Target Region Amplification Polymorphism Research Articles

Markers and mapping revisited: finding your gene

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of this highly dynamic arena of plant science.

Genetic Diversity and Relationships among 177 Public Sunflower Inbred Lines Assessed by TRAP Markers

This study investigated the genetic diversity and relationships among the 177 public sunflower (Helianthus annuus L.) inbred lines developed and released by USDA‐ARS from the 1970s to 2005 using the target region amplification polymorphism (TRAP) marker technique. Twelve primer combinations generated a total of 230 polymorphic markers that could discriminate each of these lines, suggesting that TRAP is a powerful marker technique for fingerprinting sunflower inbred lines. The pairwise genetic similarity coefficients (simple matching) based on the TRAP markers ranged from 0.30 to 0.97 with a mean of 0.58, indicating that there is substantial genetic variability in this public collection. The average similarity coefficient of 41 confection inbred lines was significantly higher than that of 136 oilseed inbred lines. Cluster analysis grouped the 177 lines into two classes, oilseed (O) class and confection (C) class with only two exceptions. The O and C classes were further categorized into two groups: fertility maintainer (OB and CB) and fertility restorer groups (OR and CR). Moreover, inbred lines in the OB, OR, CR, and CB groups were placed into four, three, two, and two clusters, respectively. These results are useful to sunflower breeders and would promote the utilization of these public inbred lines.

Genetic mapping for theRf1(fertility restoration) gene in sunflower (Helianthus annuusL.) by SSR and TRAP markers

The Rf1 gene in sunflower can effectively restore the pollen fertility of PET1 cytoplasm in male-sterile lines and has been widely used in commercial hybrid production. Identifying molecular markers tightly linked to this gene will be useful in marker-assisted selection to develop maintainer and restorer lines. Rf1 has been mapped to Linkage Group (LG) 13 of the public sunflower simple sequence repeat (SSR) map by aligning maps constructed from different populations and only one SSR marker was reported to be loosely linked to Rf1. This paper reports the result of applying target region amplification polymorphism (TRAP) and SSR markers to map and develop a sequencetagged site (STS) marker tightly linked to Rf1 using two populations derived from a cross between two U.S. public sunflower lines, RHA439 and cmsHA441. An SSR marker, ORS511, was 3.7 cM from the Rf1 gene and a TRAP marker, K11F05Sa12-160, was linked to Rf1 at a distance of 0.4 cM. This TRAP marker was converted to an STS marker for using in sunflower breeding.

Mapping quantitative trait loci (QTL) controlling seed morphology and disk diameter in sunflower (Helianthus annuus L.)

