Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea

A sample of 15 cultivars and 56 Pisum accessions from the JIC germplasm core collection has been studied using a modification of the SSAP (sequence-specific amplification polymorphisms) technique; the specific primer was designed to correspond to the polypurine tract (PPT) of PDR1, a Ty1-copia group retrotransposon of pea. Most of these SSAP products were shown to be PDR1 derived. The PDR1 SSAP markers are more informative than previously studied AFLP or RFLP markers and are distributed throughout the genome. Their pattern of variation makes them ideal for integrating genetic maps derived from related crosses. Data sets obtained with AFLP and PDR1 SSAP markers were used to construct neighbour-joining trees and for principal component analysis. These data sets give greater resolution than hitherto available for the characterisation of variation within Pisum, showing that the genus has three main groups: P. fulvum, P. abyssinicum and all other Pisum spp. P. abyssinicum is not a subgroup of cultivated P. sativum, as was previously thought, but has probably been domesticated independently. Modern cultivars are shown to form a single group within Pisum as a whole.     

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum

The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and inter-species relationships within Pisum. Received: 23 January 2000 / Accepted: 24 March 2000     

Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5′-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hot-spot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp. Received: 17 March 1999 / Accepted: 20 July 1999     

Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum

A DAPI and ethidium bromide flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum was conducted. The material included 38 accessions of P. sativum of widely different geographic origin and altogether 14 samples of P. elatius, P. abyssinicum, P. humile and P. fulvum. The relative genome size values obtained with the three staining methods were strongly correlated. No evidence for genome size variation was found among P. sativum cultivars. In particular, certain Italian cultivars, for which strongly deviating C-values have been reported, proved to be invariant. The only occasion when ambiguous evidence for marginal genome size variation was found was when all 38 accessions taxonomically affiliated with P. sativum were considered. Pisum abyssinicum and P. fulvum differed from P. sativum by about 1.066-and 1.070-fold, respectively; 1 accession of P. humile differed by 1.089-fold, and 2 of P. elatius by 1.122- and 1.195-fold, respectively (ethidiumbromide comparison), while the other accessions of these taxa were not different from P. sativum. This variation may indicate taxonomic inhomogeneity and demands further investigation. Cultivated P. sativum has long been suspected of not being constant with respect to genome size. As shown here, these findings were not based on genuine differences, but rather were technical in origin.     

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 diversity analysis in <Emphasis Type="Italic">Vicia </Emphasis>species using retrotransposon-based SSAP markers

Twelve different Ty1-copia and Ty3-gypsy group LTR retrotransposons were compared for their usefulness in SSAP marker development in two agriculturally important Vicia species. Three of the retrotransposons, PDR1, Tps19 and Tvf4, yielded useful SSAP marker systems in V. faba, and V. narbonensis. Another, Tvf1 was a good source of SSAP markers in V. narbonensis alone. The optimized SSAP marker systems were applied to the analysis of two diverse Vicia germplasm sets. Two hundred and two polymorphic Tvf1 SSAP markers were scored in 56 V. narbonensis samples and 196 polymorphic markers derived from the other three most useful retrotransposons were scored in a collection of 20 V. faba samples. The marker data were then used to construct phylogenetic trees. The trees for both species tend to show long-branch lengths, with rather little fine structure. Some V. narbonensis accessions cluster by geographical origin but many do not and a given geographical region is often represented by multiple diverse groups in the tree, suggesting a deep and ancient structure for the diversity of V. narbonensis that spans its current geographic range. The tree for the V. faba accessions also shows very limited clustering with geographical origin and no obvious correlation between diversity and morphology-based taxonomic groupings for the species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Alberto Martín Sanz, Susana Gilsanz Gonzalez and Naeem H. Syed have made equal contributions.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

