Detection and Classification of SPLCV Isolates in the U.S. Sweetpotato Germplasm Collection via a Real-Time PCR Assay and Phylogenetic Analysis.

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.

16S rDNA sequence analysis of Xylella fastidiosa strains.

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.

Detection of Ty1-copia-like reverse transcriptase sequences in Ipomoea batatas (L.) Poir.

-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.

Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation.

To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the ß-glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.

Chloroplast DNA restriction fragment length polymorphisms (RFLPs) in Musa species.

Taxonomic and phylogenetic determinations within the genus Musa are established using a numerical, morphology-based scoring system. However, within this system, the classification and relationships of some types are disputed. The application of chloroplast DNA (cpDNA) restriction fragment length polymorphism (RFLP) analysis to Musa taxonomy provided valuable, supplemental information about the classification of, and relationships between, Musa species and subspecies. Whole-cell DNA was extracted from lyophilized Musa leaf-blade tissue and digested with various restriction enzymes, Southern blotted onto nylon membranes, and probed using radioactively labeled heterologous orchid cpDNA fragments. Phylogenies were inferred from cpDNA RFLP patterns using PAUP software. The relationships between most species examined were as expected; however, some species (M. beccarii and M. basjoo) did not conform to the conventional morphology-based phylogeny.

Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tips by vitrification.

Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (-196°C) exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.

Analysis of genetic diversity in a sweetpotato (Ipomoea batatas) germplasm collection using DNA amplification fingerprinting.

A DNA amplification fingerprinting (DAF) approach was employed to develop individual-specific profiles and analyze genetic relationships among 73 plant introductions of sweetpotato (Ipomoea batatas (L.) Lam.) including unadapted lines from around the world and a few selected U.S.A. cultivars. Reliable and informative fingerprint profiles were obtained employing single octamer primers and Stoffel fragment Taq polymerase in the polymerase chain reaction, polyacrylamide-based vinyl polymer for electrophoresis, and silver staining to visualize the DNA. Using seven highly informative octamer primers, individual-specific DAF profiles were obtained for all accessions tested. The degree of polymorphism in the sweetpotato collection was very large, indicating a high level of genetic variability. Several accessions clustered together based on their geographic source. Most U.S.A. cultivars formed a separate cluster in the phenogram, while accessions from Papua New Guinea exhibited the highest genetic diversity. The wild species I. triloba and tetraploid I. batatas formed a group distinct from the cultivated sweetpotato. DAF appears to be useful in sweetpotato germplasm characterization and may be employed to identify duplicate accessions or for creation of core subsets. DAF data may also be useful for facilitating the selection of parents for a breeding program to ensure a broad genetic base.

RFLP-based phylogeny of Musa species in Papua New Guinea.

Random genomic probes were used to detect restriction fragment length polymorphisms (RFLPs) in 26 accessions of Musa representing eight species from Papua New Guinea (PNG), M. textilis, M. jackeyi and one accession of Ensete. Ninety-eight phylogenetically informative characters were scored and analyzed cladistically and phenetically. Results generally agreed with previous morphology-based phylogenetic analyses. However, the closest wild relative of the edible M. fehi (fe'i banana) appears to be M. lolodensis. Musa angustigemma is sister species with M. boman and M. jackeyi and is distinct from M. peekelii, with which it is often united. Musa boman is unambiguously placed in section Australimusa. The diploid parthenocarpic landraces of section Musa unique to PNG are closely related to, but apparently distinct from, M. acuminata ssp. banksii. The evolution of the fe'i bananas and the M. acuminata-derived diploid landraces of PNG are discussed.

Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa.

Random genomic probes were used to detect RFLPs in 19 Musa species and subspecies. A total of 89 phylogenetically informative alleles were scored and analyzed cladistically and phenetically. Results were in general agreement with morphology-based phylogenetic analyses, with the following exceptions: our data unambiguously places M. boman in section Australimusa, and indicates M. beccarii is very closely related to M. acuminata. Additionally, no support was found for the separation of section Rhodochlamys from section Musa. A comparison of morphology-based and RFLP-based phylogenetic analyses is presented.

DNA profiling of banana and plantain cultivars using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers.

Polymerase chain reaction (PCR) amplification of genomic DNA from 57 Musa cultivars with 60 random 10-mer primers generated 605 polymorphic amplification products which were useful in unambiguous cultivar identifications. Unweighted pair-group method analysis of this data grouped the cultivars into specific clusters depending on their genomic similarities. The diploid ancestral species of cultivated banana and plantains, namely Musa acuminata sp malaccensis, an A genome donor and M. balbisiana, a B genome donor, were farthest apart from each other in the phenogram. The edible fruit yielding cultivars with the genomic constitutions AA, AAA, AB, AAB, ABB, and ABBB grouped in different clusters according to overall genetic homologies. The restriction fragment length polymorphisms (RFLPs) prevalent among the cultivars were studied by hybridization of 19 random genomic clones to blots of HindIII, EcoRI and MspI digests. Cluster analysis of these data on 107 polymorphic alleles resulted in a phenogram comparable to the one obtained with random amplified polymorphic DNA (RAPD) analysis. Two multilocus probes useful in distinguishing all the 57 cultivars analyzed were also identified. The A and B types of cytoplasms in the cultivars were further distinguished by hybridization of heterologous chloroplast DNA probes. Results showed that use of different kinds of molecular markers in gene banks is essential for characterization and classification of germplasm collections.

