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Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV‐OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV‐CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium‐mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA‐B in ToLCNDV‐OM. However, the nuclear shuttle protein of DNA‐B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV‐OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV‐OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV‐CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility., Begomoviruses are usually transmitted by whiteflies and rarely by mechanical inoculation. The movement protein of tomato leaf curl New Delhi virus is identified as a determinant of mechanical transmissibility.

Sex form is one of the most important characteristics in papaya cultivation in which hermaphrodite is the preferable form. Self-pollination of H*-TSS No.7, an inbred line derived from a rare X chromosome mutant SR*, produced all-hermaphrodite progeny. The recessive lethal allele controlling the all-hermaphrodite phenomenon was proposed to be the recessive Germination suppressor (gs) locus. This study employed next-generation sequencing technology and genome comparison to identify the candidate Gs gene. One specific gene, monodehydroascorbate reductase 4 (MDAR4) harboring a unique polymorphic 3 bp deletion in H*-TSS No.7 was identified. The function of MDAR4 is known to be involved in the hydrogen peroxide (H2O2) scavenging pathway and is associated with seed germination. Furthermore, MDAR4 showed higher expression in the imbibed seeds than that in the dry seeds indicating its potential role in the seed germination. Perhaps this is the very first report providing the evidences that MDAR4 is the candidate of Gs locus in H*-TSS No.7. In addition, Gs allele-specific markers were developed which would be facilitated for breeding all-hermaphrodite lines.

Vegetable crops of the genus Cucumis are very popular worldwide and have great market value. However, their fruit quality and yield are hindered by viral diseases. C. metuliferus is considered a wild species with resistance to viral diseases that is lacking in cultivated crops of the Cucumis genus, such as melon. The C. metuliferus line L37 shows extreme resistance against Papaya ringspot virus (PRSV-HA), whereas line L35 is a susceptible line. In this study, reciprocal grafting experiments between L35 and L37 were performed, and the PRSV-HA strain was pre-inoculated in the rootstock leaves. The results revealed that the resistance signal in the L37 rootstock could transmit and provide resistance to the L35 scion. Subsequently, double sandwich grafting was performed using the pre-inoculated L35 as the rootstock, which was then grafted onto the L37 intermediate and the L35 scion. The results showed that PRSV-HA RNA accumulated in the L35 rootstock leaf, petiole, and stem tissues, whereas PRSV-HA RNA accumulated in some intermediate and scion petiole and stem tissues. No HCPro RNA was detected in the L35 scion leaves. The results showed that the suppression of the virus occurred in the leaves, and the resistance effect spread from the rootstock in the scion direction. Hence, this study has demonstrated that RNA silencing of systemic signals is responsible for L37 resistance against PRSV. C. metuliferus L37 could provide a valuable resistance source for crops of the Cucumis species against viral diseases through grafting.

Background MiRNAs play essential roles in plant development and response to biotic and abiotic stresses through interaction with their target genes. The expression level of miRNAs shows great variations among different plant accessions, developmental stages, and tissues. Little is known about the content within the plant genome contributing to the variations in plants. This study aims to identify miRNA expression-related quantitative trait loci (miR-QTLs) in the maize genome. Results The miRNA expression level from next generation sequencing (NGS) small RNA libraries derived from mature leaf samples of the maize panel (200 maize lines) was estimated as phenotypes, and maize Hapmap v3.2.1 was chosen as the genotype for the genome-wide association study (GWAS). A total of four significant miR-eQTLs were identified contributing to miR156k-5p, miR159a-3p, miR390a-5p and miR396e-5p, and all of them are trans-eQTLs. In addition, a strong positive coexpression of miRNA was found among five miRNA families. Investigation of the effects of these miRNAs on the expression levels and target genes provided evidence that miRNAs control the expression of their targets by suppression and enhancement. Conclusions These identified significant miR-eQTLs contribute to the diversity of miRNA expression in the maize penal at the developmental stages of mature leaves in maize, and the positive and negative regulation between miRNA and its target genes has also been uncovered.