AFLP and CAPS linkage maps of Cryptomeria japonica

We have used two DNA marker systems, AFLP and CAPS, in a two-way pseudo-testcross strategy applied to an F₁ population to construct genetic linkage maps of two local sugi cultivars. The AFLP markers detected about eight polymorphisms per parent per primer combination. Using 38 primer combinations, 612 AFLPs were detected in ’Haara 4’ and ’Kumotooshi’, of which 305 segregated in a 1:1 ratio (P>0.05). A total of 91 markers (83 AFLP and 8 CAPS) in ’Haara 4’ and 132 (123 AFLP and 9 CAPS) in ’Kumotooshi’ were distributed among 19 and 23 linkage groups, respectively, each of which included 2–17 markers. Maps of ’Haara 4’ and ’Kumotooshi’ spanned 1266.1 cM and 1992.3 cM, and covered approximately 50% and 80% of the sugi genome, respectively. Sequences derived from cDNA, which were previously used to construct a sugi linkage map, were also placed on our linkage maps as CAPS markers. Where a ’two-way pseudo-testcross’ is used, more than half of the sugi CAPS developed can be used to construct linkage maps for each parental family. The saturation of mapped markers, and the integration of several linkage maps derived from different mapping populations, is anticipated in the near future. Received: 15 August 1999 / Accepted: 27 August 1999     

Genetic relatedness and genetic diversity of ornamental mei (Prunus mume Sieb. et Zucc.) as analysed by AFLP markers

The genetic diversity and genetic relatedness of mei (Prunus mume; 2n = 16) were studied using amplified fragment length polymorphism (AFLP) markers. Eight EcoRI–PstI AFLP primer combinations were applied to 121 distinct genotypes of mei cultivars and related species. A total of 508 AFLP product bands were produced, of which 382 were polymorphic. The unweighted pair group method with arithmetic averages analysis was carried out based on these AFLP markers. From this analysis, “Qugeng Mei,” “Yan Mei,” “Chaodou Mei,” and mei cultivars were seen to share the same P. mume genetic stem. The AFLP data were able to clearly discriminate P. mume from other species in the genus Prunus, with P. armeniaca aligning as its closest related species. Two major groups and nine subgroups of mei flower were identified, and there was a strong coincidence of these AFLP-based groupings with the respective morphological characters of the accessions. The genetic diversity of mei accessions was greatest in the Yunnan Province and decreased toward Eastern China and Japan, so supporting the hypothesis that the southwest of China represents the genetic diversity center of the species.     

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, ‘Pink Allusion’ was selected from ‘White Butterfly’. Tissue culture of ‘Pink Allusion’ through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of ‘Pink Allusion’ obtained a cultivar, ‘Regina Red Allusion’, and tissue culture propagation of ‘Regina Red Allusion’ led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within ‘White Butterfly’ and ‘Regina Red Allusion’ groups, however, were only 1.2% and 0.4%, respectively. Jaccard's similarity coefficients among somaclonal cultivars derived from ‘White Butterfly’ and ‘Regina Red Allusion’, on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the ‘White Butterfly’ group and three out of six from the ‘Regina Red Allusion’ group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the ‘White Butterfly’ group and three from the ‘Regina Red Allusion’ group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.     

Linkage relationships among molecular markers and storage root traits of carrot (Daucus carota L. ssp. sativus)

 A 109-point linkage map consisting of three phenotypic loci (P ₁, Y ₂, and Rs), six restriction fragment length polymorphisms (RFLPs), two random amplified polymorphic DNAs (RAPDs), 96 amplified fragment length polymorphisms (AFLPs), and two selective amplification of microsatellite polymorphic loci (SAMPL) was constructed for carrot (Daucus carota L. ssp. sativus; 2n=2x=18). The incidence of polymorphism was 36% for RFLP probes, 20% for RAPD primers, and 42% for AFLP primers. The overall incidence of disturbed segregation was 18%. Linkage relationships at a LOD score of 4.0 and θ=0.25 indicated 11 linkage groups. The total map length was 534.4 cM and the map was clearly unsaturated with markers spaced at 4.9 cM. AFLP P6B15 was 1.7 cM from P ₁, AFLP P1B34 was 2.2 cM from Y ₂, and AFLP P3B30XA was 8.1 cM from Rs. Received: 2 September 1998 / Accepted: 28 November 1998     