Inheritance of egusi seed type in watermelon.

An unusual seed mutant in watermelon (Citrullus lanatus var. lanatus) has seeds with a fleshy pericarp, commonly called egusi seeds. The origin of the phenotype is unknown, but it is widely cultivated in Nigeria for the high protein and carbohydrate content of the edible seeds. Egusi seeds have a thick, fleshy pericarp that appears during the second to third week of fruit development. We studied the inheritance of this phenotype in crosses of normal seeded Charleston Gray and Calhoun Gray with two plant introduction accessions, PI 490383w and PI 560006, having the egusi seed type. We found that the egusi seed type is controlled by a single recessive gene, and the symbol eg was assigned.

Evidence for conserved tRNA genes in the 16S-23S rDNA spacer sequence and two rrn operons of Xylella fastidiosa.

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.

Improved electroporation buffer enhances transient gene expression in Arachis hypogaea protoplasts.

An electroporation medium containing 50 mM glycine or 10 mM glycylglycine (glygly), 70 mM potassium glutamate, and 0.4 M mannitol was evaluated for its ability to improve transient β-glucuronidase (GUS) expression in immature cotyledonary protoplasts of Arachis hypogaea L. GUS activity in electroporated protoplasts was 8- to 430-fold greater than that obtained using any of other four commonly employed poration media. Analysis of viability and histochemical staining of protoplasts indicated that electroporation using the glycine- or glygly-based poration medium resulted in increased protoplast viability and GUS expression when compared with other poration media. Replacement of glygly with MES or HEPES buffers significantly reduced the level of GUS expression in electroporated protoplasts.

Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz).

A size-fractionated TaqI genomic library of seashore paspalum (Paspalum vaginatum Swartz) was screened for the presence of (GA) n and (CA) n simple sequence repeats (SSRs). A total of 54 clones with a positive signal were detected among 13,000 clones screened. Forty-seven clones having repeats of n[Symbol: see text] 3 were identified, of which 85% were perfect, 13% were imperfect and 2% were compound repeat sequences. Five of ten primer pairs synthesized to amplify selected loci resulted in a product in the expected size range and were subsequently used to examine SSR polymorphisms among 46 ecotypes of P. vaginatum. The number of alleles resolved on agarose or polyacrylamide gels were similar and ranged from 6 to 16 with an average of 14 per locus. Phenetic analysis of SSR polymorphisms revealed genetic relationships among the P. vaginatum ecotypes that were in general agreement with relationships determined previously by RAPD analysis of the same plant materials. Further screening of the genomic library did not identify (AT) n , trimeric or tetrameric repeats. Hybridization of an (ATT)8 oligonucleotide probe to genomic DNA isolated from I. batatas, E. coli, Citrullis lanatus and P. vaginatum suggested that the P. vaginatum genome contained significantly fewer ATT repeats than either the I. batatas or C. lanatus genome.

Phylogenetic relationships of the pigeonpea (Cajanus cajan) based on nuclear restriction fragment length polymorphisms.

Nuclear restriction fragment length polymorphisms (RFLPs) were used to determine phylogenetic relationships in the genus Cajanus using 15 random genomic probes and six restriction enzymes. Twenty-four accessions representing 12 species of four genera (Cajanus, Dunbaria, Eriosema, and Rhynchosia) were examined to determine phylogenetic relationships in the genus Cajanus. Eriosema parviflorum was selected as the out-group. Sufficient RFLP polymorphisms were detected among species to resolve in-group taxa into distinct clusters. Topologies of trees from parsimony and similarity matrix analyses were similar but not identical, and clustering patterns agreed broadly with published phylogenies based on seed protein data and, to a lesser extent, data from cytology and breeding experiments. Accessions of cultivated C. cajan shared more DNA fragments with C. scarabaeoides than with C. cajanifolia. Inconsistencies in taxonomic relationships based on data from morphology, cytology, crossability, and RFLPs are discussed.