SUMMARY Seed morphology and disk diameter are agronomically most important traits for the confection sunflower. This paper reports the results of an analysis of quantitative trait loci (QTLs) underlying 10 seed morphological traits including seed and kernel size, shape and stripe, and disk diameter in both F 2 population and F 2:3 families derived from an oilseed by confection cross. A linkage map containing 165 target region amplification polymorphism (TRAP) and 44 simple sequence repeat (SSR) markers was constructed from 120 F 2 plants. This map contained 17 linkage groups and spanned a total genetic distance of 1784.3 cM. A total of 51 QTLs were detected and 32 of them were identified in both generations. Each QTL explained 5.1-29.3% of the phenotypic variation, suggesting these traits were controlled by multiple genes. Most of the QTLs were clustered in six chromosomal regions. Two of the three QTLs identified for disk diameter were also located in two of the six regions. Moreover, alleles from the confection line at these QTLs had positive effects on these traits. Both QTL congruence and correlation analysis revealed that different genetic bases are responsible for seed shape, stripe, and other confection traits. The information generated by this study will facilitate confection sunflower breeding. RESUMEN La morfología de las semillas y el diámetro del disco son los caracteres agronómicos más importantes para el girasol confitero. Este trabajo informa los resultados del análisis de los loci para caracteres cuantitativos (QTLs) que subyacen 10 caracteres morfológicos de las semillas, incluyendo tamaño de aquenio y pepita, forma y estriado y diámetro del disco en una población F 2 y las familias F 2:3 derivadas de un cruzamiento entre un material del tipo oleaginoso y uno confitero. Se construyó un mapa de ligamiento consistente en 165 marcadores de polimorfismo de amplificación de regiones objetivo (TRAP) y 44 marcadores de repetición de secuencias simples (SSR) a partir de 120 plantas F 2 . Este mapa contiene 17 grupos de ligamiento y explora una distancia genética total de 1784.3 cM. Se detectó un total de 51 QTLs, 32 de los cuales de identificaron en ambas generaciones. Cada QTL explicó un 5.1-29.3% de la variación fenotípica, lo que sugiere que estos atributos están controlados por múltiples genes. La mayoría de los QTLs están agrupados en seis regiones cromosómicas. Dos de los tres QTLs identificados para diámetro del disco están localizados en dos de las seis regiones cromosómicas. Además, alelos de la línea confitera en dichos QTLs presentaron efectos positivos sobre estos atributos. Tanto la congruencia de QTLs como los análisis de correlación revelaron que bases genéticas diferentes son responsables de la expresión de los atributos forma de semilla, estriado y otros caracteres de los confiteros. La información generada por este estudio facilitará el mejoramiento del girasol confitero. RÉSUMÉ La morphologie de la graine et le diamètre du capitule sont les caractères agronomiques les plus importants pour le tournesol de bouche. Cet article rapporte les résultats d’une analyse de LCQ associés à 10 caractères morphologiques parmi lesquels la taille du grain, sa forme et ses stries et le diamètre du capitule, à partir d’une population F 2 et de sa descendance F 2 :F 3 issue d’un croisement entre une lignée de type “huile” et d’une lignée de type “de bouche”. Une carte de liaison a été établie à partir de 165 marqueurs TRAP (target region amplification polymorphism) et de 44 marqueurs microsatellites (SSR), et sur 120 individus F 2 . Cette carte comporte 17 groupes de liaison et couvre une distance génétique totale de 1784.3 cM. Au total, 51 LCQ ont été mis en évidence et 32 d’entre eux l’ont été pour les deux générations. Chaque LCQ explique 5.1 à 29.3% de la variance phénotypique totale, ce qui suggère un contrôle multigénique. La plupart des LCQ se concentre sur six régions chromosomiques. Deux des trois QTL identifiés pour le diamètre du capitule co-localisent avec deux de ces six régions chromosomiques. De plus, le parent de type “de bouche” apporte les allèles favorables à ces caractères. La cohérence des résultats obtenus sur les LCQ et les analyses de corrélations mettent en évidence l’existence de bases génétiques distinctes pour la forme de la graine, ses stries, et les autres caractères d’intérêt pour le tournesol de bouche. L’information produite par cette étude facilitera l’amélioration génétique des tournesols de bouche.

Identifying Quantitative Trait Loci for Resistance to Sclerotinia Head Rot in Two USDA Sunflower Germplasms

Sclerotinia head rot is a major disease of sunflower in the world, and quantitative trait loci (QTL) mapping could facilitate understanding of the genetic basis of head rot resistance and breeding in sunflower. One hundred twenty-three F2:3 and F2:4 families from a cross between HA 441 and RHA 439 were studied. The mapping population was evaluated for disease resistance in three field experiments in a randomized complete block design with two replicates. Disease incidence (DI) and disease severity (DS) were assessed. A genetic map with 180 target region amplification polymorphism, 32 simple sequence repeats, 11 insertion-deletion, and 2 morphological markers was constructed. Nine DI and seven DS QTL were identified with each QTL explaining 8.4 to 34.5% of phenotypic variance, suggesting the polygenic basis of the resistance to head rot. Five of these QTL were identified in more than one experiment, and each QTL explained more than 12.9% of phenotypic variance. These QTL could be useful in sunflower breeding. Although a positive correlation existed between the two disease indices, most of the respective QTL were located in different chromosomal regions, suggesting a different genetic basis for the two indices.

