The United States Department of Agriculture-Agricultural Research Service sweetpotato (Ipomoea batatas) germplasm collection contains accessions that were initially collected from various countries worldwide. These materials have been maintained and distributed as in vitro plantlets since the mid-1980s. The status of viral infection by the emerging Sweet potato leaf curl virus (SPLCV) and other Begomovirus spp. in this germplasm has yet to be determined. In order to minimize the potential distribution of virus-infected clones, all accessions in the collection were tested for SPLCV using a real-time polymerase chain reaction assay. In total, 47 of 701 accessions of in vitro plantlets tested positive for SPLCV. The presence of SPLCV detected in these materials was confirmed via biological indexing using the indicator plants I. nil and I. muricata. Symptoms appeared more rapidly on I. muricata than on I. nil. Nucleotide polymorphisms among the isolates were evaluated by sequencing the AV1 coat protein gene from 24 SPLCV-infected accessions. The results revealed that the SPLCV isolates shared high sequence identity. Ten nucleotide substitutions were identified, most of which were synonymous changes. Phylogenetic analysis was conducted on those 24 SPLCV isolates in combination with six described SPLCV species and various SPLCV strains from GenBank to evaluate the relationships among viral species or strains. The results from this analysis indicated that most of the AV1 genes derived from previously classified SPLCV species clustered together, some of which formed well-supported monophyletic clades, further supporting the current taxonomy. Overall, identification of SPLCV-infected germplasm will allow approaches to be employed to eliminate the virus from the collection and limit the distribution of infected materials.

This is the first report of targeted development of B genome microsatellite markers in Musa. A total of 44 sequences with microsatellites were isolated from an enriched library of Musa balbisiana cv. 'Tani' (BB genome). Of these, 25 were polymorphic when screened on 14 diverse diploid and triploid Musa accessions. The number of alleles detected by each marker ranged between one and seven. All 25 microsatellite markers generated amplification products in all species and genome complements. These new microsatellite markers fill an important gap for diversity assessment and linkage mapping studies in plantain (AAB) and cooking banana (ABB).

An enzyme immunoassay was evaluated for its ability to quantify total capsaicinoids in fruit of 16 genotypes representing four species of Capsicum, in comparison with high-performance liquid chromatography (HPLC). Concentrations of capsaicinoids in samples ranged from 5.1 to 4284ppm, and from 0.6 to 3467ppm, as determined by enzyme immunoassay (EIA) and HPLC, respectively. Lowest concentrations of capsaicinoids occurred in sweet bell and pimento types (C. annuum L.), and the highest concentration occurred in a small-fruited ‘bird’-type accession of C. frutescens L. (PI 593924). Estimates of capsaicinoids obtained by EIA and HPLC were highly correlated (R2=0.996). Data suggest that EIA is an effective means for estimating total capsaicinoids in extracts of fresh chile fruits.

Using codominant molecular markers (microsatellites) for paternity identification was investigated in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.]. Two experimental populations (CIP and LAES), each consisting of progeny of known parentage, were scored for the presence or absence of alleles segregating at IB-316 and IB-318 microsatellite loci. Paternity was assessed using paternity exclusion and the most-likely parent methods. In the former, paternity is assigned based on the identification of incompatible parent-progeny marker data. In contrast, the latter method incorporates paternity exclusion and a log-likelihood or LOD score that weighs progeny allelic patterns as to the likelihood that they could have come from a given paternal parent. The number of correctly allocated progeny differed for the methods. Paternity exclusion correctly allocated 7% and 25% of the progeny in the LAES and CIP populations, respectively. The most-likely parent method correctly allocated 23% and 88% of the progeny in the LAES and CIP populations, respectively. The greater misassignments in the LAES population were attributed to low allelic diversity at the LAES IB-318 locus and a larger parental population. This study demonstrates the feasibility of identifying paternity in sweetpotato using a minimal number of loci.

A total of 141 Ipomoea batatas (L.) Lam. accessionsderived from botanical seed originally collected from 26 sites in 4 Provinces inPapua New Guinea, a secondary center of genetic diversity for sweetpotato, weregenetically analyzed. Two hundred Amplified Fragment Length Polymorphism (AFLP)markers were identified and utilized in the analysis. Relatedness amongaccessions was estimated by analyzing the AFLP data using the Dice coefficientof similarity and UPGMA methods. The molecular analysis revealed relativelylimited genetic diversity within and between sites. Genotypes collected in agiven region often displayed molecular marker variability similar to thatobserved over the entire sampled area. However, a subset of 14 genotypes derivedfrom seed collected from New Ireland island differed from genotypes collected onNew Guinea island. Estimates of genetic diversity-based similarity valuescalculated from the AFLP data indicated a moderate level of diversity (0.767mean coefficient of similarity) across all plant materials analyzed. Threemethods of selection were evaluated for their efficacy in capturing themolecular marker diversity within the plant materials in the form of a subset.They were random, stratified-random (geographic based), and marker-assistedselection (MAS). MAS was the most efficient. A Maximally Diverse Subset (MDS) of12 genotypes capturing 92% of the molecular marker diversity was identified.