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar

 A recombinant inbred line derived from a cross between CO39 and ‘Moroberekan’, RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F₂ individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF₃₄₈ and AF₃₄₉) linked to Pi44(t) were identified. AF₃₄₉ and AF₃₄₈ were located at 3.3±1.5 cM and 11±3.5 cM from Pi44(t), respectively. These markers were mapped on chromosome 11 using an F₂ population derived from a cross between ‘Labelle’ and ‘Black Gora’. The location of AF₃₄₈ on chromosome 11 was confirmed using another F₂ mapping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were amplified from RIL276, ‘Labelle’ and PI543851 using the same primer pairs used to amplify AF₃₄₉ and AF₃₄₈. Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes. Received: 12 May 1998 / Accepted: 13 November 1998     

A detailed linkage map of lettuce based on SSAP, AFLP and NBS markers

Molecular markers based upon a novel lettuce LTR retrotransposon and the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of disease resistance-associated genes have been combined with AFLP markers to generate a 458 locus genetic linkage map for lettuce. A total of 187 retrotransposon-specific SSAP markers, 29 NBS-LRR markers and 242 AFLP markers were mapped in an F₂ population, derived from an interspecific cross between a Lactuca sativa cultivar commonly used in Europe and a wild Lactuca serriola isolate from Northern Europe. The cross has been designed to aid efforts to assess gene flow from cultivated lettuce into the wild in the perspective of genetic modification biosafety. The markers were mapped in nine major and one minor linkage groups spanning 1,266.1 cM, with an average distance of 2.8 cM between adjacent mapped markers. The markers are well distributed throughout the lettuce genome, with limited clustering of different marker types. Seventy-seven of the AFLP markers have been mapped previously and cross-comparison shows that the map from this study corresponds well with the previous linkage map.     

AFLP and pedigree-based genetic diversity estimates in modern cultivars of durum wheat [ Triticum turgidum L. subsp. durum (Desf.) Husn.

A substantial amount of between and within cultivar genetic variation was detected in all the 13 registered modern Canadian durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husn.) cultivars based upon amplified restriction fragment polymorphism (AFLP). Of the approximately 950 detected AFLP markers, only 89 were polymorphic, with 41 between cultivars whereas the remaining 48 showed polymorphism within at least one cultivar. The ancestry of Canadian durum wheat cultivars was traced back to 125 cultivars, selections, and breeding lines including 17 landraces. Mean pair-wise genetic distance based on the kinship coefficient was 0.76. On the other hand, AFLP-based mean pair-wise genetic distance was 0.40. Even though there was a large difference between the means of the two diversity measures, a moderate positive correlation (r=0.457, p<0.002) was detected between the two distance matrices. Cluster analysis with the entire AFLP data divided all cultivars into three major groups reflecting their breeding origins. One group contained ’Pelissier’ alone, which was a selection from a landrace introduced into the US from Algeria. On the other hand such groupings among cultivars were not evident when KIN was used for genetic diversity measures instead. The level of genetic variation among individuals within a cultivar at the breeders’ seed level was estimated based on an inter-haplotypic distance matrix derived from the AFLP data. We found that the level of genetic variation within the most-developed cultivars is fairly substantial despite rigorous selection pressure aimed at cultivar purity in breeding programs. Comparison of AFLP and pedigree-based genetic diversity estimates in crop species such as durum wheat can provide important information for plant improvement. Received: 26 January 2001 / Accepted: 31 May 2001     

Genetic characterization and fine mapping of a yellow-seeded gene in Dahuang (a <Emphasis Type="Italic">Brassica rapa</Emphasis> landrace)

