Retrotransposon-based Markers Research Articles

Marker utility of transposable elements for plant genetics, breeding, and ecology: a review

Variation is the primary source for plant and animal breeding and evolution, and hence, the detection of variation is an integral part of genetics, breeding, and ecology. Various molecular marker systems have been developed to detect genetic variation. They detect sequence variations (e.g., restriction fragment length polymorphism, randomly amplified polymorphic DNA, amplified fragment length polymorphism) or simple sequence motif variations. However, a large portion of genomic variation is derived from the transposition of transposable elements (TEs), which are major denizens of most eukaryote genomes. Therefore, molecular markers derived from TEs are valuable resources for dissecting genomes in plants and animals. Because class I retrotransposons transpose by a “copy-and-paste” semi-conservative manner, retrotransposon-based markers (e.g., Inter-retrotransposon amplified polymorphism, retrotransposon-microsatellite amplified polymorphism, sequence-specific amplified polymorphism) can reveal highly accurate phylogenetic relationships among related taxa as well as among accessions within a species. Transposon display based on class II DNA transposons has also been used in various genetics fields. A large amount of fairly accurate genome sequences are now being generated, and computational biology allows us to mine the TEs on a genome-wide scale. Thus, TE-based molecular markers are adding another venue to the other marker systems used for the molecular dissection of genomes.

Use of SSR and retrotransposon-based markers to interpret the population structure of native grapevines from southern Italy.

Native grapevines are the quintessential elements of Southern Italy winemaking, and genomic characterization plays a role of primary importance for preservation and sustainable use of these unexploited genetic resources. Among the various molecular techniques available, SSR and retrotransposons-based markers result to be the most valuable for cultivars and biotypes distinctiveness. A total of 62 accessions including 38 local grape cultivars were analyzed with 30 SSR, four REMAP and one IRAP markers to assess their genetic diversity and obtain a complete genomic profiling. The use of VrZAG79, VrZAG112, VVS2, VVMD25 and VVMD5 combined with retrotransposon-based markers proved to be the most discriminating and polymorphic markers for the rapid and unambiguous identification of minority grapevines from Campania region, which is considered one of the most appreciated Italian districts for wine production. Results revealed 58 SSR marker-specific alleles, 22 genotype-specific SSR alleles, and four REMAP and IRAP private bands. Cases of synonymy and homonymy were discovered. In conclusion, we provided evidences that the integrating SSR and retrotransposon-based markers is an effective strategy to assess the genetic diversity of autochthonous grapes, allowing their easy identification.

Molecular diversity analysis of grape varieties based on iPBS markers

Thirty-five grape varieties were evaluated for their molecular diversities based on inter-primer binding site (iPBS) markers. Fifteen selected iPBS primers generated a total of 99 polymorphic DNA bands with 86.3% polymorphism. The effectiveness of iPBS marker is comparable to or even more efficient than other retrotransposon-based markers in grape. The differentiation between cultivated and wild grape varieties were clearly showed by both UPGMA cluster analysis and PCoA analysis revealing that Chinese cultivated and wild grape germplasm are highly divergent and possess abundant genetic diversities. This study also confirmed that the iPBS marker is a simple, informative, reproducible and suitable method for grape genetic diversity evaluation.

Detection of genetic relationships among spring and winter triticale (× Triticosecale Witt.) and rye cultivars (Secale cereale L.) by using retrotransposon-based markers

In the present research, we aimed to detect and evaluate the level of long terminal repeat (LTR) retrotransposons (WIS2, Wilma, Daniela, and Wham) intraspecific variability and intron polymorphism for β-amylase (BAMY) genes in 37 winter and 25 spring triticale cultivars coming from European countries and the USA and 5 Finnish rye cultivars. The triticale and rye genotypes differ significantly with respect to the patterns of the four explored LTR retrotransposons. A neighbour-joining dendrogram has separated all triticale and rye cultivars into three principal clusters: all winter triticale, all spring triticale and all rye cultivars. We have proved that retrotransposon-based markers can be used for differentiation of triticale and rye cultivars.