Population Structure in Cultivated Lettuce and Its Impact on Association Mapping

The association mapping technique is a useful tool for detecting markers linked to the genes underlying the variation of a trait among elite cultivars. To avoid false-positive results due to unrecognized population structure in the analyzed set of individuals, the subpopulations need to be identified. Fifty-four lettuce (Lactuca sativa L.) cultivars representing five horticultural types important in North America, together with six accessions from two wild species (Lactuca saligna L. and Lactuca serriola L.), were assayed for polymorphism with target region amplified polymorphism (TRAP) marker loci. The model-based clustering approach recognized three main subpopulations in cultivated lettuce that are well separated from wild species. Although the clustering based on molecular markers was generally in good agreement with horticultural types, some cultivars were classified differently or showed mixed origin. The effect of population structure on association mapping was tested on four traits with strong or weak correlation to the lettuce horticultural type and monogenic or polygenic mode of inheritance. Traits that were strongly correlated with lettuce types displayed many false-positive results when population structure was ignored, but the spurious associations disappeared when estimates of population structure or relative kinship (both based on molecular markers) were included in the statistical model. Using of horticultural types as covariate was not sufficient to control for spurious associations in the monogenic trait with strong correlation to lettuce types. The best approach to avoid spurious associations in lettuce association studies is to assess relatedness of accessions with molecular markers and to include this information into the statistical model.

Linkage mapping and genome analysis in a Saccharum interspecific cross using AFLP, SRAP and TRAP markers

Framework genetic linkage maps of two progenitor species of cultivated sugarcane, Saccharum officinarum ‘La Striped’ (2n = 80) and S. spontaneum ‘SES 147B’ (2n = 64) were constructed using amplified fragment length polymorphism (AFLP), sequence related amplified polymorphism (SRAP), and target region amplification polymorphism (TRAP) markers. The mapping population was comprised of 100 F1 progeny derived from the interspecific cross. A total of 344 polymorphic markers were generated from the female (S. officinarum) parent, out of which 247 (72%) were single-dose (segregating in a 1:1 ratio) and 33 (9%) were double-dose (segregating in a 3.3:1 ratio) markers. Sixty-four (19%) markers deviated from Mendelian segregation ratios. In the S. spontaneum genome, out of a total of 306 markers, 221 (72%) were single-dose, 43 (14%) were double-dose, and 42 markers (14%) deviated from Mendelian segregation ratios. Linkage maps with Kosambi map distances were constructed using a LOD score ≥5.0 and a recombination threshold of 0.45. In Saccharum officinarum, 146 markers were linked to form 49 linkage groups (LG) spanning 1732 cM whereas, in S. spontaneum, 121 markers were linked to form 45 LG spanning 1491 cM. The estimated genome size of S. officinarum ‘La Striped’ was 2448 cM whereas that of S. spontaneum ‘SES 147B’ was 3232 cM. Based on the two maps, genome coverage was 69% in S. officinarum and 46% in S. spontaneum. The S. officinarum parent ‘La Striped’ behaved like an auto-allopolyploid whereas S. spontaneum ‘SES 147B’ behaved like a true autopolyploid. Although a large disparity exists between the two genomes, the existence of simple duplex markers, which are heterozygous in both parents and segregate 3:1 in the progeny, indicates that pairing and recombination can occur between the two genomes. The study also revealed that, compared with AFLP, the SRAP and TRAP markers appear less effective at generating a large number of genome-wide markers for linkage mapping in sugarcane. However, SRAP and TRAP markers can be useful for QTL mapping because of their ability to target gene-rich regions of the genome, which is a focus of our future research.

