Genetic characterization of cucumber genetic resources in the NARO Genebank indicates their multiple dispersal trajectories to the East.

KEY MESSAGE: Genotyping-by-sequencing of 723 worldwide cucumber genetic resources revealed that cucumbers were dispersed eastward via at least three distinct routes, one to Southeast Asia and two from different directions to East Asia. The cucumber (Cucumis sativus) is an economically important vegetable crop cultivated and consumed worldwide. Despite its popularity, the manner in which cucumbers were dispersed from their origin in South Asia to the rest of the world, particularly to the east, remains a mystery due to the lack of written records. In this study, we performed genotyping-by-sequencing (GBS) on 723 worldwide cucumber accessions, mainly deposited in the Japanese National Agriculture and Food Research Organization (NARO) Genebank, to characterize their genetic diversity, relationships, and population structure. Analyses based on over 60,000 genome-wide single-nucleotide polymorphisms identified by GBS revealed clear genetic differentiation between Southeast and East Asian populations, suggesting that they reached their respective region independently, not progressively. A deeper investigation of the East Asian population identified two subpopulations with different fruit characteristics, supporting the traditional classification of East Asian cucumbers into two types thought to have been introduced by independent routes. Finally, we developed a core collection of 100 accessions representing at least 93.2% of the genetic diversity present in the entire collection. The genetic relationships and population structure, their associations with geographic distribution and phenotypic traits, and the core collection presented in this study are valuable resources for elucidating the dispersal history and promoting the efficient use and management of genetic resources for research and breeding in cucumber.

Elucidation of genetic variation and population structure of melon genetic resources in the NARO Genebank, and construction of the World Melon Core Collection.

Numerous genetic resources of major crops have been introduced from around the world and deposited in Japanese National Agriculture and Food Research Organization (NARO) Genebank. Understanding their genetic variation and selecting a representative subset ("core collection") are essential for optimal management and efficient use of genetic resources. In this study, we conducted genotyping-by-sequencing (GBS) to characterize the genetic relationships and population structure in 755 accessions of melon genetic resources. The GBS identified 39,324 single-nucleotide polymorphisms (SNPs) that are distributed throughout the melon genome with high density (one SNP/10.6 kb). The phylogenetic relationships and population structure inferred using this SNP dataset are highly associated with the cytoplasm type and geographical origin. Our results strongly support the recent hypothesis that cultivated melon was established in Africa and India through multiple independent domestication events. Finally, we constructed a World Melon Core Collection that covers at least 82% of the genetic diversity and has a wide range of geographical origins and fruit morphology. The genome-wide SNP dataset, phylogenetic relationships, population structure, and the core collection provided in this study should largely contribute to genetic research, breeding, and genetic resource preservation in melon.

Identification of quantitative trait loci for powdery mildew resistance in highly resistant cucumber (Cucumis sativus L.) using ddRAD-seq analysis.

Powdery mildew, caused by Podosphaera xanthii (syn. Sphaerotheca fuliginea ex Fr. Poll.), is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber parental line 'Kyuri Chukanbohon Nou 5 Go', developed from weedy cucumber line CS-PMR1, is highly resistant to powdery mildew and is promising breeding material. We performed quantitative trait locus (QTL) analysis using double-digest restriction-site-associated DNA sequencing (ddRAD-Seq) in a population from a cross between 'Kyuri Chukanbohon Nou 5 Go' and the Japanese native cultivar 'Kaga-aonaga-fushinari', which is susceptible to powdery mildew. The resistance of the population and its parents was evaluated using leaf disc assays and image analysis. We detected one major QTL on Chr. 5 that was effective at both 20°C and 25°C and one minor QTL on Chr. 1 effective at 20°C. We detected two additional QTLs in subpopulation: one on Chr. 3 effective at 20°C and one on Chr. 5 effective at both 20°C and 25°C in a position different from the major QTL. The resistance alleles at all four QTLs were contributed by 'Kyuri Chukanbohon Nou 5 Go'. The results of this study can be used to develop practical DNA markers tightly linked to genes for powdery mildew resistance.

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.

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 T(3), T(4), and T(5) 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 T(1) and T(2) generations, and that the transgenic T(3) generation was homozygous for the CP gene. Segregation of the neomycin phosphotransferase II (npt II) gene was about 3:1 in the T(1) generation, but the full length npt II gene was not detected in the T(2) or T(3) generation. The segregation pattern of the CP and npt II genes in the T(1) 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 T(1) plant selected in our study did not have the npt II gene. DNA sequences flanking T-DNA insertions in the T(2) 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.

Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression.

Melon exhibits substantial natural variation especially in fruit ripening physiology, including both climacteric (ethylene-producing) and non-climacteric types. However, genomic mechanisms underlying such variation are not yet fully understood. Here, we report an Oxford Nanopore-based high-grade genome reference in the semi-climacteric cultivar Harukei-3 (378 Mb + 33,829 protein-coding genes), with an update of tissue-wide RNA-seq atlas in the Melonet-DB database. Comparison between Harukei-3 and DHL92, the first published melon genome, enabled identification of 24,758 one-to-one orthologue gene pairs, whereas others were candidates of copy number variation or presence/absence polymorphisms (PAPs). Further comparison based on 10 melon genome assemblies identified genome-wide PAPs of 415 retrotransposon Gag-like sequences. Of these, 160 showed fruit ripening-inducible expression, with 59.4% of the neighboring genes showing similar expression patterns (r > 0.8). Our results suggest that retrotransposons contributed to the modification of gene expression during diversification of melon genomes, and may affect fruit ripening-inducible gene expression.

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.