Previous work by our group has detected the presence of a heterogeneous population of Ty1-copia-like reverse transcriptase retrotransposon sequences in the sweetpotato genome. Recently, we detected the presence of putatively active Ty1-copia-like reverse transcriptase sequences from a virus-infected `Beauregard' sweetpotato clone. In the current study, we report the differential detection of putatively stress-activated sequences in clones from seedling 91-189. The clones were infected with different combinations of virus isolates followed by extraction of leaf RNA samples at three sampling dates (weeks 2, 4, and 6) after inoculation. After repeated DNAse treatments to eliminate contaminating DNA, the RNA samples were subjected to first strand cDNA synthesis using random decamer primers followed by PCR analysis utilizing Ty1-copia reverse transcriptase-specific primers. Through this approach, we detected amplified fragments within the expected size range (280-300 bp) from clones infected with isolates of sweetpotato leaf curl (SPLC) and feathery mottle viruses (FMV) (week 2 and 6) and FMV (week 4). We were unable to detect PCR products from the noninfected clones or the other infected samples. The data suggests that specific viruses may be involved in the expression of these Ty1-copia-related reverse transcriptase sequences. It also appears that sampling at various dates is necessary to detect putative activity over time. This preliminary information is essential before proceeding to the construction and screening of cDNA libraries to isolate and fully characterize the putatively active sweetpotato Ty1-copia-like retrotransposon sequences. Through the partial or complete characterization of sweetpotato Ty1-copia elements, sequences that correspond to cis-regulatory element(s) can be identified and further studied for their roles in responding to specific stress factors.

The 16S-23S rDNA spacer of the type strain (ATCC 35879) of Xylella fastidiosa was amplified by PCR, cloned, and sequenced. The spacer sequence (455 bp) contains two tRNA (tRNA(ala) and tRNA(ile)) genes. Identical tRNA genes were also found in the 16S-23S spacer sequences of all the 51 strains of X. fastidiosa retrieved from the GenBank database. At this particular locus, the gene order of tRNA(ala) - tRNA(ile) is conserved among all the studied strains of Xylella and Xanthomonas, and different from those of other bacteria. Sequence analysis showed that Xanthomonas is the most closely related genus. Results from restriction endonuclease analysis suggested the presence of two rrn operons in the genome of a Xylella fastidiosa Pierce's disease strain.

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.

Genetic relationships among specimens of the 15 currently recognized species in Rhododendron L. section Pentanthera G. Don were derived from sequence comparisons of the internal transcribed spacer (ITS) region. Sequences of the entire ITS region including ITS1, ITS2, and the 5.8S subunit were generated by direct sequencing of polymerase chain reaction (PCR) amplified fragments. Rhododendron vaseyi A. Gray, Rhododendron section Rhodora (L.) G. Don was used as an outgroup. Aligned sequences of the 16 taxa resulted in 688 characters. The region contained 38 variable sites and eight phylogenetically informative characters. A bootstrap analysis was performed and a dendrogram was constructed with MEGA. Divergence values among the taxa were extremely low ranging from 0.00 to 3.51%, providing support to traditional views of section Pentanthera as a group of very closely related species.

The cultivated peanut, also known as groundnut, (Arachis hypogaea L.) is an important oilseed crop widely grown in the tropical, subtropical, and warmer temperate areas of the world. Peanuts are mainly utilized for human consumption and livestock feed, but they also provide important raw materials for edible oil, confectionery products, and high protein-content foods and meals (Bajaj 1984). In some countries, such as China and the USA, peanut is a major export commodity that generates annual revenues of hundreds of millions of dollars (Fletcher et al. 1992; Zhang et al. 1994).

The internal transcribed spacer regions (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA from the four recognized species of Citrullus (C. lanatus var. lanatus (Thunb.) Matsum. & Nakai., C. lanatus var. citroides (Bailey) Mansf., C. colocynthis (L.) Schrad., C. ecirrhosus Cogn., and C. rehmii De Winter) and Acanthosicycos naudinianus (Sond.) C. Jeffrey were amplified by PCR, and direct sequenced. Within the taxa examined, the length of ITS1 varied from 216 bp to 219 bp, and ITS2 varied from 239 bp to 249 bp. The average %CG content ranged from 59 to 64% and from 62 to 66% for ITS1 and ITS2, respectively. The greater length variation observed in ITS2 was primarily attributable to the occurrence of a (CC)n microsatellite. Cladistic (PAUP) and phenetic (MEGA) analyses resulted in highly resolutive trees. ITS sequence analysis placed the recently described C. rehmii adjacent to the cultivated watermelon and supported the validity of the species classification of this taxa.

