Yoichi Kawazu

Isolation and functional characterization of the FLOWERING LOCUS T homolog, the LsFT gene, in lettuce.

High temperature-induced bolting of lettuce is undesirable agriculturally, making it important to find the mechanism governing the transition from vegetative to reproductive growth. FLOWERING LOCUS T (FT) genes play important roles in the induction of flowering in several plant species. To clarify floral induction in lettuce, we isolated the FT gene (LsFT) from lettuce. Sequence analysis and phylogenetic relationships of LsFT revealed considerable homology to FT genes of Arabidopsis, tomato, and other species. LsFT induced early flowering in transgenic Arabidopsis, but was not completely effective compared to AtFT. LsFT mRNA was abundant in the largest leaves under flowering-inducible conditions (higher temperatures). Gene expression was correlated with flower differentiation of the shoot apical meristem. Our results suggest that LsFT is a putative FT homolog in lettuce that regulates flower transition, similar to its homolog in Arabidopsis. This is the first information on the lettuce floral gene for elucidating regulation of the flowering transition in lettuce.

Transgenic resistance to Mirafiori lettuce virus in lettuce carrying inverted repeats of the viral coat protein gene.

Mirafiori lettuce virus (MiLV), a plant RNA virus belonging to the genus Ophiovirus, is considered to be a causal agent of lettuce big-vein disease. In this study, inverted repeats of a fragment of the coat protein (CP) gene of MiLV in a binary vector pBI121 were transferred via Agrobacterium tumefaciens-mediated transformation into lettuce (Lactuca sativa L.) in order to generate MiLV-resistant lettuce. Forty T1 lines were analyzed for resistance to MiLV by detecting MiLV in leaves, and two lines (lines 408 and 495) were selected as resistant to MiLV. Both lines were susceptible to Lettuce big-vein associated virus (LBVaV), and line 495 showed higher resistance to MiLV than line 408. Further analysis indicated that line 495 showed resistance to big-vein symptoms expression. Small interfering RNA (siRNA) molecules derived from the transgene were detected in plants of line 495. MiLV was detected in roots but not in leaves of line 495 plants after MiLV inoculation, suggesting that resistance to MiLV is less effective in roots than in leaves.

Detailed characterization of Mirafiori lettuce virus-resistant transgenic lettuce

Lettuce big-vein disease is caused by Mirafiori lettuce virus (MiLV), which is vectored by the soil-borne fungus Olpidium brassicae. A MiLV-resistant transgenic lettuce line was developed through introducing inverted repeats of the MiLV coat protein (CP) gene. Here, a detailed characterization study of this lettuce line was conducted by comparing it with the parental, non-transformed ‘Kaiser’ cultivar. There were no significant differences between transgenic and non-transgenic lettuce in terms of pollen fertility, pollen dispersal, seed production, seed dispersal, dormancy, germination, growth of seedlings under low or high temperature, chromatographic patterns of leaf extracts, or effects of lettuce on the growth of broccoli or soil microflora. A significant difference in pollen size was noted, but the difference was small. The length of the cotyledons of the transgenic lettuce was shorter than that of ‘Kaiser,’ but there were no differences in other morphological characteristics. Agrobacterium tumefaciens used for the production of transgenic lettuce was not detected in transgenic seeds. The transgenic T3, T4, and T5 generations showed higher resistance to MiLV and big-vein symptoms expression than the resistant ‘Pacific’ cultivar, indicating that high resistance to lettuce big-vein disease is stably inherited. PCR analysis showed that segregation of the CP gene was nearly 3:1 in the T1 and T2 generations, and that the transgenic T3 generation was homozygous for the CP gene. Segregation of the neomycin phosphotransferase II (npt II) gene was about 3:1 in the T1 generation, but the full length npt II gene was not detected in the T2 or T3 generation. The segregation pattern of the CP and npt II genes in the T1 generation showed the expected 9:3:3:1 ratio. These results suggest that the fragment including the CP gene and that including the npt II gene have been integrated into two unlinked loci, and that the T1 plant selected in our study did not have the npt II gene. DNA sequences flanking T-DNA insertions in the T2 generation were determined using inverse PCR, and showed that the right side of the T-DNA including the npt II gene had been truncated in the transgenic lettuce.