A novel linkage map of sugarcane with evidence for clustering of retrotransposon-based markers.

BackgroundThe development of sugarcane as a sustainable crop has unlimited applications. The crop is one of the most economically viable for renewable energy production, and CO2 balance. Linkage maps are valuable tools for understanding genetic and genomic organization, particularly in sugarcane due to its complex polyploid genome of multispecific origins. The overall objective of our study was to construct a novel sugarcane linkage map, compiling AFLP and EST-SSR markers, and to generate data on the distribution of markers anchored to sequences of scIvana_1, a complete sugarcane transposable element, and member of the Copia superfamily.ResultsThe mapping population parents (‘IAC66-6’ and ‘TUC71-7’) contributed equally to polymorphisms, independent of marker type, and generated markers that were distributed into nearly the same number of co-segregation groups (or CGs). Bi-parentally inherited alleles provided the integration of 19 CGs. The marker number per CG ranged from two to 39. The total map length was 4,843.19 cM, with a marker density of 8.87 cM. Markers were assembled into 92 CGs that ranged in length from 1.14 to 404.72 cM, with an estimated average length of 52.64 cM. The greatest distance between two adjacent markers was 48.25 cM. The scIvana_1-based markers (56) were positioned on 21 CGs, but were not regularly distributed. Interestingly, the distance between adjacent scIvana_1-based markers was less than 5 cM, and was observed on five CGs, suggesting a clustered organization.ConclusionsResults indicated the use of a NBS-profiling technique was efficient to develop retrotransposon-based markers in sugarcane. The simultaneous maximum-likelihood estimates of linkage and linkage phase based strategies confirmed the suitability of its approach to estimate linkage, and construct the linkage map. Interestingly, using our genetic data it was possible to calculate the number of retrotransposon scIvana_1 (~60) copies in the sugarcane genome, confirming previously reported molecular results. In addition, this research possibly will have indirect implications in crop economics e.g., productivity enhancement via QTL studies, as the mapping population parents differ in response to an important fungal disease.

Utility of retrotransposon-derived marker systems for differentiation of presumed clones of the apricot cultivar Velkopavlovická

Apricot cultivars may be composed of a variety of clones differing in minor genetic and phenotypic characteristics. Transposable elements might be considered, among other factors, a potential source of mutations that lead to clonal variation. Therefore, genetic markers derived from transposable regions should be suitable for differentiation of clones. The aims of the present work were to verify the utility of sequence-specific amplified polymorphism (SSAP) markers and inter-primer binding sites (iPBS) as universal retrotransposon-derived marker systems for differentiation of clones on the basis of their different DNA fingerprints and to compare the utility of SSAP and iPBS systems with other, more commonly used marker systems (SSR and AFLP). The results obtained with SSAP and iPBS marker systems were similar to those with the conventional AFLP method. Thus, the hypothesis that these retrotransposon-based markers should be more suitable for clonal differentiation was not confirmed. An additional objective of the present study was to map the level of genetic diversity within the genotypes described as clones of the cultivar Velkopavlovická collected from different locations in the Czech Republic and to compare these clones with clones originating from other countries. The results indicated that clones grown in the Czech Republic and other European countries have similar origins.

Comparison of a retrotransposon-based marker with microsatellite markers for discriminating accessions of Vitis vinifera