Target Region Amplification Polymorphism (TRAP) as a Tool for Detecting Genetic Variation in the Genus Pelargonium

Pelargonium was a priority genera collected by the Ornamental Plant Germplasm Center (OPGC) until a recent reorganization. To preserve genetic diversity for future breeders, OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection at OPGC consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Target region amplification polymorphism (TRAP) has the advantage of producing a large number of markers through use of sequence information that is already available. Our first goal was to determine the feasibility of TRAP for the analysis of this large collection, so that in the future the most diverse genotypes may be retained. To achieve this goal, we first modified existing DNA extraction techniques to account for the high levels of phenolic compounds present in some Pelargonium species by combining several washes to remove the phenolics with the addition of high levels of antiphenolic compounds. Second, we evaluated the TRAP procedure using the DNA isolated from 46 accessions. For 44 accessions, one or two primer combinations generated enough fragments to discriminate each of the accessions, and similar clades were produced by cluster analysis of the polymorphic fragments amplified by different primer combinations. All the scorable fragments were polymorphic, for one primer combination there were 148 markers from one image and the other produced 160 markers on two images. These results demonstrate that TRAP is an effective method for molecular characterization of ornamental collections.

Application of target region amplification polymorphism (TRAP) technique to Porphyra (Bangiales, Rhodophyta) fingerprinting

L. Qiao, H. Liu, J. Sun, F. Zhao, B. Guo, M. Weng, T. Liu, J. Dai, and B. Wang. 2007. Application of target region amplification polymorphism (TRAP) technique to Porphyra (Bangiales, Rhodophyta) fingerprinting. Phycologia 46: 450–455. DOI: 10.2216/07-08.1To demonstrate the applicability of the target region amplification polymorphism (TRAP) marker technique to Porphyra, 15 Porphyra lines, representing three species, were fingerprinted with 32 primer-combinations. Ten primer-combinations that gave a stable and reproducible amplification pattern were selected. In total 432 fragments from 50 to 500 base pairs (bp) in length were amplified with these 10 selected primer-combinations, and all the fragments were polymorphic. The 432 fragments were scored respectively and used to construct a dendrogram of the 15 Porphyra lines with unweighted pair-group method arithmetic average (UPGMA). These Porphyra lines were divided into two major groups at the 0.71 similarity level. This result is basically consistent with the conventional Porphyra taxonomy. Twelve of the 432 fragments, which were amplified by two primer-combinations, Tps/1F and Tps/3F, were chosen and used to develop the DNA fingerprints of these Porphyra lines. The developed DNA fingerprints then were converted into binary codes and resulted in one unique binary code for each of the 15 Porphyra lines. This paper is the first report concerning the application of TRAP technique to seaweeds. Our results suggest that TRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the identification, classification and resource protection of Porphyra lines.

High‐density Linkage Map of Cultivated Allotetraploid Cotton Based on SSR, TRAP, SRAP and AFLP Markers

AbstractA high‐density linkage map was constructed for an F2 population derived from an interspecific cross of cultivated allotetraploid species between Gossypium hirsutum L. and G. barbadense L. A total of 186 F2 individuals from the interspecific cross of “CRI 36 × Hai 7124” were genotyped at 1 252 polymorphic loci including a novel marker system, target region amplification polymorphism (TRAP). The map consists of 1 097 markers, including 697 simple sequence repeats (SSRs), 171 TRAPs, 129 sequence‐related amplified polymorphisms, 98 amplified fragment length polymorphisms, and two morphological markers, and spanned 4 536.7 cM with an average genetic distance of 4.1 cM per marker. Using 45 duplicated SSR loci among chromosomes, 11 of the 13 pairs of homologous chromosomes were identified in tetraploid cotton. This map will provide an essential resource for high resolution mapping of quantitative trait loci and molecular breeding in cotton.(Handling editor: Yong‐Biao Xue)

Assessment of Genetic Diversity and Relationships Among Caladium Cultivars and Species Using Molecular Markers