The development of yellow-seeded cultivars in Brassica rapa (B. rapa) would improve the quality and quantity of available oil. The identification and mapping of the seed coat color gene may aid in the development of yellow-seeded cultivars and facilitate introgression of the yellow-seeded gene into desirable Brassica napus (B. napus) lines through marker-assisted selection. In the current study, we investigated the inheritance of a yellow-seeded landrace in B. rapa, “Dahuang”, originating from the Qinghai-Tibetan plateau. Genetic analysis revealed that the phenotype of the yellow-seeded trait in Dahuang is controlled by one recessive gene, termed Brsc1. Mapping of the Brsc1 gene was subsequently conducted in a BC₁ population comprised 456 individuals, derived from (Dahuang × 09A-126) × Dahuang. From a survey of 256 amplified fragment length polymorphism (AFLP) primer combinations, 10 tightly linked AFLP markers were obtained. The closest AFLP markers flanking Brsc1, Y10 and Y06, were 0.2 and 0.4 cM away, respectively. Subsequently, using simple sequence repeat (SSR) markers in the reference map, the Brsc1 gene was mapped on A09 in B. rapa. Blast analysis revealed that seven AFLP markers showed sequence homology to A09 of B. rapa, wherein six AFLP markers in our map were in the same order as those in A09 of B. rapa. The two closest markers, Y10 and Y06, delimited the Brsc1 gene within a 2.8 Mb interval. Furthermore, Y05 and Y06, the two closest AFLP markers on one side linked to Brsc1, were located in scaffold000059 on A09 of B. rapa, whereas the closet AFLP marker on the opposite side of Brsc1, Y10, was located in scaffold000081 on A09 of B. rapa. Molecular markers developed from these studies may facilitate marker-assisted selection (MAS) of yellow-seeded lines in B. rapa and B. napus and expedite the process of map-based cloning of Brsc1.     

Evaluation of genetic variation in the daylily (Hemerocallis spp.) using AFLP markers

The daylily (Hemerocallis spp.) is one of the most economically important ornamental plant species in commerce. Interestingly, it is also one of the most heavily bred crops during the past 60 years. Since the American Hemerocallis Society began acting as the official registry of daylily cultivars in 1947, more than 40 000 registrations have been processed. In order to determine the effects of intensive breeding on cultivar development, and to study relationships among different species, genetic variation in the daylily was estimated using AFLP markers. Nineteen primary genotypes (species and early cultivars) and 100 modern cultivars from different time periods were evaluated using 152 unambiguous bands (average 79% polymorphism rate) derived from three AFLP primer combinations. Overall, pairwise similarity estimates between entries ranged between 0.618 and 0.926 (average=0.800). When comparing cultivar groups from different time periods (1940–1998), genetic similarity was initially increased, compared to the primary diploid genotypes, remained constant from 1940 to 1980, and then steadily increased as breeding efforts intensified and hybridizers began focusing on a limited tetraploid germplasm pool derived by colchicine conversion. Among modern (1991–1998) daylily cultivars, genetic similarity has increased by approximately 10% compared to the primary genotypes. These data were also used to evaluate recent taxonomic classifications among daylily species which, with a few minor exceptions, were generally supported by the AFLP data. Received: 15 March 2000 / Accepted: 13 June 2000     

High-resolution mapping of loci conferring resistance to sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers

Sugarcane mosaic virus (SCMV) is one of the most important virus diseases of maize in Europe. Genetic analysis on backcross five (BC5) progeny derived from the cross FAP1360A (resistant) × F7 (susceptible) confirmed that at least two dominant genes, Scm1 and Scm2, are required for resistance to SCMV in the progeny of this cross. With the aid of RFLP and SSR marker analyses, Scm1 was mapped in the region of 8.7 cM – between the nucleolus organizer region (nor) and RFLP marker bnl6.29 on the short arm of chromosome 6, while Scm2 was mapped to an interval of 26.8 cM flanked by the RFLP markers umc92 and umc102 near the centromere region of chromosome 3. Both chromosome regions were further enriched for AFLP markers by successful application of a bulked segregant analysis to this oligogenic trait. A total of 23 linked AFLP markers were identified, clustered in chromosome regions adjacent to either Scm1 or Scm2. Seven AFLP markers linked to Scm1 resided within the nor-bnl6.29 interval, and one of them, E3M8-1, showed no recombination with Scm1. Three AFLP markers linked to Scm2 are located between umc92 and umc102. Received: 13 October 1998 / Accepted: 18 January 1999     

An integrated high-density RFLP-AFLP map of tomato based on two Lycopersicon esculentum×L. pennellii F2 populations