Identification and knowledge concerning genetic diversity are fundamental for efficient management and use of grapevine germplasm. Recently, new types of molecular markers have been developed, such as retrotransposon-based markers. Because of their multilocus pattern, retrotransposon-based markers might be able to differentiate grapevine accessions with just one pair of primers. In order to evaluate the efficiency of this type of marker, we compared retrotransposon marker Tvv1 with seven microsatellite markers frequently used for genotyping of the genus Vitis (VVMD7, VVMD25, VVMD5, VVMD27, VVMD31, VVS2, and VZAG62). The reference population that we used consisted of 26 accessions of Vitis, including seven European varieties of Vitis vinifera, four North American varieties and hybrids of Vitis labrusca, and 15 rootstock hybrids obtained from crosses of several Vitis species. Individually, the Tvv1 and the group of seven SSR markers were capable of distinguishing all accessions except 'White Niagara' compared to 'Red Niagara'. Using the Structure software, the retrotransposon marker Tvv1 generated two clusters: one with V. vinifera plus North American varieties and the other comprising rootstocks. The seven SSR markers generated five clusters: V. vinifera, the North American varieties, and three groups of rootstock hybrids. The percentages of variation explained by the first two components in the principal coordinate analysis were 65.21 (Tvv1) and 50.42 (SSR markers) while the Mantel correlation between the distance matrixes generated by the two types of markers was 42.5%. We conclude that the Tvv1 marker is useful for DNA fingerprinting, but it lacks efficiency for discrimination of structured groups.

Effectiveness of AFLPs and Retrotransposon-Based Markers for the Identification of Portuguese Grapevine Cultivars and Clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.

Genetic Diversity in Old Portuguese Durum Wheat Cultivars Assessed by Retrotransposon-Based Markers

Retrotransposon-based markers, such as the inter-retrotransposon-amplified polymorphism (IRAP) and the retrotransposon microsatellite-amplified polymorphism (REMAP) are highly informative, multilocus, and reveal insertional polymorphisms among plant individuals. These markers have been used for evolutionary studies, genetic diversity assessment, DNA fingerprinting, genetic mapping linkage, and for the detection of genetic rearrangements induced by polyploidization. In this study, we used IRAP and REMAP markers to assess the genetic diversity among 51 old Portuguese durum wheat cultivars belonging to 27 botanical varieties and to define their genetic relationships. Five IRAP and four REMAP primer combinations were used. IRAP markers revealed 66.3% of polymorphism and an average of 18.4 bands per primer combination which ranged in size from 450 to 3,100 bp. The REMAP technique allowed the detection of 86.36% of inter-cultivar polymorphism and an average of 11 bands per primer combination. The molecular weight of the REMAP bands ranged from 250 to 2,750 bp. The durum wheat cultivars analyzed here belong to 27 botanical varieties of the subspecies Triticum turgidum subsp. turgidum L. [syn. T. turgidum] and Triticum turgidum L. subsp. durum [syn. T. durum] (Desf.) Husn.. Our results showed that the genetic variability assessed by both the IRAP and REMAP markers did not allow the clustering of the durum wheat cultivars according to their taxonomical criteria (subspecies or botanical variety) or homonymy. Nonetheless, these markers were useful for the assessment of genetic diversity at the individual level, for the definition of genetic relationships among cultivars, and for estimation of the genetic structure of the Old collection under analysis. The achieved data could be valuable for future experiments of DNA fingerprinting, genetic improvement, and germplasm conservation in wheat.

Retrotransposon- and microsatellite sequence-associated genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride

Allopolyploidization is considered an essential evolutionary process in plants that could trigger genomic shock in allopolyploid genome through activation of transcription of retrotransposons, which may be important in plant evolution. Two retrotransposon-based markers, inter-retrotransposon amplified polymorphism and retrotransposon-microsatellite amplified polymorphism and a microsatellite-based marker, inter simple sequence repeat were employed to investigate genomic changes in early generations of a newly synthesized allotetraploid Cucumis×hytivus Chen & Kirkbride (2n=4x=38) which was derived from crossing between cultivated cucumber C. sativus L. (2n=2x=14) and its wild relative C. hystrix Chakr. (2n=2x=24). Extensive genomic changes were observed, most of which involved the loss of parental DNA fragments and gain of novel fragments in the allotetraploid. Among the 28 fragments examined, 24 were lost while four were novel, suggesting that DNA sequence elimination is a relatively frequent event during polyploidization in Cucumis. Interestingly, of the 24 lost fragments, 18 were of C. hystrix origin, four were C. sativus-specific, and the remaining two were shared by both species, implying that fragment loss may be correlated with haploid DNA content (genome size) of diploid parents. Most changes were observed in the first generation after polyploidization (S(1)) and stably inherited in the subsequent three generations (S(2)-S(4)), indicating that genomic changes might be a rapid driving force for the stabilization of allotetraploids. Sequence analysis of 11 of the 28 altered DNA fragments showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted into genome randomly and had a genome-wide effect on the allotetraploid evolution. Fluorescence in situ hybridization (FISH) analysis revealed a unique distribution of retrotransposon and/or microsatellite flanking sequences in mitotic and meiotic chromosomes, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.

Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae)

In the study, we developed new markers for phylogenetic relationships and intraspecies differentiation in Coffea. Nana and Divo, two novel Ty1-copia LTR-retrotransposon families, were isolated through C. canephora BAC clone sequencing. Nana- and Divo-based markers were used to test their: (1) ability to resolve recent phylogenetic relationships; (2) efficiency in detecting intra-species differentiation. Sequence-specific amplification polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and retrotransposon-based insertion polymorphism (RBIP) approaches were applied to 182 accessions (31 Coffea species and one Psilanthus accession). Nana- and Divo-based markers revealed contrasted transpositional histories. At the BAC clone locus, RBIP results on C. canephora demonstrated that Nana insertion took place prior to C. canephora differentiation, while Divo insertion occurred after differentiation. Combined SSAP and REMAP data showed that Nana could resolve Coffea lineages, while Divo was efficient at a lower taxonomic level. The combined results indicated that the retrotransposon-based markers were useful in highlighting Coffea genetic diversity and the chronological pattern of speciation/differentiation events. Ongoing complete sequencing of the C. canephora genome will soon enable exhaustive identification of LTR-RTN families, as well as more precise in-depth analyses on contributions to genome size variation and Coffea evolution.

Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70-100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.

Phylogeny, phylogeography and genetic diversity of the Pisum genus

The tribeFabeae(formerlyVicieae) contains some of humanity's most important grain legume crops, namelyLathyrus(grass pea/sweet pea/chickling vetches; about 160 species);Lens(lentils; 4 species);Pisum(peas; 3 species);Vicia(vetches; about 140 species); and the monotypic genusVavilovia.Reconstructing the phylogenetic relationships within this group is essential for understanding the origin and diversification of these crops. Our study, based on molecular data, has positionedPisumgenetically betweenViciaandLathyrusand shows it to be closely allied toVavilovia.A study of phylogeography, using a combination of plastid and nuclear markers, suggested that wild pea spread from its centre of origin, the Middle East, eastwards to the Caucasus, Iran and Afghanistan, and westwards to the Mediterranean. To allow for direct data comparison, we utilized model-based Bayesian Analysis of Population structure (BAPS) software on 4429Pisumaccessions from three large world germplasm collections that include both wild and domesticated pea analyzed by retrotransposon-based markers. An analysis of genetic diversity identified separate clusters containing wild material, distinguishingPisum fulvum,P. elatiusandP. abyssinicum, supporting the view of separate species or subspecies. Moreover, accessions of domesticated peas of Afghan, Ethiopian and Chinese origin were distinguished. In addition to revealing the genetic relationships, these results also provided insight into geographical and phylogenetic partitioning of genetic diversity. This study provides the framework for defining globalPisumgermplasm diversity as well as suggesting a model for the domestication of the cultivated species. These findings, together with gene-based sequence analysis, show that although introgression from wild species has been common throughout pea domestication, much of the diversity still resides in wild material and could be used further in breeding. Moreover, although existing collections contain over 10,000 pea accessions, effort should be directed towards collecting more wild material in order to preserve the genetic diversity of the species.

