Sequence-specific Amplification Polymorphism Markers Research Articles

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.

Development and application S-SAP molecular markers for the identification of apple (Malus × domestica Borkh.) sports

SummaryApple (Malus _ domestica Borkh.) is a widely-cultivated, economically important fruit crop with nutritional and medicinal value. Apple has emerged as a model horticultural crop in the post-genomic era. Apple sport resources are important, develop gradually in the apple industry, and provide genetic diversity. In this study, we have successfully developed a sequence-specific amplification polymorphism (S-SAP) marker system based on Ty1-copia retrotransposons in the apple genome. Among the 34 primer combinations used to develop the S-SAP marker system, two primer combinations produced profiles with clear and different peak patterns. Based on these primer combinations, 15 apple sport accessions (four ‘Gala’, six ‘Fuji’, and five ‘Delicious’) were grouped into three clusters based on a genetic variation coefficient value of 0.78. In addition, 27 ‘Delicious’ apple sport accessions were also selected to perform S-SAP marker analysis using the same two primer combinations. The results showed that all ‘Delicious’ apple sport accessions tested were clearly distinguished. The S-SAP data shared many consistencies with known relationships among apple sport accessions. The available evidence showed that these S-SAP markers had a high level of polymorphism in these apple accessions, which could be applied efficiently to determine genetic diversity, systematic evolution, and for variety identification.

S-SAP MARKERS - A VALUABLE TOOL FOR GENETIC DISTANCE ANALYSIS IN BRASSICA RAPA L.

Because transposable elements contribute significantly to diversification between and within plant species, transposon-based molecular markers could have special benefits for phylogenetic studies. Sequence-specific amplification polymorph-ism (S-SAP) technique uses transposon-related primers combined with AFLP primers. Among the Class II TEs CACTA elements are of special interest, having highly conserved ends and highly variable internal sequences. For our analysis Bot1-related TIR sequences were used. Combinations with different AFLP primers were tested for number of bands, polymorphism degree and reproducibility. With selected primer combinations the VIR Brassica rapa core collection of 96 accessions encompassing a wide range of morphological types and geographical origins was investigated. Twelve primer combinations generated 123 polymorphic markers. The mean PIC value of these markers was 1.47-fold that of 149 SSR alleles tested in the same core collection. The set of S-SAP markers was used for distance analysis to generate a phylogenetic tree as basis for association studies with morphological traits, metabolite content and resistance traits.

Phylogenetic analysis of pigeonpea (<i>Cajanus cajan</i>) genotypes using <i>Panzee </i>retrotransposon based SSAP markers

In the present study, twenty one pigeonpea [Cajanus cajan (L.) Millspaugh] genotypes representing three different gene pools were analysed for phylogenetic relationships using sequence specific amplification polymorphism markers. Four primers were screened involving selective AFLP primers in combination with primer specific to 5'LTR region of Panzee retrotransposon. A total of 183 bands were scored, out of which 166 (90.71%) bands were polymorphic with an average of 41.5 polymorphic bands per primer combination. Polymorphism information content (PIC) values ranged from 0.15 [Panzee-LTR/ EcoRI (+AAC)] to 0.26 [Panzee-LTR/ EcoRI (+AAG)] for four primer combinations. Resolving power (RP) values ranged from 10.14 [Panzee-LTR/ EcoRI (+AAC)] to 24.92 [Panzee-LTR/ MseI (+CAC)], whereas marker index (MI) values ranged from 5.25 [Panzee-LTR/ EcoRI (+AAC)] to 14.25 [Panzee-LTR/ MseI (+CAC)] exhibiting positive correlation (r2=0.99 P<0.05). Primer Panzee-LTR/ MseI (+CAC) with higher values of RP (24.92) and MI (14.25) was found most informative for analysing the phylogenetic relationships. NJ tree based on 166 polymorphic SSAP markers exhibited concordant relationships among pigeonpea genotypes supported by high bootstrap values. The genotypes belonging to tertiary gene pools and primary gene pool were placed at extremes, whereas one accession of C. cajanifolius (ICP 15632) was placed closer to primary gene pool, confirming C. cajanifolius is the closest progenitor of cultivated pigeonpea. SSAP markers produced high rate of polymorphism and revealed phylogenetic relationships among pigeonpea genotypes.

SSAP markers based on a novelTy1-copialike element are a powerful tool for the assessment of genetic diversity in rye (Secale cerealeL.) inbred lines

A partial sequence of a Ty1-copia retrotransposon was isolated from the genome of the rye (Secale cereale L.) inbred line L318 using degenerate primers targeted at the RNaseH sequence. Its putative 3’LTR region exhibited similarity to LTRs of known plant retrotransposons such as BARE-1, while no significant similarity to known rye retrotransposon sequences was found. A Sequence Specific Amplification Polymorphism (SSAP) protocol employing primer designed to correspond to the putative polypurine tract was developed and used for analysis of 30 rye inbred lines. Values of the parameters calculated to evaluate the efficiency of the method as well as the results of cluster analysis demonstrate that it is a powerful tool for genetic diversity analyses of rye.