Genetic relationships and variation among ecotypes of seashore paspalum (Paspalum vaginatum) determined by random amplified polymorphic DNA markers.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic relationships and variation among ecotypes of the turfgrass seashore paspalum (Paspalum vaginatum Swartz). Vegetative tissues or seeds of 46 seashore paspalum ecotypes were obtained from various locations in the United States, Argentina, and South Africa. Leaf DNA extracts were screened for RAPD markers using 34 10-mer random primers. A total of 195 reproducible RAPD fragments were observed, with an average of six fragments per primer. One hundred and sixty-nine fragments (87% of the total observed) were polymorphic, among which 27 fragments (16%) were present in three or less ecotypes, indicating the occurrence of a high level of genetic variation among the examined accessions of this species. Cluster analysis (UPGMA) and principal coordinates analysis were performed on the RAPD data set. The results illustrate genetic relationships among the 46 ecotypes, and between ecotypes and their geographical origins. Ecotypes from southern Africa could be differentiated from the U.S. and most of the Argentinean ecotypes. With a few exceptions, ecotypes collected from Argentina, Hawaii, Florida, and Texas were separated into distinct clusters.

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens.

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t1 seed by T0 plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 3∶1 Mendelian ratio in most T1 generation plants.

Use of 16S rDNA sequences as signature characters to identify Xylella fastidiosa.

The nucleotide sequences of 16S rDNAs (coding for the small subunit ribosomal RNAs) were used to identify Xylella fastidiosa, a nutritionally fastidious plant pathogenic bacterium. The near-complete 16S rDNAs from nine strains of Xyl. fastidiosa, including seven pathotypes and one strain of Xanthomonas campestris pv. campestris, were amplified through PCR with two conserved primers (forward primer 5'-AGA GTT TGA TCC TGG CTC AG-3' and reverse primer 5'-AAG GAG GTG ATC CAG CC-3') and sequenced. The 16S sequences were compared with all eukaryote and prokaryote DNA entries in GenBank database. A Xyl. fastidiosa 16S rDNA sequence, M26601, was determined to be the most similar to all the near-complete (1537 bp) and partial 5' end sequences from Xyl. fastidiosa, but not those from the Xanthomonas strain. A 20-bp oligonucleotide (5'-TTG GTA GTA ATA CCA TGG GT-3') was found to be highly characteristic of Xyl. fastidiosa. Since the 16S rDNA of Xyl. fastidiosa strains are highly homologous and characteristically different from other bacteria, including the most closely related Xanthomonas, 16S rDNA sequences can be used as signature characters to identify this bacterium.

Simple sequence repeats in watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai).

Simple sequence repeat length polymorphisms were utilized to examine genetic relatedness among accessions of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai). A size-fractionated TaqI genomic library was screened for the occurrence of dimer and trimer simple sequence repeats (SSRs). A total of 96 (0.53%) SSR-bearing clones were identified and the inserts from 50 of these were sequenced. The dinucleotide repeats (CT)n and (GA)n accounted for 82% of the SSRs sequenced. PCR primer pairs flanking seven SSR loci were used to amplify SSRs from 32 morphologically variable watermelon genotypes from Africa, Europe, Asia, and Mexico and a single accession of Citrullus colocynthis from Chad. Cluster analysis of SSR length polymorphisms delineated 4 groups at the 25% level of genetic similarity. The largest group contained C. lanatus var. lanatus accessions. The second largest group contained only wild and cultivated "citron"-type or C. lanatus var. citroides accessions. The third group contained an accession tentatively identified as C. lanatus var. lanatus but which perhaps is a hybrid between C. lanatus var. lanatus and C. lanatus var. citroides. The fourth group consisted of a single accession identified as C. colocynthis. "Egusi"-type watermelons from Nigeria grouped with C. lanatus var. lanatus. The use of SSRs for watermelon germplasm characterization and genetic diversity studies is discussed.

Specific detection of Xylella fastidiosa Pierce's disease strains.

Pierce's disease (PD, Xylella fastidiosa) of grapevine is the primary pathogen limiting vinifera grape production in Florida and other regions of the southeastern United States. Quick and accurate detection of PD strains is essential for PD studies and control. A unique random amplified polymorphic DNA (PD1-1-2) was isolated from a PD strain from Florida. Fragment PD1-1-2 was cloned, sequenced, and found to be 1005 bp in length. PCR primers were designed to utilize these sequence data for PD strain detection. One primer set (XF176f-XF954r) amplified a 779-bp DNA fragment from 34 PD strains including seven pathotypes of X. fastidiosa, but not from strains of Xanthomonas campestris pv. campestris, Xan. vesicatoria or Escherichia coli. A second primer set (XF176f and XF686r) amplified a 511-bp fragment specific to 98 PD strains, but not from strains of citrus variegated chlorosis, mulberry leaf scorch, oak leaf scorch, periwinkle wilt, phony peach, or plum leaf scald. Sequence analysis indicated that RAPD fragment PD1-1-2 contains a Ser-tRNA gene. The PD-specific region includes a TaqI restriction site (TCGA) and is 150 bp downstream of the Ser-tRNA gene.