Caladium (Caladium ×hortulanum Birdsey) is an important aroid widely used in the ornamental plant industry. Concerns have been raised about possible loss of genetic diversity due to a drastic decline in the number of cultivars in the last century. This study assessed genetic diversity and relationships among caladium cultivars and species accessions. Forty-five major cultivars and 14 species accessions were analyzed based on 297 DNA fragments produced by the target-region amplification polymorphism marker system. A low level of diversity (44.4% polymorphism) was exhibited in cultivars, while a high level of diversity (96.8% polymorphism) was present among seven accessions of Caladium bicolor (Aiton) Vent., Caladium marmoratum Mathieu, Caladium picturatum C. Koch, and Caladium schomburgkii Schott. A small percentage (7.6%) of DNA fragments was present in cultivars but absent in the seven species accessions, while a high percentage (32.2%) of DNA fragments was present in the seven species accessions but absent in cultivars. Cultivars shared a higher level of similarity at the molecular level with an average Jaccard coefficient at 0.802, formed a large group in cluster analysis, and concentrated in the scatter plot from a principal-coordinate analysis. Two accessions of C. bicolor and C. schomburgkii were very similar to cultivars with Jaccard similarity coefficients from 0.531 to 0.771, while the rest of the species accessions had small similarity coefficients with cultivars (0.060 to 0.386). Caladium steudnirifolium Engler and Caladium lindenii (André) Madison were very dissimilar to C. bicolor, C. marmoratum, C. picturatum, and C. schomburgkii, with Jaccard similarity coefficients from 0.149 to 0.237 (C. steudnirifolium) and from 0.060 to 0.118 (C. lindenii). There is a limited amount of molecular diversity in caladium cultivars, but the great repertoire of unique genes in species accessions could be used to enhance the diversity in future cultivars and reduce potential genetic vulnerability.

Genetic diversity of 38 spinach (Spinacia oleracea L.) germplasm accessions and 10 commercial hybrids assessed by TRAP markers

Target region amplification polymorphism (TRAP) markers were used to assess genetic variability among 38 germplasm accessions and 10 commercial hybrids of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Germplasm accessions with different geographic origins and 10 commercial hybrids were examined. For assessing genetic diversity within accessions, DNA was extracted from 12 individual seedlings from six germplasm accessions and two hybrids. A relatively high level of polymorphism was found within accessions based on 59 polymorphic TRAP markers generated from one fixed primer derived from the Arabidopsis-like telomere repeat sequence and two arbitrary primers. For evaluating interaccession variability, DNA was extracted from a bulk of six to 13 seedlings of each accession. Of the 492 fragments amplified by 12 primer combinations, 96 (19.5%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice) was 57.5% with a range from 23.2 to 85.3%. A dendrogram indicated that the genetic relationships among the accessions were not highly associated with the geographic locations in which the germplasms were collected. The seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities tended to reduce the genetic variability. This preliminary study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability among spinach germplasms.

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines

The aneuploid stocks of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat (T. aestivum L.) have been developed mainly in 'Langdon' (LDN) and 'Chinese Spring' (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.

Saturation and comparative mapping of a major Fusarium head blight resistance QTL in tetraploid wheat

Fusarium head blight (FHB) is a devastating disease of cultivated wheat worldwide. Partial resistance to FHB has been identified in common wheat (Triticum aestivum L.). However, sources of effective FHB resistance have not been found in durum wheat (T. turgidum L. var. durum). A major FHB resistance quantitative trait loci (QTL), Qfhs.ndsu-3AS, was identified on chromosome 3A of T. dicoccoides, a wild relative of durum wheat. Here, we saturated the genomic region containing the QTL using EST-derived target region amplified polymorphism (TRAP), sequence tagged site (STS), and simple sequence repeat (SSR) markers. A total of 45 new molecular marker loci were detected on chromosome 3A and the resulting linkage map consisted of 55 markers spanning a genetic distance of 277.2 cM. Qfhs.ndsu-3AS was positioned within a chromosomal interval of 11.5 cM and is flanked by the TRAP marker loci, Xfcp401 and Xfcp397.2. The average map distance between the marker loci within this QTL region was reduced from 4.9 cM in the previous study to 3.5 cM in the present study. Comparative mapping indicated that Qfhs.ndsu-3AS is not homoeologous to Qfhs.ndsu-3BS, a major FHB QTL derived from the common wheat cultivar Sumai 3. These results facilitate our efforts toward map-based cloning of Qfhs.ndsu-3AS and utilization of this QTL in durum wheat breeding via marker-assisted selection.