Sex form is one of the most important characters in papaya cultivation in which hermaphrodite is preferable sex form. However, sex determination is impossible until flowering. This study has developed SCAR markers derived from the SVP-like gene and can truly discriminate the three sex forms: female, male, and hermaphrodite papaya. Molecular markers were applied to study the mechanisms responsible for the all-hermaphrodite phenomenon in the self-pollinated progeny of “H*-TSS No.7,” an inbred line derived from a rare X chromosome mutant SR*. This study has provided evidence to support that “H*-TSS No.7” contained a recessive lethal allele, ml, on the X chromosome, and the X*X* homozygous genotype (ml/ml) was detected only in seeds but not in seedlings. Consequently, lost germination ability rather than seed abortion was responsible for missing female progeny in the selfed “H*-TSS No.7” population. The lethal effect in genotypes of YY, YYh, and YhYh papaya could be explained by at least one functional X chromosome being necessary for seed development. This study has demonstrated that “H*-TSS No.7” with a lethal allele (ml) on its X* chromosome is the cause of the defect of germinating ability in its homozygous female progeny.

Non-pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F1 hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non-pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non-pungent bell pepper line GC3 was conducted by introgression of S-type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC1F1 containing S-type cytoplasm and four lines from BC2F2 containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non-pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC1F1 and BC1F2 populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.

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.

Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO4 treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed proline catabolism gene, proline dehydrogenase (PDH). However, in the experiment with tobacco leaf discs floating on CuSO4 solutions, the excess copper decreased proline content and suppressed the expression of the P5CS, OAT and PDH genes. Therefore, proline accumulation in the potted tobacco plants treated with excess Cu treatment might not be the consequence of the increased copper content in tobacco leaves but rather by the accompanied decrease in water content and/or increased ABA content.

Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

Proline accumulation is responsible for stress adaptation in many plants. To distinguish the involvement of two proline synthetic pathways, the virus induced gene silencing (VIGS) system that silenced the expression of genes encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC:1.5.1.12) and ornithine-δ-aminotransferase (OAT; EC 2.6.1.13) was performed, separately or concomitantly, in four-week-old Nicotiana benthamiana. Leaf discs of VIGS-treated tobacco were subjected to the treatment of drought, abscisic acid (ABA), or polyethylene glycol (PEG). The treated leaf discs were then collected for the determination of mRNA, chlorophyll, proline and polyamine level. Under drought stress or PEG treatment, most proline accumulation was inhibited in P5CS-silenced plants and only a small portion was inhibited in OAT-silenced plants under drought stress and no inhibition was observed under PEG treatment. Under ABA treatment, proline accumulation was inhibited completely in P5CS-silenced plants but unaffected in OAT-silenced plants. The degradation of chlorophyll was enhanced in P5CS-silenced plants but retarded in OAT-silenced plants under PEG treatment. Under ABA treatment, the degradation of chlorophyll was unaffected in both P5CS-silenced and OAT-silenced plants. The increase of polyamine level was unaffected in P5CS-silenced plants but increased in OAT-silenced plants under PEG treatment. Under ABA treatment, the increase of polyamine level was unaffected in P5CS-silenced plants but the polyamine level was increased later in OAT-silenced plants. Therefore, P5CS plays a major role in proline accumulation under drought, PEG, or ABA treatment, while OAT plays a minor role in drought or PEG treatment and does not participate in ABA treatment. OAT appears to have a close relationship with the regulation of polyamine levels in PEG and ABA treatments.

Tomato-infecting begomoviruses, a member of whitefly-transmitted geminivirus, cause the most devastating virus disease complex of cultivated tomato crops in the tropical and subtropical regions. Numerous strategies have been used to engineer crops for their resistance to geminiviruses. However, nearly all have concentrated on engineering the replication-associated gene (Rep), but not on a comprehensive evaluation of the entire virus genome. In this study, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting begomovirus in Taiwan, was subjected to the investigation of the viral gene fragments conferring resistance to geminiviruses in transgenic plants. Ten transgenic constructs covering the entire ToLCTWV genome were fused to a silencer DNA, the middle half of N gene of Tomato spot wilt virus (TSWV), to induce gene silencing and these constructs were transformed into Nicotiana benthamiana plants. Two constructs derived from IRC1 (intergenic region flanked with 5' end Rep) and C2 (partial C2 ORF) were able to render resistance to ToLCTWV in transgenic N. benthamiana plants. Transgenic plants transformed with two other constructs, C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3' end of the C1 ORF), significantly delayed the symptom development. Detection of siRNA confirmed that the mechanism of resistance was via gene silencing. This study demonstrated for the first time the screening of the entire genome of a monopartite begomovirus to discover viral DNA fragments that might be suitable for conferring virus resistance, and which could be potential candidates for developing transgenic plants with durable and broad-spectrum resistance to a DNA virus via a gene silencing approach.

