Retrotransposon-based Molecular Markers Research Articles

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches' broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this promp- ted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter- retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymor- phism (REMAP) techniques were used to study the variability of 70M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geo- graphical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.

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.

Isolation of Ty1-copia retrotransposon in myrtle genome and development of S-SAP molecular marker

Long terminal repeat (LTR)-retrotransposons are mobile genetic elements that are ubiquitous in plants and constitute a major portion of their nuclear genomes. LTR- retrotransposons possess unique properties that make them appropriate for investigating relationships between populations, varieties and closely related species. Myrtus communis L. is an evergreen shrub growing spontaneously throughout the Mediterranean area. Accessions show significant variations for agriculturally important traits, so the development of specific molecular markers for conservation and characterization of myrtle germplasm is desirable to conserve biodiversity. In this study, we isolated the first retrotransposon Ty1-copia-like element (Tmc1) in Myrtus communis L. genome and used this as a molecular marker. We successfully employed the S-SAP marker system to specifically characterize four myrtle accessions belonging to different areas in the province of Caserta (Italy). The high level of polymorphism detected in isolated LTRs, make Tmc1 a good molecular marker for this species. Our findings confirm that retrotransposon-based molecular markers are particularly valuable tools for plant molecular characterization studies.

Molecular characterization of novel Ty1-copia-like retrotransposons in pear (Pyrus pyrifolia)

Retrotransposons are present in all plant genomes and play important roles in genome size, genome structure remodeling, gene function, and genome evolution. Eight novel long terminal repeat retrotransposons were identified from a bacterial artificial chromosome library of Japanese pear (Pyrus pyrifolia). On the basis of the order of gene arrangement within the gag and pol domains (protease, integrase, reverse transcriptase, and RNase H), these newly identified retrotransposons appear to be closely related to Ty1-copia retrotransposons. They were designated Ppcrt1–8 and classified into two groups based on the presence or absence of a 142-amino-acid deletion within the group-specific antigen DNA-binding domain. Ppcrt1–8 were grouped with the copia-like retrotransposons RIRE1 and BARE-1 by phylogenetic analysis based on the amino acid sequences encoded by the gag and pol domains. Fluorescence in situ hybridization analysis showed that sequences homologous to Ppcrt4 were dispersed throughout more than half of the pear chromosomes. Southern blot analysis suggested that many copies of Ppcrt retrotransposons exist in the pear genome. Sequence information from these eight retrotransposons should be useful for the development of retrotransposon-based molecular marker systems in Japanese pear.

The use of retrotransposon-based molecular markers to analyze genetic diversity

Summary: Molecular markers play an essential role in all aspects of genetics, modern plant breeding, in human forensics, for map-based cloning of genes, ranging from the identification of genes responsible for the desired traits to the management of backcrossing programs. Retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their “copy and paste” life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Following the demonstration that retrotransposons are ubiquitous, active, and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of genetic diversity in plants and the way for the rapid isolation of retrotransposon termini.

Analysis of plant diversity with retrotransposon-based molecular markers

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.

Genetic and epigenetic instability of amplification-prone sequences of a novel B chromosome induced by tissue culture in Plantago lagopus L.

Gene amplification is prevalent in many eukaryotes and has been found linked to various phenomena such as ontogenesis, carcinogenesis, in vitro culturing, neoplasia and drug resistance. Earlier, we reported a novel B chromosome in Plantago lagopus L., which was found to have arisen as a result of massive amplification of 5S rDNA. In addition, the chromosome is also composed of 45S rDNA and transposable elements. While the importance of gene amplification cannot be underestimated, its mechanism of origin is still unclear. Therefore, the aim of the present study was to determine whether amplification can be reactivated in the novel B chromosome. For this purpose, in vitro culture was used as stress. Three modes of tissue culture, i.e., direct, indirect and somatic embryogenesis were used for raising in vitro cultures. The variations due to genetic and epigenetic mechanisms were assessed in regenerants using molecular techniques, namely, PCR-RFLP, SSAP and MSAP. The retrotransposon-based molecular markers were applied to detect the polymorphism within transposable elements of in vitro regenerated and mother plants. We detected the variations that may be due to genetic changes either because of element recombination or activation of transposable elements which can lead to increase in the copy number. MSAP analysis revealed the differences in the DNA methylation pattern of the regenerants derived from novel chromosome bearing mother plants. Some regenerated plants were associated with increase and decrease in DNA methylation of both internal and external cytosine of the CCGG sequence.