Development of marker-free transgenic lettuce resistant to Mirafiori lettuce big-vein virus.

Lettuce big-vein disease caused by Mirafiori lettuce big-vein virus (MLBVV) is found in major lettuce production areas worldwide, but highly resistant cultivars have not yet been developed. To produce MLBVV-resistant marker-free transgenic lettuce that would have a transgene with a promoter and terminator of lettuce origin, we constructed a two T-DNA binary vector, in which the first T-DNA contained the selectable marker gene neomycin phosphotransferase II, and the second T-DNA contained the lettuce ubiquitin gene promoter and terminator and inverted repeats of the coat protein (CP) gene of MLBVV. This vector was introduced into lettuce cultivars ‘Watson’ and ‘Fuyuhikari’ by Agrobacterium tumefaciens-mediated transformation. Regenerated plants (T0 generation) that were CP gene-positive by PCR analysis were self-pollinated, and 312 T1 lines were analyzed for resistance to MLBVV. Virus-negative plants were checked for the CP gene and the marker gene, and nine lines were obtained which were marker-free and resistant to MLBVV. Southern blot analysis showed that three of the nine lines had two copies of the CP gene, whereas six lines had a single copy and were used for further analysis. Small interfering RNAs, which are indicative of RNA silencing, were detected in all six lines. MLBVV infection was inhibited in all six lines in resistance tests performed in a growth chamber and a greenhouse, resulting in a high degree of resistance to lettuce big-vein disease. Transgenic lettuce lines produced in this study could be used as resistant cultivars or parental lines for breeding.

Quantitative trait locus analysis of cucumber fruit morphological traits based on image analysis

Fruit morphology is one of the most important traits in cucumber because it directly affects its commercial value. The cucumber line CS-PMR1 is a promising breeding material for improving disease resistance, but its fruit morphology is not ideal for the Japanese market, and this hampers breeding. We used recombinant inbred lines derived from a cross between CS-PMR1 and the Japanese cultivar ‘Santou’ for genetic analysis of fruit morphological traits. Fruit shape variation was evaluated in detail by means of image analysis, and quantitative trait locus (QTL) analyses were performed by composite interval mapping or interval mapping with a nonparametric model. We detected 41 QTLs: 5 for length, 6 for diameter, 5 for the ratio of length to diameter, 4 for the ratio of placenta diameter to fruit diameter, 16 for principal component scores of elliptic Fourier descriptors related to fruit shape, 2 for wart size and 3 for wart density. Some QTLs were detected in the same chromosomal regions at different fruit stages and for different morphological traits. In particular, several QTLs with large effects on fruit size were located in nearly the same regions as genes for resistance to powdery mildew and downy mildew, indicating that the linkage between fruit shape and the resistance genes is likely to limit breeding efficiency. The results of this study will contribute to the development of informative DNA markers tightly linked to genes for requisite fruit morphological traits, which are urgently required for efficient breeding of new cultivars.

DNA Markers in Cucurbitaceae Breeding

Cucumber (Cucumis sativus), melon (Cucumis melo), watermelon (Citrullus lanatus), and pumpkins and squash (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo) are important vegetable crops of the Cucurbitaceae family. Marker-assisted selection has earned an important position in crop breeding. Genetic linkage maps constitute a useful infrastructure for it. Many attempts have been undertaken to construct genetic linkage maps and consensus maps with phenotypic traits and QTLs to develop DNA markers that are useful for marker-assisted selection in Cucurbitaceae breeding. Recently, draft genome sequences of cucumber, melon, and watermelon have been produced using high-throughput sequencing technologies. Massive sequencing has led to the discovery of a large amount of DNA markers such as SSRs and SNPs, which are highly polymorphic, codominant, transferable, and high-throughput molecular markers. These markers are anticipated as the underlying basis for high-density genetic linkage map and gene identification. Furthermore, synteny conserved among Cucurbitaceae family enables the use of genetic information among related species.