(276) Assessing Genetic Diversity of Spinach Germplasm using TRAP Markers

Target region amplified polymorphism (TRAP) markers were used to evaluate genetic variability among 48 accessions of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Thirty-eight accessions collected and preserved by the USDA National Plant Germplasm System (NPGS) and 10 commercial hybrids were used in the current study. For assessing genetic diversity within accessions, DNA samples were prepared from nine to 12 individual seedlings from six germplasm accessions and two hybrids. Relatively high levels of polymorphism was found within accessions based on 61 polymorphic TRAP markers generated with two fixed primers derived from the Arabidopsis-type telomere repeat sequence and two arbitrary primers. For evaluating inter-accession variability, DNA was extracted from a bulk of six to 10 seedlings of each accession. Of the 1092 fragments amplified by 14 primer combinations, 96 (8.8%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice, Nei) was 57.5% with a range from 23.2 to 85.3%. A dendrogram was constructed based on the similarity matrix. It was found that the genetic relationships were not highly correlated with the geographic locations in which the accessions were collected. However, seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities have effect on the genetic variability. This study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability of spinach germplasm.

Defining the sunflower (Helianthus annuus L.) linkage group ends with the Arabidopsis-type telomere sequence repeat-derived markers

The target region amplification polymorphism (TRAP) marker technique was employed to define sunflower (Helianthus annuus L.) linkage group ends. In combination with eight arbitrary primers, nine fixed primers containing the Arabidopsis-type telomere repeat sequences worked successfully in generating polymorphic markers in the mapping population of 92 F(7) recombinant inbred lines (RIL) derived from the cross RHA 280 x RHA 801. This population was used in the construction of the densest sunflower linkage map of 577 simple sequence repeat (SSR) markers. With 18 sets of PCR reactions, 226 polymorphic TRAP markers were amplified from the two parental lines and 92 RIL. The computer program, Mapmaker, placed 183 markers into the established 17 linkage groups of the SSR map. Although most of the added markers spread across the genome, 32 markers were mapped to the outermost positions of the linkage groups, defining 21 of the 34 linkage group ends of the sunflower linkage map. The telomeric origin of a few of these markers was confirmed by sequence analyses. These telomere-associated markers will provide an accurate assessment of the completeness of a linkage group and a better estimate of the actual genetic lengths. The potential application of the telomere mapping to sunflower improvement is discussed.

Recent Developments in Primer Design for DNA Markers in Higher Plants

Primer design is a critical step in the application of PCR-based technologies in genetic diversity analysis of horticultural plants. As more plant genomes have been sequenced in recent years, the emphasis of primer design strategy has shifted to genome-wide and high-throughput direction. This poster summarizes recent advances in primer design for profiling of DNA polymorphism in higher plants, including new primers for the classical plant DNA marker system such as simple sequence repeat (SSR) and single-nucleotide polymorphism (SNPs), as well as newly developed DNA marker systems such as sequence-related amplified polymorphism (SRAP), sequence-specific amplification polymorphism (SSAP), target region amplification polymorphism (TRAP), and universal rice primer (URP). Although most of these primers were designed for agronomical crops, they could be applied to horticultural plants because plant genomes are evolutionarily related. Also, these new primer design strategies could help horticultural researchers develop better primers specifically for profiling of polymorphism in a variety of horticultural crops, invasive weeds, or medicinal plants. We will present examples of their utilization in these diverse systems.