Isolation of two novel complete Ty1-copia retrotransposons from apple and demonstration of use of derived S-SAP markers for distinguishing bud sports of Malus domestica cv. Fuji

Retrotransposon-based molecular markers are a powerful tool for mapping and diversity studies. The scarcity of retrotransposon long terminal repeat (LTR) sequences limits the application of retrotransposon-based molecular marker systems. Here, we isolated two novel complete Ty1-copia retrotransposons (CTcrm1 and CTcrm2) in apple using a genome walking strategy. The CTcrm retrotransposons are nearly 5 kb long, and they have all the features of Ty1-copia retrotransposons. The differences in gene organization and nucleotide sequence length between the CTcrm retrotransposons and other reported complete retrotransposons in apple showed that CTcrm1 and CTcrm2 are the first two distinct complete Ty1-copia retrotransposons in the apple genome. To investigate the potential utility of the two retrotransposons as molecular markers, primers complementary to the CTcrm LTRs were designed to develop sequence-specific amplification polymorphism markers for discriminating bud sports of Fuji apple. Multiple polymorphisms corresponding to CTcrm1 and CTcrm2 were detected and could easily be used to discriminate bud sports from their Fuji progenitor, as well as from each other.

Genetic variability in sunflower (Helianthus annuus L.) and in the Helianthus genus as assessed by retrotransposon-based molecular markers

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon's and Jaccard's indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.

Comparison of four molecular markers for genetic analysis in Diospyros L. (Ebenaceae)

Four molecular markers, including inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplified polymorphism (SSAP), and amplified fragment length polymorphism (AFLP), were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 28 genotypes in the genus Diospyros. The results were as follows: (1) the highest level of detected polymorphism were observed for IRAP; (2) AFLP was the most efficient marker system due to the simultaneous detection of abundant polymorphism markers per single reaction; (3) the marker index (MI) value was lower for SSAP than for AFLP, but SSAP had a higher level of detected polymorphism than AFLP; (4) the correlation coefficients of similarity were statistically significant for all four marker systems; (5) the four molecular markers yielded similar phylogenetic trees. To our knowledge, this was the first detailed report of a comparison of performance among three retrotransposon-based molecular markers (IRAP, REMAP, SSAP) and the AFLP technique (DNA-based molecular marker) on a set of samples of Diospyros. The results provide guidance for future efficient use of these molecular methods in the genetic analysis of Diospyros.

Development of retrotransposon primers and their utilization for germplasm identification in Diospyros spp. (Ebenaceae)

Retrotransposons play an important role in plant genetic instability and genome evolution. Retrotransposon-based molecular markers are valuable tools to reveal the behavior of retrotransposons in their host genome. In this study, suppression polymerase chain reaction was used, for the first time, to develop retrotransposon long terminal repeat (LTR) and polypurine tract (PPT) primers in Japanese persimmon (Diospyros kaki Thunb.), which were then employed for germplasm identification by means of interretrotransposon-amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP) and retrotransposon-microsatellite-amplified polymorphism (REMAP) molecular markers. The results showed that 16 out of 26 primers produced expected amplifications and abundant polymorphisms by IRAP in 28 genotypes of Diospyros. Moreover, some of these primers were further successfully used in REMAP and SSAP analysis. Each type of molecular markers produced unique fingerprint in 28 genotypes analyzed. Among the primers/primer combinations, two IRAP primers and four SSAP primer combinations could discriminate all of the germplasm solely. Further comparative analysis indicated that IRAP was the most sensitive marker system for detecting variability. High level of retrotransposon insertion polymorphisms between bud sports were detected by IRAP and SSAP, and the primers/primer combinations with powerful discrimination capacity for two pairs of bud sports lines were further obtained. Additionally, possible genetic relationships between several Japanese persimmon were discussed. To our knowledge, this is the first report on the development of retrotransposon LTR and PPT primers in Diospyros, and the retrotransposon primers developed herein might open new avenue for research in the future.

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.

Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley.

Net blotch, which is caused by the fungus Pyrenophoral teres Drechs. f. teres Smedeg., presents a serious problem for barley production worldwide, and the identification and deployment of sources of resistance to it are key objectives for many breeders. Here, we report the identification of a major resistance gene, accounting for 65% of the response variation, in a cross between the resistant line C19819 and the susceptible cv. Rolfi. The resistance gene was mapped to chromosome 6H with the aid of two recently developed systems of retrotransposon-based molecular markers, REMAP and IRAP. A total of 239 BARE-1 and Sukkula retrotransposon markers were mapped in the cross, and the 30-cM segment containing the locus with significant resistance effect contained 26 of the markers. The type and local density of the markers should facilitate future map-based cloning of the resistance gene as well as manipulation of the resistance through backcross breeding.