 Two independent F₂ populations of Lycopersicon esculentum×L. pennellii which have previously been investigated in RFLP mapping studies were used for construction of a highly saturated integrated AFLP map. This map spanned 1482 cM and contained 67 RFLP markers, 1078 AFLP markers obtained with 22 EcoRI+MseI primer combinations and 97 AFLP markers obtained with five PstI+MseI primer combinations, 231 AFLP markers being common to both populations. The EcoRI+MseI AFLP markers were not evenly distributed over the chromosomes. Around the centromeric region, 848 EcoRI+ MseI AFLP markers were clustered and covered a genetic distance of 199 cM, corresponding to one EcoRI+ MseI AFLP marker per 0.23 cM; on the distal parts 1283 cM were covered by 230 EcoRI+MseI AFLP markers, corresponding to one marker per 5.6 cM. The PstI/MseI AFLP markers showed a more even distribution with 16 PstI/MseI AFLP markers covering a genetic distance of 199 cM around the centromeric regions and 81 PstI/MseI AFLP markers covering a genetic distance of 1283 cM on the more distal parts, corresponding to one marker per 12 and 16 cM respectively. In both populations a large number of loci showed a significant skewed segregation, but only chromosome 10 loci showed skewness that was similar for both populations. This ultra-dense molecular-marker map provides good perspectives for genetic and breeding purposes and map-based cloning. Received: 3 September 1998 / Accepted: 27 October 1998     

Characterization and comparative analysis of sequence-specific amplified polymorphisms based on two subfamilies of IRRE retrotransposons in <Emphasis Type="Italic">Iris missouriensis</Emphasis> (Iridaceae)

DNA markers based on transposable-element polymorphisms are potentially useful alternatives to anonymous fragment-length polymorphisms (AFLPs). We developed the retrotransposon sequence-specific amplified polymorphism (retrotransposon SSAP) technique for the angiosperm Iris missouriensis (Iridaceae) in order to evaluate its use in generating population-genetic markers. Our cloning strategy identified two groups of long-terminal repeat retrotransposons of the IRRE family. Primers homologous to conserved regions of these elements generated repeatable and polymorphic markers. In comparison, the AFLP protocol failed to produce useful markers in our hands in this species. To investigate the distribution and evolutionary tempo of the two retrotransposons, we developed a phylogeny of representative species of subgenus Limniris based on chloroplast sequence. Sequences of both groups of retrotransposons were widespread in Limniris, but we also found evidence of substantial sequence and copy-number evolution since the divergence of I. missouriensis from other Limniris species. We corroborated these results with quantitative real-time PCR estimates of relative copy number. Importantly, the geographic structure of retrotransposon SSAP was strikingly different for the two groups of retrotransposons, indicating that different mutational dynamics and/or selective pressures govern their distribution. Although these primers should be useful for population-genetic studies of Iris missouriensis and other Limniris species, our findings reinforce the need for caution in evaluating transposable-element markers used to analyze the relatedness of populations or cultivars, as very different conclusions may be reached depending on the sequence amplified.     

Classification of rice germplasm: III. High-resolution fingerprinting of cytoplasmic genetic male-sterile (CMS) lines with AFLP

 The cytoplasmic genetic male-sterile (CMS) lines developed at the International Rice Research Institute are valuable in producing tropical rice hybrids. Efficient use of CMS lines in hybrid rice production will depend on their level of genetic diversity. Aside from morphological characterization, molecular analysis based on DNA markers can provide information on the genetic diversity of the germplasm. The Amplified Fragment Length Polymorphism (AFLP) technique was used to fingerprint 71 CMS lines and four rice cultivars, ‘IR64’, ‘Azucena’, ‘IR74’, and ‘FR13A’. Eleven primer pair combinations specific to the enzymes PstI and MseI were used to generate 530 AFLP markers, 176 of which were polymorphic. Each CMS line revealed a distinct fingerprint. The AFLP marker-based dendrogram depicted genetic variation among the CMS lines. The CMS lines developed in japonica background grouped with ‘Azucena’, a japonica cultivar. None of the CMS lines clustered with ‘FR13A’, a flood-tolerant traditional indica variety. ‘IR64’ was found to be distinct from the other indica CMS lines and clustered with lines developed in its background. The grouping of CMS lines into a few groups is useful for breeders in selecting genetically diverse CMS lines for hybrid rice production and in avoiding test crossing every CMS line empirically. This study demonstrated that AFLP is a powerful and reliable tool in determining the genetic relationships and in producing distinct fingerprints of rice cultivars. Received: 20 December 1996 / Accepted: 9 October 1997