Genetic assessment of the genus Pisum L. based on sequence specific amplification polymorphism data

The present study was conducted to analyze genetic diversity among 56 accessions of Pisum sativum, Pisum elatius, Pisum pumilio, Pisum fulvum and Podandrogyne brevipedunculata from Syria (42) and a set of 16 reference accessions from Israel (3), Ethiopia (2), Greece (2), Italy (2), Turkey (3), Tunisia (1) and Georgia (1). The study was based on the analysis of multiple data sets generated using sequence specific amplification polymorphism (SSAP) and associated morphological and ecogeographic data. The diversity analysis based on SSAP markers resulted in 83 scorable fragments in P. sativum, P. elatius (63), P. fulvum (62), P. pumilio (53), P. brevipedunculata (31) and P. abyssinicum (29). Genetic variability was measured as Nei’s gene diversity and maximum polymorphism was found in P. elatius (33.87%) and P. fulvum (32.74%) whereas minimum diversity was observed in P. brevipedunculata (12.48%). Pair wise band sharing among Pisum species was also calculated, significant band sharing was observed between P. fulvum and P. elatius, P. elatius and P. sativum and P. pumilio and P. elatius. The relationship among species as revealed by SSAP data could not significantly be correlated with those based on the agro-morphologic characters, suggesting that the two inferences are independent and estimates of genetic relations differently among Pisum taxa. The ecogeographic data associated with Pisum species (latitude, longitude) were also plotted using Arc View GIS 3.2 in order to show the distribution of species in Syria.

Development of Ty1-copia retrotransposon-based S-SAP markers in strawberry (Fragaria × ananassa Duch.)

The most popular retrotransposon-based molecular marker system at the present time is the sequence-specific amplification polymorphism (S-SAP) system, which is widely used for various applications including phylogenetic analysis. In this study, we successfully developed a new S-SAP marker system based on FaRE1 retrotransposon in strawberry genome. Among 34 primer combinations used to develop the S-SAP marker system, seven primer combinations produced high quality and reliability banding patterns. A total of 288 polymorphic bands from 722 amplified bands were detected in 13 strawberry accessions, with a polymorphic ratio of 39.8%. Phylogenetic analysis showed that genetic coefficient ranged from 0.67 to 1.00 for 13 strawberry accessions. An 187bp S-SAP band was detected from 50 strawberry plantlets regenerated from leaves which were cultured on MS media supplemented with 1.0mg/l thidiazuron (TDZ) and 0.1mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The available evidences proved S-SAP marker had high level of polymorphism in strawberry accessions.

Highly variable AFLP and S-SAP markers for the identification of 'Malbec' and 'Syrah' clones

The retrotransposon-based sequence-specific amplification polymorphism (S-SAP) and the amplified fragment length polymorphism (AFLP) marker systems were used to assess the clonal variation of 14 ‘Syrah’ and 22 ‘Malbec’ ( Vitis vinifera L.) clones. The utility of S-SAP markers was compared to that of AFLP markers. On the basis of our results, S-SAP is more informative marker system and showed higher average number of polymorphic bands per cultivar group than AFLP. Relationships among clones were analyzed by cluster analysis using unweighted pair-groups using arithmetic averages and in both cases revealed well defined groups of clones, in which ‘Malbec’ clones were separated from ‘Syrah’ clones. High variability of some clones could also be seen within these clusters. The different levels of polymorphism for ‘Malbec’ and ‘Syrah’ obtained in this study suggest that ‘Malbec’ exhibits a higher mutation frequency than ‘Syrah’. Our results indicate that higher proportion of polymorphic bands in S-SAP makes it a less labour-intensive and more efficient approach for developing markers for clonal identification.

Comparison of Genetic Structure Assessed by Amplified Fragment Length Polymorphism and Retrotransposon-based Sequence-specific Amplification Polymorphism for Portuguese Populations of Pinus pinea L.

AbstractIn order to assess genetic diversity within and among populations of Pinus pinea L. (stone pine), seven Portuguese populations originating from three Provenance Regions were selected and genotyped using two marker systems. We compared the genetic variation of these populations using retrotransposon-based sequence-specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). In total, 105 trees were screened with three primer enzyme combinations (PEC), producing 232 SSAP and 132 AFLP loci. Where SSAP yielded approximately twice-the number of polymorphic fragments compared to AFLP. Differentiation was slightly higher for SSAP, than for AFLP (FST = 0.105 for SSAP and 0.074 for AFLP), and both significantly different from zero, P < 0.01. The levels of average genetic diversity within-population found with the two types of marker were not significantly different between SSAPs and AFLPs (26.6% and 22.8%, respectively). The populations that displayed the highest and lowest genetic diversity scores were the same for both markers, and only two populations had significantly different He estimates. The neighbor-joining tree based on the Nei’s genetic distance displayed some geographic pattern. With the AFLP markers the populations grouped according to the provenance regions where they were sampled, resulting in one well supported cluster with the Southern populations, but with SSAP the pattern was not so coherent. In this study SSAP generated more polymorphic fragments and higher estimates of genetic diversity than AFPL did, due, probably, to the higher mutation rate of retrotransposition relative to base mutation. Nevertheless, congruence was found between estimates obtained with both markers, which is very interesting, for, in general, SSAP markers have lower costs compared to AFLPs, and they might be an interesting alternative marker system, when higher resolution is requested.

