IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

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 insertional polymorphism in sunflower (Helianthus annuus L.) lines revealed by IRAP and REMAP markers

Retrotransposons are ubiquitous components of plants genomes, making them useful molecular markers for genetic diversity studies. We used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers to assess genetic diversity and survey activity of LTR retrotransposon elements in 106 sunflower (Helianthus annuus L.) genotypes from different research centers. We found 118 (out of 128) and 113 (out of 120) polymorphic loci using 14 IRAP and 14 REMAP primers, respectively. The Mantel test between IRAP and REMAP cophenetic matrices revealed low correlation (r = 0.55) between them. Dice similarities based on combined (IRAP + REMAP) data ranged from 0.34 to 0.93 among (“11 × 12” and “F1250/03”) and (“HA335B” and “TMB51”) genotypes, respectively. Classification of genotypes using the Dice similarity matrix derived from IRAP+REMAP data based on the un-weighted pair-group method using the arithmetic average algorithm resulted in nine distinct groups. The studied genotypes were divided into seven groups considering their origins (research centers). Classification of genotypes can be useful to assess the genetic variation and gene flow between and within research centers. Analysis of molecular variance based on IRAP+REMAP data revealed a higher level of genetic variation within (94%) than between (6%) research centers. A high amount of gene flow was detected among USDA, ASGROW, and ENSAT groups. Because environmental factors have no influence on molecular markers, the construction of heterotic groups based on retrotransposon markers will be useful for the selecting of parents with a high probability of producing superior hybrids.     

Retrotransposon Insertional Polymorphism in Iranian Bread Wheat Cultivars and Breeding Lines Revealed by IRAP and REMAP Markers

Inter-retrotransposon amplified polymorphisms (IRAPs) and retrotransposon-microsatellite amplified polymorphisms (REMAPs) were used to detect retrotransposon integration events and genetic diversity in 101 Iranian bread wheat (Triticum aestivum L.) cultivars and breeding lines. The 9 IRAP primers amplified 128 loci, and 20 REMAP primers amplified 263 loci. Percentage of polymorphic loci, average expected heterozygosity, number of effective alleles, and Shannon’s information index for the REMAP markers were slightly higher than those for the IRAP markers. The same estimated parameters calculated for native and nonnative retrotransposons were not considerably different. A Mantel test between IRAP and REMAP cophenetic matrices evidenced no significant correlation. Cluster analysis based on the Dice similarity coefficient and complete linkage algorithm using IRAP+REMAP loci identified five groups among the genotypes studied that could be applied as crossing parents in T. aestivum breeding programs.     

TRIM retrotransposons occur in apple and are polymorphic between varieties but not sports

Retrotransposon markers have been demonstrated to be powerful tools for investigating linkage, evolution and genetics diversity in plants. In the present study, we identified and cloned three full-size TRIM (terminal-repeat retrotransposon in miniature) group retrotransposon elements from apple (Malus domestica) cv. ‘Antonovka’, the first from the Rosaceae. To investigate their utility as markers, we designed primers to match the long terminal repeats (LTRs) of the apple TRIM sequences. We found that PCR reactions with even a single primer produced multiple bands, suggesting that the copy number of these TRIM elements is relatively high, and that they may be locally clustered or nested in the genome. Furthermore, the apple TRIM primers employed in IRAP (inter-retrotransposon amplified polymorphism) or REMAP (retrotransposon-microsatellite amplified polymorphism) analyses produced unique, reproducible profiles for 12 standard apple cultivars. On the other hand, all seven of the sport mutations in this study were identical to their mother cultivar. Genetic similarity values calculated from the IRAP/REMAP analyses or the STMS (sequence tagged microsatellite sites) analysis were generally comparable. PAUP cluster analysis based on IRAP and REMAP markers in apple and Japanese quince generated an NJ tree that is in good accordance with both a tree based on SMTS markers and the origin of the studied samples. Our results demonstrate that, although they do not encode the proteins necessary to carry out a life cycle and are thereby non-autonomous, TRIMs are at least as polymorphic in their insertion patterns as conventional complete retrotransposons. Kristiina Antonius-Klemola, Ruslan Kalendar are the first two authors contributed equally to this work     

Retrotransposon based genetic status of North-West Himalayan Zingiber officinale revealed high heterogeneity

Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were successfully applied, for the first time, to analyze genetic diversity among 92 ginger landraces collected from north-western Himalayan region of India. Six IRAP primer/combinations generated 75 loci with an average of 12 loci/primer displaying an overall polymorphism of 95.95 %. On the other hand, twenty five REMAP primer combinations produced 414 loci with 96.5 % polymorphism. IRAP showed maximum Rp (5.39) and PIC (0.28) values, while the same in REMAP was observed to be 10.92 and 0.34. Cluster analysis using Jaccard’s similarity coefficient for IRAP and REMAP data ranged between 0.21 to 1.0 and 0.21 to 0.85, respectively distinguishing all the genotypes with diverse genetic makup. The results also confirmed the presence of sukkula retrotransposon (RT6) in the ginger genome which effectively acted as genetic marker revealing high regional genetic diversity in the ginger gene pool. The study will help in giving insight to the genetic constitution of vegetatively grown ginger crop and for its further utilization in improvement, conservation and management programmes.     

Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley (Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families (BARE-1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.     

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 Vinh?o 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.     

