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The phylogenetic relationships of Dioscorea, including sections Botryosicyos, Combilium, Enantiophyllum, Lasiophyton, Opsophyton, Shannicorea and Stenophora, are reconstructed with chloroplast trnL-F, matK, rbcL and atpB-rbcL sequence data, covering a total of 72 accessions including 48 ingroup species and five outgroup species from East and Southeast Asia. The seven Asian sections do reflect the genetic relationships among the species that they include. In summary, D. sect. Combilium and sect. Shannicorea are recognized as closely related sections with moderate support. The results also support Burkills treatment of subsect. Euopsophyton (D. bulbifera) as sect. Opsophyton and subsect. Macrourae (D. sansibarensis) as sect. Macroura. In addition, there is a well-supported sister relationship between D. sect. Lasiophyton and sect. Botryosicyos. The level of morphological characters and molecular divergence within D. sect. Enantiophyllum is low, but the members of this group could still be distinguished from each other.

KEY MESSAGE: miR319 was identified as a dwarf-inducing gene from Shiokari and its dwarf near isogenic line, and its transgenic rice showed a reduced plant height. This finding reveals the potential application of miR319 in future molecular breeding. It is well known that microRNAs (miRNAs) play important roles in plant physiology, especially in development and stress responses. However, little is known about the role of miRNAs in plant height. In this study, the rice cultivar Shiokari and its dwarf near isogenic line Shiokari-d6 were analysed to identify and characterize plant height-associated miRNAs. This anatomic and morphological investigation revealed that the major cause of the shorter height of Shiokari-d6 is the significantly dis-elongated internodes, particularly the second internode and those underneath it. The results of miRNA microarray profiling and real-time RT-PCR indicated that miR319 is expressed at a significantly higher level in Shiokari-d6 than in Shiokari. Transgenic rice overexpressing miR319 in Oryza sativa L. cv. Tainung 67 generated through Agrobacterium-mediated transformation had a stable dwarf phenotype regardless of whether the plants were from the T1 or T2 generation. We also found that the internodes of miR319-overexpressing rice are shortened, particularly the third internode and those underneath it. Furthermore, we identified three putative miR319 target genes that were previously uncharacterized with expression levels that were negatively correlated with the expression of miR319. In conclusion, miR319 is the first miRNA proposed to be involved in plant height regulation, and its function may influence the elongation of internodes, which leads to decreased plant height.

Mungbean yellow mosaic Indian virus (MYMIV) and bruchid infestation are severe production constraints of mungbean in South Asia, a major global mungbean production area. Marker-assisted selection for resistance against these disorders while maintaining or even improving agronomic traits is an important step toward breeding elite mungbean varieties. This study employed recombinant inbred lines (F12) derived from a cross between MYMIV-tolerant Vigna radiata NM92 and bruchid-resistant V. radiata ssp. sublobata TC1966 to identify chromosomal locations associated with disease and insect pest resistance and seed traits. A linkage map comprising 11 linkage groups was constructed with random amplified polymorphic DNA (RAPD), sequence characterized amplified regions (SCAR), cleaved amplified polymorphic DNA (CAP), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. Quantitative trait loci (QTLs) for MYMIV and bruchid resistance, 100 seed weight and seed germination rate were identified. Three major QTLs for MYMIV and one major bruchid resistance locus were mapped on LG 9. The resistance alleles were contributed by the MYMIV tolerant parent NM92 and the bruchid resistant parent TC1966 respectively. One of the MYMIV QTLs was tightly linked in repulsion phase to the bruchid resistance locus. In addition, three minor QTLs for MYMIV resistance were found, where the resistance alleles were contributed by TC1966. Lines combining MYMV resistance alleles from both parents have greater resistance to MYMIV than the tolerant parent. Two minor bruchid resistance QTLs were identified in TC1966. Furthermore, three QTLs each for 100 seed weight and germination rate were detected. The markers defining the QTLs identified in this study will be useful in marker-assisted breeding of improved mungbean varieties in the future.

This study was undertaken to establish for the first time an efficient regeneration and transformation system for Cucumis metuliferus line PI292190, which is the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic virus 1, WMV-1). Different combinations of growth regulators were evaluated for the regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal culture conditions, a total of 7 to 10 weeks was necessary to obtain C. metuliferus plantlets from cotyledons. Furthermore, transgenic plants were successfully obtained using an Agrobacterium tumefaciens-mediated transformation method as shown by polymerase chain reaction analysis and histochemical β-glucuronidase (GUS) assay. A total of nine transgenic plants were developed from 360 cotyledon explants, giving a transformation frequency of 2.5%.

