAP marker data, the 87 inbred lines were divided into groups I, II, and admixed based on the membership probability threshold of 0.8. Using combined marker data, the population structure was K = 3 and was divided into groups I, II, III, and admixed. This study represents a comparative analysis of SSR and SSAP marker data for the study of genetic diversity and genetic relationships in super sweet corn inbred lines. Our results would be useful for maize-breeding programs in Korea.

Cultivar Identity, Intravarietal Variation, and Health Status of Native Grapevine Varieties in Croatia and Montenegro

Croatia and Montenegro (located in southeast Europe) host numerous native (autochthonous) minor grapevine (Vitis vinifera L.) varieties that might irreversibly disappear in the near future because of limited conservation efforts and lack of virus-free propagation material. To prevent their disappearance and help sustain these native populations, a collaborative project was launched to gather and evaluate extremely rare and neglected native grapevine genotypes by establishing true-to-type and virus-tested stock material, ready to use for nurseries. During several field expeditions, 284 grapevine accessions collected in 22 different locations in Croatia and Montenegro were subjected to ampelographic in situ description, genetic identification by nine simple sequence repeat (SSR) markers, serological testing for the presence of common grape viruses, and intravarietal variability assessment by sequence-specific amplification polymorphism (S-SAP) markers. We observed 53 unique SSR profiles, of which 25 genotypes have not been previously reported and are thus worthy of further research. Comparison of these genetic profiles with those from some very rare and poorly described accessions within the European Vitis Database and other published studies revealed several previously unknown synonyms and homonyms, which helped properly name some of the profiles and provided novel information on the history of these cultivars and their distribution in the region. Virus testing revealed high frequencies of infection with Grapevine leafroll-associated viruses 1 and 3 and indicated the urgent need for sanitary selection. Assessment of intravarietal genetic variation indicated the presence of accumulated mutations, which strongly suggests a need for clonal selection.

Genetic and epigenetic diversity among dent, waxy, and sweet corns

Maize is an important crop species that has been subjected to intensive breeding programs. Understanding the genetic diversity in maize is thus vital for crop improvement and breeding studies. We performed four different multi-allele detecting marker systems amplified fragment length polymorphism (AFLP), sequence-specific amplified polymorphism (SSAP), methylation-sensitive amplified polymorphism (MSAP), and transposon display methylation amplified polymorphism (TD-MSAP) to analyze genetic and epigenetic variation and diversity among dent, waxy, and sweet corn. The (epi) genetic diversity, (epi) genetic distance, gene flow, genetic frequency, cluster analysis, PCoA, and AMOVA analysis were calculated. Cultivar differentiation is well-established in maize, and the results suggest that not only genetic but also epigenetic variation is responsible for the differences obtained in maize cultivars. Estimates of genetic relationship were significantly correlated between the AFLP, SSAP, and MSAP datasets. The current study is the first report on the systematic comparison of TD-MSAP with the other marker systems. The genetic variation detected by TD-MSAP was higher than that calculated by AFLP, SSAP, and MSAP but was in discordance with accepted genetic relationships. These dissimilarities between TD-MSAP and other marker systems can be informative for understanding the genetic relationships between the species.

Identification, characterization and diversification of non-autonomous hAT transposons and unknown insertions in Brassica

Transposons, the mobile genetics elements played an important role in genome evolution and duplication. Several DNA transposons superfamilies are described in plants and animal genomes; CACTA, hAT, Mariner Mutator and Harbinger being the most diverse among plants. The hAT transposons are proliferating in many plant and animal species. The dot plot comparison, computational and molecular approaches were used for the characterization and diversification of non-autonomous hAT families in Brassica and 494 and 30 full length elements were collected with estimated copies of ~5052 and ~4110 from whole Brassica rapa and Brassica oleracea genomes respectively. The analysis of terminal inverted repeats (TIRs) of these hAT sequences classified them into 13 families on the basis of TIRs sequence similarities. The WebLogo of TIRs of hATs confirmed that few family based TIRs are highly conserved, while other showed variable nucleotide sequences. Transposons based sequence specific amplification polymorphism markers revealed the insertion preference of hATs in diverse Brassica genomes and found many elements as polymorphic across Brassica accessions. Some elements were A or C-genome specific, while most of them are present in Brassica diploids and allopolyploids revealing their presence before separation of A, B and C-Brassica genomes around 8 MYA. Several other unknown mobile insertions were identified with or without TIRs and TSDs of varied lengths, not common to known transposon superfamilies. The detailed study of these insertions revealed their distribution and mobile nature, which although less in numbers and small in sizes are playing a role in genome evolution.