Molecular markers based on LTR retrotransposons BARE-1 and Jeli uncover different strata of evolutionary relationships in diploid wheats

Molecular markers based on retrotransposon insertions are widely used for various applications including phylogenetic analysis. Multiple cases were described where retrotransposon-based markers, namely sequence-specific amplification polymorphism (SSAP), were superior to other marker types in resolving the phylogenetic relationships due to their higher variability and informativeness. However, the patterns of evolutionary relationships revealed by SSAP may be dependent on the underlying retrotransposon activity in different periods of time. Hence, the proper choice of retrotransposon family is essential for obtaining significant results. We compared the phylogenetic trees for a diverse set of diploid A-genome wheat species (Triticum boeoticum, T. urartu and T. monococcum) based on two unrelated retrotransposon families, BARE-1 and Jeli. BARE-1 belongs to Copia class and has a uniform distribution between common wheat (T. aestivum) genomes of different origin (A, B and D), indicating similar activity in the respective diploid genome donors. Gypsy-class family Jeli was found by us to be an A-genome retrotransposon with >70% copies residing in A genome of hexaploid common wheat, suggesting a burst of transposition in the history of A-genome progenitors. The results indicate that a higher Jeli transpositional activity was associated with T. urartu versus T. boeoticum speciation, while BARE-1 produced more polymorphic insertions during subsequent intraspecific diversification; as an outcome, each retrotransposon provides more informative markers at the corresponding level of phylogenetic relationships. We conclude that multiple retroelement families should be analyzed for an image of evolutionary relationships to be solid and comprehensive.

Molecular characterization of the Sasanda LTR copia retrotransposon family uncovers their recent amplification in Triticum aestivum (L.) genome

Retrotransposons constitute a major proportion of the Triticeae genomes. Genome-scale studies have revealed their role in evolution affecting both genome structure and function and their potential for the development of novel markers. In this study, family members of an LTR copia retrotransposon which mediated the duplication of the gene encoding the high molecular weight glutenin subunit Bx7 in cultivar Glenlea were characterized. This novel element was named Sasanda_EU157184-1 (TREP3516). High density filters of the Glenlea hexaploid wheat BAC library were screened with a Sasanda long terminal repeat (LTR)-specific probe and approximately 1,075 positive clones representing an estimated copy number of 347 elements per haploid genome were identified. The 242 BAC clones with the strongest hybridization signal were selected. To maximize isolation of complete elements, this subset of clones was screened with a reverse transcriptase (RT) domain probe and DNA was isolated from the 133 clones that produced a strong hybridization signal. Left (5') and right (3') LTRs as well as the RT domains were PCR amplified and sequencing was carried out on the final subset of 121 clones. Evolutionary relationships were inferred from a data set consisting of 100 RT, 102 5' LTR and 100 3' LTR sequences representing 233, 451 and 495 informative sites for comparison, respectively. Neighbour-joining tree indicated that the element is at least 1.8 million years old and has evolved into a minimum of five sub-families. The insertion times of the 89 complete elements were estimated based on the divergence between their LTRs. Corroborating the inference from the RT domain, analysis of the LTR domains also indicated bursts of amplification from 2.6 million years ago (MYA) to now, except for one member dated to 4.6 +/- 0.7 MYA, which corresponds to the interval of divergence of Triticum and Aegilops (3 MYA) and divergence of Triticum and Rye (7 MYA). In 44 elements, the 5' and 3' LTRs were identical indicating recent transposition activity. The element can be used to develop retrotransposon-based markers such as sequence-specific amplified polymorphism, retrotransposon microsatellite amplified polymorphism and inter-retrotransposon amplified polymorphism, all of which are well suited for genotyping studies.

Potato variety identification by molecular markers based on retrotransposon analyses

We analyzed a set of twenty most grown potato (Solanum tuberosum L.) varieties listed in the Czech Variety List using the PCR-IRAP (Inter-Retrotransposon Amplified Polymorphism) method in order to distinguish fast and unambiguously the varieties. In total, 62 polymorphic alleles were amplified using the three primers P-Tst-1, P-Tst-3 and P-Tst-6. The recorded pattern of markers was stable and reproducible. The analyses were repeated three times and identical results were always obtained. The level of polymorphism varied from 11% to 79% depending on the respective primer. All analysed varieties could be reliably distinguished after multivariate statistics have been applied to the data obtained by the PCO and UPGMA analyses. The best resolution of individual varieties was obtained if all three primers were evaluated as a complex. The use of retrotransposon-based markers appears to be suitable for the differentiation of large sets of potato samples and should be an eligible complement to other molecular markers used in potato variety identification such as Simple Sequence Repeats (SSR) and Amplified Fragment Length Polymorphisms (AFLP).