Pear plants (Pyrus pyrifolia var. Hengshen) showing symptoms of chlorotic spots on leaves were observed in orchards in central Taiwan in 2004. The sap of diseased leaves reacted positively to Apple chlorotic leaf spot virus (ACLSV) antiserum. A purified virus isolate (LTS1) from pear was characterized by host range, electron microscopy, phylogenetic analyses, serological property, and back-inoculation experiments to pear. Fifteen of 28 species of tested plants were susceptible to this virus after mechanical inoculation. Pathogenicity of ACLSV isolate LTS1 was verified by back-inoculating to pear seedlings. Filamentous virions of ≈12 × 750 nm were observed in the preparations of purified virus. Virus particles accumulated in the cytoplasm were observed in the ultrathin sections of LTS1-infected pear leaf tissue. Sequence analyses of the coat protein (CP) gene of LTS1 and the CP gene of LTS2, which originated from a distinct symptomatic pear sample, shared 81.4% to 92.6% nucleotide and 87.6% to 98.4% amino acid identities with those of the CP of 35 ACLSV isolates available in GenBank. ACLSV isolates were grouped into four clusters, i.e., Asia I, II, III, and Europe, and isolates LTS1 and LTS2 were classified as members of cluster Asia II and Asia I, respectively, based on phylogenetic data. Moreover, the variability of amino acid sequences of the CP gene of 37 ACLSV isolates showed geographically associated clustering in the phylogenetic tree. To our knowledge, this is the first study on the characterization of ACLSV causing the leaf chlorotic spot disease of pear in Taiwan. This study also provides the phylogenetic relationships among ACLSV populations based on amino acid sequences of CPs, which are correlated with their geographic origins.

Simple sequence repeat (SSR) markers are widely applied in studies of plant molecular genetics due to their abundance in the genome, codominant nature, high repeatability, and transferability in cross-species applications. To investigate the possibility of applying rice SSR markers in bamboo, we selected 120 rice SSR markers that are evenly distributed on rice chromosomes and assessed these for their transferability to 21 different bamboo species. A total of 4847 bands of 2196 alleles were obtained from 82 SSR markers that were able to amplify products in the bamboo genome; the transferability was 68.3%. Seven markers specifically amplified individual bamboo species and are consequently valuable markers for species identification. SSR markers located on rice chromosome 7 and 1 showed the highest and lowest transferability, respectively to the bamboo genome. SSR markers located on some regions of the rice chromosomes could not be amplified in bamboo, suggesting that regional divergence occurred between rice and bamboo during evolution. A dendrogram was constructed. The dendrogram classified bamboo species into two major groups which coincided with rhizome type, runner, and clumper. The results of this study demonstrate that rice SSR markers can be a valuable source of markers for those genomes lacking useful marker systems.

Bruchid, Callosobruchus spp. (Coleoptera: Bruchidae), is a serious pest during storage of seeds of mungbean (Vigna radiata (L.) Wilczek) and other Vigna species. A source of resistance to this pest has been identified in Vigna sublobata (Roxb.) Bairig. accession TC1966. Two hundred recombinant inbred lines at the F12 generation have been developed for molecular mapping of bruchid resistance (Br) gene in TC1966. Through bulked segregant analysis (BSA), ten randomly amplified polymorphic DNA (RAPD) markers associated with the bruchid resistance gene were successfully identified. A total of four closely linked RAPDs were cloned and transformed into sequence characterized amplified region (SCAR) and cleaved amplified polymorphism (CAP) markers. Seven CAPs developed from the identified RAPD markers showed tighter linkage with the Br gene than the original RAPD. Through transformation of RAPDs into CAPs, codominant markers for bruchid resistance were successfully obtained. Homozygous genotypes of these PCR-based markers were estimated to contribute 85% of the variance for seed damage when the insect assay was performed under favorable growth conditions for bruchid.

Molecular-marker analysis of a cross between yellow pear, a tomato variety bearing small, pear-shaped fruit, and the round-fruited, wild species, Lycopersicon pimpinellifolium LA1589, revealed that pear-shaped fruit is determined largely by a major QTL on chromosome 2 and, to a lesser extent, a minor QTL on chromosome 10. The locus on chromosome 2 was also detected in a cross between yellow pear and the round-fruited introgression line (IL2–5) which carried the distal portion of chromosome 2 from the Lycopersicon pennellii genome. Based on its map position, we propose that the locus detected on chromosome 2 is the same as a locus referred to as ovate in the early tomato literature (Linstrom 1926, 1927). The fruit-shape index (length/diameter) and neck constriction were highly correlated in both populations suggesting that ovate exerts control over both traits or that the genes for these traits are tightly linked on chromosome 2. Using two-way ANOVA test, the minor QTL on chromosome 10 showed no significant interaction with the ovate locus on chromosome 2 with respect to the fruit-shape index. For ovate round fruit was dominant to elongated fruit in the L. pimpinellifolium populations, but additive in the IL2–5 population. Thus far, no genes controlling fruit shape have been cloned. The molecular mapping of the ovate locus may ultimately lead to its isolation via map-based cloning.