Ty1-copiaretrotransposon-based SSAP marker development and its potential in the genetic study of cucurbits

Three long terminal repeat (LTR) sequences of Ty1-copia retrotransposons were identified in cucumber (Cucumis sativus L.) and named Tcs 1, Tcs 2, and Tcs 3. A sequence-specific amplification polymorphism (SSAP) marker system based on these LTR sequences displayed a higher level of polymorphism than AFLPs in cucumber. This marker system could also detect loci in other Cucumis species for genetic diversity analysis. The three Tcs LTRs existed within the exons of genes because of the effective amplification band patterns from the cDNA templates. The potential usefulness of the SSAP marker system in studies of the evolution of genes or genomes was verified after exploring loci changes in first and second generations of a synthetic allotetraploid in Cucumis. This study is the first report of the development of a retrotransposon-based marker system and the SSAP technique in cucurbits.

Isolation and characterization of Ty1- copia retrotransposon sequences in the blue agave ( Agave tequilana Weber var. azul) and their development as SSAP markers for phylogenetic analysis

Agave tequilana Weber variety “azul” is the only variety of blue agave which is accepted for the production of tequila, but the phylogenetic relationships between this variety and other agave germplasm is still poorly understood. Retrotransposon-based molecular markers have proven to be highly informative in determining phylogenetic relationships in other crop systems. We report the isolation of a population of Ty1- copia retrotransposon terminal sequences from A. tequilana and develop three of these as sequence-specific amplification polymorphism (SSAP) molecular genetic markers. SSAP analysis shows high levels of retrotransposon polymorphism throughout agave varieties and species and identifies the tequila agaves as a distinct phylogenetic group. The retrotransposon SSAP markers presented here represent a powerful new tool for germplasm assessment in the agaves.

Ty1-copia retrotransposon-based SSAP marker development in cashew (Anacardium occidentale L.)

The most popular retrotransposon-based molecular marker system in use at the present time is the sequence-specific amplification polymorphism (SSAP) system . This system exploits the insertional polymorphism of long terminal repeat (LTR) retrotransposons around the genome. Because the LTR sequence is used to design primers for this method, its successful application requires sequence information from the terminal region of the mobile elements . In this study, two LTR sequences were isolated from the cashew genome and used successfully to develop SSAP marker systems. These were shown to have higher levels of polymorphism than amplified fragment length polymorphic markers for this species.

Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR

The retrotransposon-based sequence-specific amplification polymorphism (SSAP) marker system was used to assess the genetic diversities of collections of tomato and pepper industrial lines. The utility of SSAP markers was compared to that of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. On the basis of our results, SSAP is most informative of the three systems for studying genetic diversity in tomato and pepper, with a significant correlation of genetic relationships between different SSAP datasets and between SSAP, AFLP and SSR markers. SSAP showed about four- to ninefold more diversity than AFLP and had the highest number of polymorphic bands per assay ratio and the highest marker index. For tomato, SSAP is more suitable for inferring overall genetic variation and relationships, while SSR has the ability to detect specific genetic relationships. All three marker results for pepper showed general agreement with pepper types. Additionally, retrotransposon sequences isolated from one species can be used in related Solanaceae genera. These results suggest that different marker systems are suited for studying genetic diversity in different contexts depending on the group studied, where discordance between different marker systems can be very informative for understanding genetic relationships within the study group.

Transposable elements reveal the impact of introgression, rather than transposition, in Pisum diversity, evolution, and domestication.

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.

Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, recently derived from the Amplified Fragment Length Polymorphism (AFLP) technique, produces amplified fragments containing a retrotransposon LTR sequence at one end and a host restriction site at the other. We report the application of this procedure to the LTR of the Tms1 element from Medicago sativa L. Genomic dot-blot analysis indicated that Tms1 LTRs represent about 0.056% of the M. sativa genome, corresponding to 16 x 10(3) copies per haploid genome. An average of 66 markers were amplified for each primer combination. Overall 49 polymorphic fragments were reliably scored and mapped in a F(1) population obtained by crossing diploid M. falcata with M. coerulea. The utility of the LTR S-SAP markers was higher than that of AFLP or SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) markers. The efficiency index of the LTR S-SAP assay was 28.3, whereas the corresponding values for AFLP and SAMPL markers were 21.1 and 16.7, respectively. The marker index for S-SAP was 13.1, compared to 8.8 for AFLP and 9.5 for SAMPL. Application of the Tms1 LTR-based S-SAP to double-stranded cDNA resulted in a complex banding pattern, demonstrating the presence of Tms1 LTRs within exons. As the technique was successfully applied to other species of the genus Medicago, it should prove suitable for studying genetic diversity within, and relatedness between, alfalfa species.