Retrotransposon-based markers from potato monoploids used in somatic hybridization

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.

Molecular markers to assess genetic diversity

Molecular markers play an essential role today in all aspects of plant breeding, ranging from the identification of genes responsible for desired traits to the management of backcrossing programs. The emergence of marker systems has, for the last 30 years, closely tracked developments in biochemistry and molecular biology. Following the demonstration that retrotransposons are ubiquitous, active, and abundant in plant genomes, markers were developed that are based on the insertional polymorphism they create upon replication. They virtually all exploit the joint that is formed, during retrotransposon integration, between genomic DNA and the long terminal repeats at either end of retrotransposon. The various retrotransposon marker systems differ in the nature of the second primer used in the amplification reactions. All except one of these marker methods is dominant (RBIP is co-dominant). Hence, the availability of effective means to generate doubled haploid populations through gametophytic embryogenesis is valuable for the efficient use of these markers. Over the last eight years, retrotransposon-based markers have been developed for crop species and trees across the plant kingdom, as well as for fungi and insects. Many retrotransposons features make them appealing as the basis of molecular marker systems. They are usually dispersed throughout the genome and produce large genetic changes at the point of insertion that can be detected with family-specific amplification primers. The ancestral state of a retrotransposon insertion is known, and subsequent changes at the locus are not subject to homoplasy. Retrotransposon markers are useful in accelerating backcrossing programs, tagging genes of interest, tracking germplasm, verifying and producing pedigrees, and examining crop evolution.

Genome constitution and classification using retrotransposon-based markers in the orphan crop banana

We have exploited the repetitive and dispersed nature of many long terminal repeat (LTR)-retrotransposon families for characterizing genome constitutions and classifying cultivars of the genusMusa. Insertional polymorphisms of the elements were studied using seven published and two newly designed primers facing outwards from the LTRs and reverse transcriptase (RT) domain of the retrotransposon. The primers generated specific amplification patterns showing the universal applicability of this marker type. The Inter-Retrotransposon Amplified Polymorphism (IRAP) markers distinguished the A and B genomes of the banana species (Musa acuminata Col la andMusa balbisiana Colla) and between banana cultivars. The IRAP markers enabled phylogenetic analysis of 16 Malaysian banana cultivars and determination of the genome constitution of hybrid banana (AAB, ABB, AABB, and AAAB), and gave information about ancestral genotypes of the hybrids. In addition, the IRAP detected new retrotransposon insertions into the genome of tissue culture regenerants. This PCR-based IRAP assay is amenable to large-scale throughput demands in screening breeding populations and is applicable for any crop.

Active retrotransposons are a common feature of grass genomes.

A large fraction of the genomes of grasses, members of the family Graminae, is composed of retrotransposons. These elements resemble animal retroviruses in their structure and possess a life cycle similar to theirs that includes transcription, translation, and integration of daughter copies. We have investigated if retrotransposons are generally transcribed in the grasses and other plants, and whether the various families of elements are translationally and integrationally active in multiple grass species. A systematic search of 7.8 x 10(5) publicly available expressed sequence tags from plants revealed widespread retrotransposon transcripts at a frequency of one in 1,000. Monocot retrotransposons found relatively more expressed sequence tags from non-source species than did those of dicots. Antibodies were raised to the capsid protein, GAG, of BARE-1, a transcribed and translated copia-like retrotransposon of barley (Hordeum vulgare). These detected immunoreactive proteins of sizes identical to those of the BARE-1 GAG and polyprotein, respectively, in other species of the tribe Triticeae as well as in oats (Avena sativa) and rice (Oryza sativa). Retrotransposon-based markers showed integrational polymorphisms for BARE-1 in different subfamilies of the Graminae. The results suggest that grasses share families of transcriptionally, translationally, and integrationally active retrotransposons, enabling a comparative and integrative approach to understanding the life cycle of retrotransposons and their impact on the genome.