fs8.1 is a major fruit-shape QTL differentiating fresh-market and processing tomatoes. Mature fruits from plants with the wild-type fresh-market alleles are round, whereas those with alleles from processing variety E6203 are elongated (sometimes referred to as blocky or square tomatoes). Fine mapping was undertaken en to determine whether the effect is due to a single gene or several tightly linked genes. RAPD and RFLP linkage analysis, and substitution mapping of nearly isogenic lines (NILs) segregating for the 22.8 cM-TG176-CT92 interval at the top of chromosome 8 in tomato were used for high-resolution mapping. For the 1212 gametes screened in F-2 and F-3 families, it was determined that fs8.1 maps as a single locus near the centromere of chromosome 8. A comparative developmental study of fs8.1 NILs revealed that fs8.1 alleles exert their effects on fruit shape early in carpel development at least 6 days before anthesis. Field evaluations of the NILs indicate that fs8.1 affects not only fruit shape, fruit length, and fruit weight but also the number of flowers and fruits per inflorescence, and the harvest index. The date of first flower and fruit diameter were not significantly affected.

Pineapple (Ananas comosus) is one of the major fruit crops in Taiwan, accounting for 275 million U.S. dollars in 2006, following betel nut and citrus production in crop value. Tainung No. 17 is the most important cultivar, accounting for more than 70% of pineapples planted. Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple. Pineapple mealybug wilt-associated virus-1 (PMWaV-1), PMWaV-2, and PMWaV-3 were identified as three distinct species in Ampelovirus from diseased Hawaiian pineapple (1,2). In November of 2007, pineapples (cv. Tainung No. 17) planted in Pingtung County of southern Taiwan showed symptoms similar to MWP. Mealybugs (Dysmicoccus brevipes) were also found. Three primer pairs, 225/226, 223/224, and 263/264 described previously specific for the HSP70h genes of PMWaV-1 (1), -2, and -3 (2), respectively, were used to detect the presence of these three viruses by reverse transcription (RT)-PCR. Expected DNA fragments of 590, 610, and 499 nt were obtained from the total RNA isolated from the leaves of diseased pineapples with primer pairs 225/226, 223/224, and 263/264, respectively. The RT-PCR amplified fragments were cloned, sequenced, and analyzed. The 590-nt fragment (Accession No. EU769113) shared 91.6 to 99.5% nucleotide and 96.8 to 99.5% amino acid identity to those of five isolates of PMWaV-1 available in the GenBank; one each from Hawaii (Accession No. AF414119) and Thailand (Accession No. EF620774) and three from Australia (Accession Nos. EF488752, EF467923, and EF467925). The 610-nt fragment (Accession No. EU769115) showed 98.7 and 99.7% nucleotide and 98% and 100% amino acid identity to those of PMWaV-2 from Hawaii (Accession No. AF283103) and Thailand (Accession No. EU016675), respectively. The 499-nt fragment (Accession No. FJ209047) shared 86.8 to 99.0% nucleotide and 94.0 to 100.0% amino acid identity to those of five PMWaV-3 isolates available in the GenBank; one from Hawaii (Accession No. DQ399259) and four from Australia (Accession Nos. EF467918, EF467919, EF488754, and EF488755). Using primer pairs FJ08-1 (5′-ATGGCTGATTCGAGC)/FJ08-2 (5′-TTATTTGCGTCCACC), FJ08-7 (5′-AGTGAGATTGATCGT)/FJ08-8 (5′-TGCAGGTATCCGCTG), and FJ08-35 (5′-AACGACCGAACTCGC)/FJ08-36 (5′-ATACTACAGATATTG) specific to the coat protein (CP) genes of PMWaV-1, -2, and -3, respectively, expected DNA fragments of 774, 909, and 789 nt were amplified by RT-PCR. The 774-nt CP gene of PMWaV-1 (Accession No. EU769114) shared 99% nucleotide and 98.4% amino acid identity to those of Hawaiian isolate (Accession No. AF414119). The 909-nt CP gene of PMWaV-2 (Accession No. EU769116) shared 99.0 and 99.1% nucleotide identity with isolates from Hawaii (Accession No. AF283103) and Cuba (Accession No. DQ225114), respectively, and 99.3% amino acid identity with both. The 789-nt CP gene of PMWaV-3 (Accession No. FJ209048) shared 99.1% nucleotide and 98.1% amino acid identity to those of the Hawaiian isolate (Accession No. DQ399259). One to two viruses among PMWaV-1, -2, and -3 were detected in all 40 samples collected from diseased pineapples. To our knowledge, this is the first report to identify three PMWaVs in the most important and widely planted pineapple cultivar in Taiwan, Tainung No. 17, by molecular characterization of the HSP70h and CP genes. References: (1) D. M. Sether et al. Plant Dis. 85:856, 2001. (2) D. M. Sether et al. Plant Dis. 89:450, 2005.