A Phylogeny of Pelargonium Based on TRAP Markers

Pelargonium is one of the priority genera collected by the Ornamental Plant Germplasm Center (OPGC). In order to protect future breeders from a loss of genetic diversity, the OPGC collects heirloom cultivars, breeding lines, and wild species. The current Pelargonium collection consists primarily of cultivars originating from P. ×hortorum and P. ×domesticum. Our project was designed to analyze the current collection in order to facilitate the maintenance of a more-diverse core collection. We have expanded our TRAP (Target Region Amplified Polymorphism) analysis from 120 plants with one primer set to include 780 plants with four primer sets. Each primer set consists of a labeled arbitrary primer paired with a gene-specific primer, and two different fluorescent labels were used to allow multiplexed PCR reactions. We scored about 90 markers in each of the first two primer sets and about 60 markers in each of the second two. In comparisons between the phylogeny and the morphology and taxonomy of these plants, we show some matching clusters that may be explained by the breeding history of the plants.

(278) Genetic Variability and Relationship of Caladium Cultivars As Revealed with Molecular Markers

Cultivated caladiums are valued for their bright colorful leaves and are widely used in containers and landscapes. More than 1500 named cultivars have been introduced during the past 150 years, yet currently only about 100 cultivars are in commercial propagation in Florida. Caladium tubers produced in Florida account for 95% of the world supplies. Loss of caladium germplasm or genetic diversity has been a concern to future improvement of this plant. In addition, the relationship among the available cultivars, particularly those of close resemblance, has been lacking. This study was conducted to assess the genetic variability and relationship in commercial cultivars and species accessions. Fifty-seven major cultivars and 15 caladium species accessions were analyzed using the target region amplification polymorphism marker technique. This marker system does not involve DNA restriction or adaptor linking, but shares the same high throughput and reliability with the amplified fragment length polymorphism system (AFLP). Eight primer combinations amplified 379 scorable DNA fragments among the caladium samples. A high level of polymorphism was detected among the species accessions as well as among cultivars. These markers allowed differentiation of all the cultivars tested, including those hardly distinguishable morphologically. Clustering analysis based on these DNA fingerprints separated the cultivars into five clusters and Caladium lindenii far from other caladium species. The availability of this information will be very valuable for identifying and maintaining the core germplasm resources and will aid in selecting breeding parents for further improvement.

Genetic characterization and molecular mapping of Hessian fly resistance genes derived from Aegilops tauschii in synthetic wheat

Two synthetic hexaploid wheat lines (xAegilotriticum spp., 2n = 6x = 42, genomes AABBDD), SW8 and SW34, developed from the crosses of the durum wheat cultivar Langdon (Triticum turgidum L. var. durum, 2n = 4x = 28, genomes AABB) with two Aegilops tauschii Cosson accessions (2n = 2x = 14, genome DD), were determined to carry Hessian fly [Mayetiola destructor (Say)] resistance genes derived from the Ae. tauschii parents. SW8 was resistant to the Hessian fly biotype Great Plains (GP) and strain vH13 (virulent to H13). SW34 was resistant to biotype GP, but susceptible to strain vH13. Allelism tests indicated that resistance genes in SW8 and SW34 may be allelic to H26 and H13 or correspond to paralogs at both loci, respectively. H26 and H13 were localized to chromosome 4D and 6D, respectively, in previous studies. Molecular mapping in the present study, however, assigned the H26 locus to chromosome 3D rather than 4D. On the other hand, mapping of the resistance gene in SW34 verified the previous assignment of the H13 locus to chromosome 6D. Linkage analysis and physical mapping positioned the H26 locus to the chromosomal deletion bin 3DL3-0.81-1.00. A linkage map for each of these two resistance genes was constructed using simple sequence repeat (SSR) and target region amplification polymorphism (TRAP) markers.