-like sequences were PCR amplified from sweetpotato [Ipomoea batatas (L.) Poir.] L87-95 genomic DNA samples by using Ty1-copia reverse transcriptase-specific primers. PCR fragments within the expected size range were isolated, cloned, and sequenced. Inferred amino acid sequences of two randomly selected cloned fragments were found to be highly homologous to Ty1-copia-like reverse transcriptase sequences in the GenBank database. Subsequent sequencing of an additional 22 cloned fragments revealed a high level of reverse transcriptase sequence diversity (sequence divergence ranged from 2% to 73%). Southern blot hybridization analysis indicated that these sequences are present in the genome of I. batatas and taxonomic relatives in high copy numbers. PCR amplification from leaf cDNA obtained from a sweetpotato clone using Ty1-copia reverse transcriptase-specific primers yielded a Ty1-copia-like fragment. This is the first known report of the presence of genomic, and putatively expressed, Ty1-copia-like reverse transcriptase sequences in I. batatas.

The objectives of the present study were to evaluate the inheritance and nucleotide sequence profiles of microsatellite genetic markers in hexaploid sweetpotato [ Ipomoea batatas (L.) Lam.] and its putative tetraploid and diploid ancestors, and to test possible microsatellite mutation mechanisms in polyploids by direct sequencing of alleles. Sixty three microsatellite loci were isolated from genomic libraries of I. batatas and sequenced. PCR primers were designed and used to characterize microsatellite loci in two hexaploid I. batatas populations, a tetraploid Ipomoea trifida population, and a diploid I. trifida population. Nine out of the sixty three primer pairs tested yielded a clearly discernible, heritable banding pattern; five showed Mendelian segregation. All other primer pairs produced either smeared banding patterns, which could not be scored, or no bands at all in I. batatas. All of the primers which produced discernible banding patterns from I. batatas also amplified products of similar size in tetraploid and diploid I. trifida accessions. The sequence analysis of several alleles in the three species showed differences due to mutations in the repeat regions consistent with small differences in the repeat number. However, in some cases insertions/deletions and base substitutions in the microsatellite flanking regions were responsible for polymorphisms in both polyploid and diploid species. These results provide strong empirical evidence that complex genetic mechanisms are responsible for SSR allelic variation in Ipomoea. Four I. batatas microsatellite loci showed polysomic segregation fitting tetraploid segregation ratios. To our knowledge this is the first report of segregation ratios for microsatellites markers in polyploids.

Progress in the breeding of plantain and banana has been restricted by the complex genetic structure and behaviour of cultivated polyploid Musa. Genetic improvement has been hindered due to the large amount of space required for growth and maintenance of plant populations, in addition to the long growth cycle and the low levels of fertility and seed viability characteristic of cultivated genotypes. Molecular marker assisted breeding has the potential to dramatically enhance the pace and efficiency of genetic improvement in Musa. This study was conducted to compare different PCR-based marker systems (RAPD, VNTR and AFLP) for the analysis of breeding populations generated from two diverse Musa breeding schemes. All three assays detected a high level of polymorphism between parental genotypes and within progeny populations. As expected, AFLP assays had by far the highest multiplex ratio while VNTR analysis detected the highest levels of polymorphism. AFLP analysis of a full-sib tetraploid hybrid population confirmed previous reports based on VNTR analysis, of a high frequency of recombination during 2n (3x) gamete formation by a triploid plantain landrace. In addition, both VNTR and RAPD analyses of a full-sib triploid hybrid population suggested a high frequency of homoeologous recombination during n (2x) gamete formation by tetraploid hybrids. In general, there was a poor correlation between estimates of genetic similarity based on different types of marker. The implications of these findings for the molecular breeding of Musa crops are discussed.