Molecular Diversity in some Ghanaian Cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. (Walp)] Accessions

Cowpea [Vigna unguiculata L. (Walp)] is grown mainly for its protein-rich grains and is consumed in various forms in sub-Saharan Africa. Average grain yield in farmers’ fields is generally low due to a number of biotic and abiotic stresses. One hundred and six cowpea accessions from Ghana, which had previously been evaluated for seedling drought tolerance, were used for this study. This paper attempts to use three multi-locus PCR-based molecular markers; simple sequence repeats (SSR), inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphisms (REMAP), to analyse genetic diversity in the cowpea accessions. Analysis of the polymorphic bands data indicated that 101 alleles were amplified among 121 cowpea genotypes (83.4%) from 16 SSR primer pairs out of a total of 30 SSR primer pairs. Likewisely, a total of 66 (54.5%) polymorphic bands were obtained from IRAP and a total of 114 (94.2%) highly polymorphic bands obtained from REMAP analysis. The outcome indicated the highly polymorphic nature of the DNA markers, as small groups of these molecular markers were found to be able to identify each of the accessions used. Microsatellite markers (SSRs) and retrotransposon-based markers, like IRAP and REMAP, were found to be highly polymorphic and informative, suggesting that genomic fingerprinting has a major role in characterizing populations.     

Comparison of ISSR,IRAP and REMAP markers for assessing genetic diversity in different species of <Emphasis Type="Italic">Brassica</Emphasis> sp.

Molecular markers provide facilities in order to study genetic diversity and relationship among genotypes. In this study, genetic diversity among 35 genotype of Brassica sp. (belonging B. napus, B. juncea, B. rapa, B. nigra) were determined using 13 ISSR, 3 IRAP markers and 18 REMAP (primer combinations of ISSR and retrotransposon primer). The percentage of polymorphism for ISSR, IRAP and REMAP was 96.38, 94 and 96%, respectively. By comparison between markers, ISSRs indicated the highest expected heterozygosity (He) and Shannon’s information index (I) with value of 0.34 and 0.51, respectively, while REMAP marker had by far the highest number of polymorphic bands (340) and marker index (7.1) among all fragments scored over all markers. In pattern of clustering based on Bayesian methods, K = 8 was resulted for combined data clustering that was more organized clustering for genotypes compared to others. This research suggests the combined data of ISSR, IRAP and REMAP markers are most reliable than each solely marker whilst have been clustered genotypes in their taxonomic classification of Brassica without any mixture. Principle coordinate analysis (PCoA) separated 35 genotypes in four groups which all of genotypes were clustered correctly based on their taxonomic classification. The findings of this study provide the valuable insight into the Brassica species relationships in terms of similarity among genotypes which can be helpful in breeding programs, and also demonstrate that retrotransposon markers are legible for genetic diversity and next genetic analysis in Brassica genus.     

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.     

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.     

Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica C.E. Hubbard (Poaceae)

Spartina x townsendii arose during the end of the 19th century in England by hybridization between the indigenous Spartina maritima and the introduced Spartina alterniflora, native to the eastern seaboard of North America. Duplication of the hybrid genome gave rise to Spartina anglica, a vigorous allopolyploid involved in natural and artificial invasions on several continents. This system allows investigation of the early evolutionary changes that accompany stabilization of new allopolyploid species. Because allopolyploidy may be a genomic shock, eliciting retroelement insertional activity, we examined whether retrotransposons present in the parental species have been activated in the genome of S. anglica. For this purpose we used inter-retrotransposon amplified polymorphism (IRAP) and retrotransposons-microsatellite amplified polymorphism (REMAP) markers, which are multilocus PCR-based methods detecting retrotransposon integration events in the genome. IRAP and REMAP allowed the screening of insertional polymorphisms in populations of S. anglica. The populations are composed mainly of one major multilocus genotype, identical to the first-generation hybrid S. x townsendii. Few new integration sites were encountered in the young allopolyploid genome. We also found strict additivity of the parental subgenomes in the allopolyploid. Both these findings indicate that the genome of S. anglica has not undergone extensive changes since its formation. This contrasts with previous results from the literature, which report rapid structural changes in experimentally resynthesized allopolyploids.     

Genome merger: from sequence rearrangements in triticale to their elimination in wheat–rye addition lines

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat–rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat–rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale × wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat–rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat–rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.     

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.     

植物反转录转座子及其分子标记

反转录转座子（retrotransposon）是真核生物中一类可移动因子,可分为LTR反转录转座子和非LTR反转录转座子。反转录转座子以高拷贝在植物界广泛分布,可以通过纵向和横向分别在世代之间和不同种之间进行传递,同一家族的反转录转座子具有高度的异质性. 在一些生物的和非生物的逆境条件下,反转录转座子的转录可以被激活。由于反转录转座子的特点,使其作为一种分子标记得以应用。S-SAP、IRAP、REMAP和RBIP等分子标记相继发展起来,在基因作图、生物遗传多样性与系统进化、品种鉴定等方面具有广泛的应用前景。     

植物反转录转座子及其分子标记

反转录转座子(retrotransposon)是真核生物中一类可移动因子,可分为m反转录转座子和非LTR反转录转座子。反转录转座子以高拷贝在植物界广泛分布,可以通过纵向和横向分别在世代之间和不同种之间进行传递,同一家族的反转录转座子具有高度的异质性．在一些生物的和非生物的逆境条件下,反转录转座子的转录可以被激活。由于反转录转座子的特点,使其作为一种分子标记得以应用。SSAP、IRAP、REMAP和RBIP等分子标记相继发展起来,在基因作图、生物遗传多样性与系统进化、品种鉴定等方面具有广泛的应用前景。     

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.