Hsin-Mei Ku

Identifying the loci responsible for natural variation in fruit size and shape in tomato

Abstract 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.

The genetic basis of pear-shaped tomato fruit

Abstract 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.

Development of a molecular marker for a bruchid (Callosobruchus chinensis L.) resistance gene in mungbean

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 Vignasublobata (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.

Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct

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 R0 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 R1 progeny of the two resistant R0 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 R1 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.

Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct.

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.

Identification and characterization of a mechanical transmissible begomovirus causing leaf curl on oriental melon

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.

Transferability of rice SSR markers to bamboo

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.

Evaluation of DNA fragments covering the entire genome of a monopartite begomovirus for induction of viral resistance in transgenic plants via gene silencing

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.

Tagging Rice Drought-related QTL with SSR DNA Markers

Drought is one of the major limited factors that affect the growth of rice in Taiwan. The chromosomal regions associated with drought tolerance in rice using SSR mapping system were detected in this study. A F2 mapping population was generated from the cross between Taichung 189 (japonica type), a local drought susceptible rice, and Milyang 23 (indica type), a drought tolerant line. A total of 525 SSR DNA markers were used to screen polymorphisms between parents. There were 121 SSR DNA markers showed polymorphisms and were mapped on the F2 population. A total of 4 SSR DNA markers, dis4.1, lr8.1, lr10.1, and lr12.1 on chromosomes 4, 8, 10, and 12 were associated with drought tolerance. Other QTL affecting agronomically important traits were also reported. Five QTL RM136 (y6.1, chromosome 6), RM537 (y4.1, chromosome 4), RM5443 (y1.1, chromosome 1), RM3231 (y8.1, chromosome 8), and RM3 (y6.2, chromosome 6) were associated with yield. Applying these SSR DNA markers that are closely linked with drought tolerance will facilitate early selection of drought tolerant lines and shorten breeding period.

Analysis by virus induced gene silencing of the expression of two proline biosynthetic pathway genes in Nicotiana benthamiana under stress conditions

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.