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.

Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato spotted wilt virus (TSWV) are two major tomato viruses that cause serious economic losses. In this study, a partial C2 gene from ToLCTWV and the middle half of the N gene of TSWV were fused as a chimeric transgene to develop multiple virus resistance in transgenic plants. This construct was introduced into Nicotiana benthamiana and tomato by Agrobacterium-mediated transformation. Several transgenic lines showed no symptom post agro-inoculation with ToLCTWV and displayed high resistance to TSWV. The detection of siRNAs indicated that the resistance was via RNA silencing. This study demonstrated that linkage of gene segments from two viruses with distinct genomic organization, one DNA and the other RNA, can confer multiple virus resistance in transgenic plants via gene silencing.

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.

Oriental melon plants, Cucumis melo var. makuwa cv. Silver Light, showing virus-induced symptoms of mosaic, leaf curl and puckering were observed in the fields of eastern Taiwan in 2007. A virus culture, designated as SL-1, isolated from the diseased melon was established in systemic host plants, Nicotiana benthamiana and oriental melon, by mechanical inoculation. SL-1 did not react to the antisera against common cucurbit-infecting RNA viruses. Viral DNAs extracted from the diseased plant were amplified with the degenerate primers for begomoviruses. The full-length genomic DNA-A and DNA-B of SL-1 were sequenced and found to be closest, with 97.7% and 90.6% nucleotide identity, respectively, to Tomato leaf curl New Delhi begomovirus (ToLCNDV) cucumber isolate from a group of cucurbit-infecting begomoviruses. The virus SL-1 was designated as ToLCNDV oriental melon isolate (ToLCNDV-OM). The pathogenicity of ToLCNDV-OM was confirmed by agroinfection. Progeny virus from the agroinfected N. benthamiana plants was able to infect oriental melon by mechanical inoculation and caused symptoms similar to the original diseased melon in the field. The ToLCNDV-OM also infected five other species of cucurbitaceous plants by mechanical inoculation. This is the first report of a new ToLCNDV isolate causing severe disease on oriental melon in Taiwan.

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS 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.

Although tomato has been the subject of extensive quantitative trait loci (QTLs) mapping experiments, most of this work has been conducted on transient populations (e.g., F2or backcross) and few homozygous, permanent mapping populations are available. To help remedy this situation, we have developed a set of inbred backcross lines (IBLs) from the interspecific cross between Lycopersicon esculentum cv. E6203 and L. pimpinellifolium (LA1589). A total of 170 BC2F1plants were selfed for five generations to create a set of homozygous BC2F6lines by single-seed descent. These lines were then genotyped for 127 marker loci covering the entire tomato genome. These IBLs were evaluated for 22 quantitative traits. In all, 71 significant QTLs were identified, 15% (11/71) of which mapped to the same chromosomal positions as QTLs identified in earlier studies using the same cross. For 48% (34/71) of the detected QTLs, the wild allele was associated with improved agronomic performance. A number of new QTLs were identified including several of significant agronomic importance for tomato production: fruit shape, firmness, fruit color, scar size, seed and flower number, leaf curliness, plant growth, fertility, and flowering time. To improve the utility of the IBL population, a subset of 100 lines giving the most uniform genome coverage and map resolution was selected using a randomized greedy algorithm as implemented in the software package MapPop (http://www.bio.unc.edu/faculty/vision/lab/mappop/). The map, phenotypic data, and seeds for the IBL population are publicly available (http://soldb.cit.cornell.edu) and will provide tomato geneticists and breeders with a genetic resource for mapping, gene discovery, and breeding.Key words: tomato, Lycopersicon esculentum, IBLs, QTL, mapping.