SSAP analysis reveals candidate genes associated with deastringency in persimmon (Diospyros kaki Thunb.) treated with 40 °C water

Warm water treatment is a simple and effective deastringency method for the persimmon fruit. The astringency in the ‘Eshi 1’ persimmon, a Chinese pollination-constant and non-astringent (C-PCNA) cultivar, can be quickly removed in 40 °C water coupled with the activation of the retrotransposon DkRE1. In this study, transcriptional level changes in ‘Eshi 1’ persimmon treated with 40 °C water were studied with a sequence-specific amplification polymorphism (SSAP) approach to investigate the genes associated with deastringency. The complementary DNA (cDNA)-SSAP analysis revealed that 283 differentially expressed transcript-derived fragments (TDFs) potentially involved in ‘Eshi 1’ persimmon deastringency were successfully cloned and sequenced. In total, 116 differentially expressed TDFs were annotated as genes with known function, including four aldehyde metabolism genes and two pectin-related genes. Our data suggested that the tannin-pectin complex formation and the aldehyde-mediated coagulation effect contributed to the deastringency of the persimmon fruit treated with 40 °C water. Notably, three gene fragments, triosephosphate isomerase (TDF 175-3), pyruvate kinase (TDF 284-2), and neomenthol dehydrogenase (TDF 238-2), which are potentially involved in the 40 °C water coagulation effect were identified. To our knowledge, this report is the first to identify these gene fragments in the persimmon. The SSAP method was innovatively applied to gene discovery based on high-density polymorphisms. Expression analysis and transcriptome data confirmed the validity and accuracy of the cDNA-SSAP method for discovering new candidate genes. These successfully isolated genes could facilitate understanding of astringency removal in persimmon.

DKRE1—The first full-length Ty1-copia-like retrotransposon in persimmon: Isolation, characteristic and potential involvement in occurrence of bud mutations

The focus of this study revolved around persimmon (Diospyros kaki Thunb.) retrotransposable element 1 (DKRE1), a complete long terminal repeat (LTR) retrotransposon (RTN) that was isolated from persimmons. Its structure, phylogenetic classification, copy number, active characteristics and potential roles involved in the occurrence of bud mutations in persimmons were investigated. Having a length of 6, 235bp, DKRE1 possessed all of the sequence features associated with Ty1-copia-like retrotransposons. Phylogenetic analysis showed significant, but not strong, identity in amino acid sequences with BARE1 (barley retrotransposable element-1), RIRE1 (rice retrotransposable element-1) and Ppcrt (Pyrus pyrifolia copia retrotransposon) groups. The estimated copy number of 45 copies per haploid genome suggested that DKRE1 possesses a low constitution in the persimmon genome. Furthermore, the transcription of DKRE1 could be detected in leaf tissue treated with salicylic acid (SA), methyl jasmonate (MJA), and abscisic acid (ABA), indicating its active characteristics when subjected to exogenous phytohormones. Polymorphisms were revealed between several pairs of bud mutations by way of either inter-retrotransposon amplified polymorphism (IRAP) or sequence-specific amplified polymorphism (SSAP) retrotransposon-based molecular markers, showing a potential involvement in their occurrence. To our knowledge, this is the first report of the isolation of a complete LTR retrotransposon with transcriptional activity in persimmons. This study is valuable for future research on the mechanisms of bud mutation and development of retrotransposon-based molecular markers for genetic analysis.