Rhododendron section Pentanthera has traditionally been viewed as a group of closely related species due to the relative lack of distinctive morphological characteristics separating species and numerous reports in the literature of artificial and natural fertile hybrids that produce fertile progeny. Significant variation within species has created difficulties in efforts to assemble these taxa into well-defined groups. Genetic relationships among cultivated specimens of the 15 currently recognized species in Rhododendron section Pentanthera were derived from sequence comparisons of the internal transcribed spacer (ITS) region. Sequences of the entire ITS region including ITS1, ITS2, and the 5.8S subunit were generated by direct sequencing of polymerase chain reaction (PCR) amplified fragments. Rhododendron vaseyi A.Gray, Rhododendron section Rhodora (L.) G.Don was used as an outgroup. Aligned sequences of the 16 taxa resulted in 690 characters. A distance matrix of sequence divergence was calculated using Kimura's two parameter model. A bootstrap analysis was performed and a phenogram was constructed using MEGA. A phenetic assay rather than a phylogenetic analysis was performed because the ITS region contained only eight (1.16%) phylogenetically informative sites. The entire ITS region contained 41 variable sites (5.94%). Five species had identical ITS sequences. Seven additional species differed only by one or two base pair substitutions and/or the addition or deletion of one or two base pairs. R. luteum Sweet, R. occidentale A. Gray, R. molle (Blume) G.Don, and the outgroup, R. vaseyi had noticeable variation (base substitutions, additions, and deletions) compared to the other species. Divergence values among the taxa were extremely low, ranging from 0.00% to 3.51%. This molecular data, and previous hybridization studies, do not support the accepted taxonomic treatment of the section.

The triploid genome of plantain (Musa spp., AA group) has been considered intractable to genetic improvement because of the production of putatively homogeneous 2n (= 3x) gametes. Plantain breeding schemes have been based on the hybridization of 2n megaspores from the triploid plantain and haploid microspores from a wild diploid banana. The resultant fuli‐sib populations of tetraploid hybrids exhibited extreme variation in phenotypic characteristics. This study was conducted to analyze the genetic constitution of tetraploid hybrids in order to characterize the nature of the gametes from which they were derived. Parental genotypes were screened with 31 primer pairs specific to different Musa microsatellite loci. Primers which detected polymorphisms between the parental genotypes were used to screen a population of 14 full‐sib tetraploid hybrids. A subset of primers was also used to screen a population generated by self‐pollination of the parental diploid banana. During analysis of the tetraploid hybrid population, 75% of the alleles donated by the maternal genotype (Obino l'Ewai) were observed to segregate. These data demonstrate the occurrence of recombination during the formation of 2n megaspores in triploid plantain. Analysis of the paternal diploid banana accession (Calcutta 4) and its selfed progeny suggests that this accession is highly heterozygous. These data demonstrate the importance of genetic characterization to Musa breeding, and also indicate that microsatellite markers are well suited for marker‐assisted selection systems in Musa.

Pierce's disease (PD) strains of Xylella fastidiosa were identified by random amplified polymorphic DNA (RAPD) fingerprinting. Two random primers including OPA-03 (agtcagccac) and OPA-11 (caatcgccgt) were found to be efficient for differentiating PD strains isolated from a vineyard in North Florida in 1996 (129 strains) and 1997 (29 strains) from non-PD strains of X. fastidiosa (citrus variegated chlorosis, mulberry leaf scorch, periwinkle wilt, plum leaf scald, and phony peach) and strains from Xanthomonas campestris pv. vesicatoria and Escherichia coli. This study shows that RAPD fingerprinting is a useful tool to supplement the conventional symptoms-colony morphology-slow growth identification procedure routinely used to identify the PD pathogen.

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (‘Coker 797’ * 4 / ‘Hamlet’) and differed by the presence or absence of H21 (on 2RL) derived from ‘Chaupon’ rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of 'streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

Plant growth retardants were evaluated for their ability to reduce the growth rate of sweetpotato (Ipomoea batatas (L.) Lam.) in vitro. Nodal sections of cv. Jewel were cultured for 30 days on medium containing NDA, ancymidol, phosfon, TIBA, difenzoquat, chlormequat, ACC, mepiquat chloride, or daminozide at 0, 10−4, 10−5, 10−6, 10−7, or 10−8m. Difenzoquat, NDA, phosfon, and TIBA, at 10−4m, were lethal to axillary bud explants. A low concentration (10−8m) of chlorflurenol or NDA stimulated shoot elongation. The effective concentration range for most growth retardants was 10−5 to 10−6m. Small (2- to 4-mm diameter) storage root-like swellings were observed on roots in cultures containing TIBA or ancymidol. The growth-inhibiting effects of ancymidol and NDA were transitory and did not persist through a 180-day culture period. Shoots cultured on medium containing 10−5m phosfon, TIBA, or difenzoquat were significantly shorter than control plants after a 180-day culture period. Culture on medium containing TIBA, NDA, ancymidol, or ACC resulted in abnormal leaf and stem development. Plants derived from nodal explants cultured on medium containing either phosfon or chlormequat were near normal in appearance but with some plants exhibiting interveinal chlorosis and reduced root system development.