Fruit size and shape are two major factors determining yield, quality and consumer acceptability for many crops. Like most traits important to agriculture, both are quantitatively inherited. Despite their economic importance none of the genes controlling either of these traits have been cloned, and little is known about the control of the size and shape of domesticated fruit. Tomato represents a model fruit-bearing domesticated species characterized by a wide morphological diversity of fruits. The many genetic and genomic tools available for this crop can be used to unraveal the molecular bases of the developmental stages which presumably influence fruit architecture, size and shape. The goal of this review is to summarize data from the tomato QTL studies conducted over the past 15 years, which together allow the identification of the major QTLs responsible for fruit domestication in tomato. These results provide the starting point for the isolation of the genes involved in fruit-size/shape determination in tomato and potentially other fruit-bearing plants.

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.

In two previous quantitative trait locus (QTL) mapping studies conducted inLycopersicon esculentum x L. pimpinellifolium BC1 and BC2 populations we had localized a major QTL for fruit shape,fs8.1, to a ca. 20 cM interval on the short arm of chromosome 8, flanked by markers TG176 and CT92. At this QTL the allele from the wild species reduces the length of fruit, giving round-shaped fruit. In order to define more precisely the location offs8.1, near-isogenic lines (NILs) segregating for the region of interest were developed. The results from substitution mapping show that no recombination occurred betweenfs8.1 and the marker CD40 in 322 meioses. The gene action forfs8.1 was determined in a BC4F3 population to be partial dominance. The main effect offs8.1 is exerted on fruit length while fruit diameter is not significantly affected. A highly significant correlation (r=0.89;P

A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future.

High-resolution genetic and physical maps were constructed for the region of chromosome 2 containing the major fruit-shape locus ovate. A total of 3000 NIL F2and F3NILs derived from Lycopersicon esculentum cv. Yellow Pear (TA503) × L. pennellii (a wild tomato) were used to position ovate adjacent to the marker TG645 and flanked by markers TX700 and BA10R (a 0.03-cM interval). BAC libraries and a BIBAC library were screened with the closest marker, TG645. Genetic mapping with the ends of isolated BAC clones revealed that two BAC clones (100 and 140 kb) both contained the ovate locus. Screening of sequences from these BAC clones revealed synteny between this segment of tomato chromosome 2 and the chromosome-4 region of Arabidopsis containing the BAC clone ATAP22. Microsynteny between the two genomes was exploited to find additional markers near the ovate locus. The placement of ovate on a BAC clone will now allow cloning of this locus and, hence, may open the door to understanding the molecular basis of fruit development and also facilitate the genetic engineering of fruit-shape characteristics. This also represents the first time that microsynteny with Arabidopsis has been exploited for positional cloning purposes in a different plant family.Key words: map, fruit development, colinearity, positional cloning.

A 105-kilobase bacterial artificial chromosome (BAC) clone from the ovate- containing region of tomato chromosome 2 was sequenced and annotated. The tomato BAC sequence was then compared, gene by gene, with the sequenced portions of the Arabidopsis thaliana genome. Rather than matching a single portion of the Arabidopsis genome, the tomato clone shows conservation of gene content and order with four different segments of Arabidopsis chromosomes 2–5. The gene order and content of these individual Arabidopsis segments indicate that they derived from a common ancestral segment through two or more rounds of large-scale genome duplication events—possibly polyploidy. One of these duplication events is ancient and may predate the divergence of the Arabidopsis and tomato lineages. The other is more recent and is estimated to have occurred after the divergence of tomato and Arabidopsis ≈112 million years ago. Together, these data suggest that, on the scale of BAC-sized segments of DNA, chromosomal rearrangements (e.g., inversions and translocations) have been only a minor factor in the divergence of genome organization among plants. Rather, the dominating factors have been repeated rounds of large-scale genome duplication followed by selective gene loss. We hypothesize that these processes have led to the network of synteny revealed between tomato and Arabidopsis and predict that such networks of synteny will be common when making comparisons among higher plant taxa (e.g., families).

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.