This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication.
The Horticulture Journal Preview e Japanese Society for doi: 10.2503/hortj.UTD-292 JSHS Horticultural Science http://www.jshs.jp/
Genetic Diversity Assessment of Cucurbita Genetic Resources in Japan by Nuclear and Organelle DNA Markers
Ruikun Chen1, Kaede Takamura2, Keita Sugiyama3, Daisuke Kami3, Koichiro Shimomura4 and Yosuke Yoshioka1*
1Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan 2Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan 3Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization, Sapporo 062-8555, Japan 4Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization, Tsukuba 305-8517, Japan
Genetic diversity analysis of worldwide Cucurbita genetic resources preserved in the Japanese Genebank can provide valuable information for breeding. In this study, 612 Cucurbita accessions of six species, including 40 accessions with no identification information, were genotyped with 30 SSR markers; 378 alleles were detected (12.6 alleles per marker; range, 4–24). By cluster analysis, the 40 unidentified and 53 likely misidentified accessions were (re)identified. The identification was verified by cluster analysis based on the sequence of the mitochondrial atp4–ccmC region. After correction of the identification information, C. pepo accessions had the highest diversity indices among the species analyzed and thus showed potential as an ideal genetic resource for breeding. Among the three major species (C. moschata, C. maxima, and C. pepo), the diversity indices of accessions from Japan were lower than those of overseas accessions, indicating that the overseas accessions preserved in Japan are more genetically diverse and can be used as materials for the development of new cultivars. STRUCTURE and principal coordinate analyses of C. moschata revealed that several Japan accessions constituted an indispensable part of global crop genetic resources owing to their differences from overseas accessions. Commercial cultivars of C. maxima showed genetic similarity to each other in principal coordinate analysis, suggesting that they may have similar breeding properties. This study corrects some identification errors in the Genebank and could help improve the breeding of Cucurbita vegetables.
Key Words: mitochondrial marker, pumpkin, squash, SSR marker.
moschata, C. maxima, and C. pepo are the major culti‐ Introduction vated species (Wang et al., 2011; OECD, 2016). They Cucurbita L. (2n = 40) is a genus in the Cucur‐ are thought to have originated in North and South bitaceae family, which includes many edible cash crops America and are now widely cultivated on all conti‐ (OECD, 2016). The fruits of this genus are known as nents (Nee, 1990). In addition to the fruits, stems, squash, pumpkin, zucchini, or gourd, and by some other leaves, and seeds are also important nutrient sources in names such as “Japanese pumpkin” (C. moschata) and some countries. In recent years, about 27.4 million tons “Kabocha” (C. maxima) that refer to the cultivars from of Cucurbita fruits have been produced annually for use Japan. The genus is considered to have 12 to 27 species, as food, in folk medicine, and as decorations (OECD, of which five, C. ficifolia, C. maxima, C. mixta, 2016). A large number of seedlings are used as root‐ C. moschata, and C. pepo, are cultivated. Cucurbita stocks because some Cucurbita species exhibit strong growth and high resistance to diseases (Schulz et al., 2004; Xiao et al., 2010; Kong et al., 2014; FAO, 2020). Received; February 28, 2021. Accepted; May 6, 2021. In Japan, the earliest record of Cucurbita plants dates First Published Online in J-STAGE on July 21, 2021. back to 1548, when a Portuguese trading vessel brought This work was supported by a grant (PGRAsia project phase 1 and 2) Cucurbita fruits from Cambodia to Kyushu Island from the Ministry of Agriculture, Forestry and Fisheries, Japan. * Corresponding author (E-mail: [email protected]. in western Japan (Rural Culture Association Japan, ac.jp). 2004). Later, the Japanese named this new vegetable
© 2021 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 2 R. Chen, K. Takamura, K. Sugiyama, D. Kami, K. Shimomura and Y. Yoshioka
“Kabocha” after the Cambodian name (Rural Culture high variability, low analysis cost, and good repeatabili‐ Association Japan, 2004; de St. Maurice, 2017; ty (Guan et al., 2017; Abbasov et al., 2018; Chen et al., Ministry of Agriculture, Forestry and Fisheries (Japan), 2020). The applicability of SSR markers to study the 2020). The majority of Cucurbita crops cultivated in genetic diversity of various Cucurbita species has been Japan before the 1960s were C. moschata, and many confirmed (Formisano et al., 2012; Kong et al., 2014; local cultivars were developed. However, over time, Martins et al., 2015; Kaźmińska et al., 2017; Verdone C. maxima became more popular with Japanese con‐ et al., 2018). When using molecular markers to study sumers because of its sweeter taste and softer texture the diversity of genetic resources of various animal and (Rural Culture Association Japan, 2004). In addition, plant species, many researchers have found that species the unripe fruits of zucchini (C. pepo) have become identification was incorrect because it was based only increasingly popular in Japan since the 1980s, with on the phenotype, such as phenological and morpholog‐ annual production close to 10,000 tons (Food and ical characteristics, which are sometimes similar among Agricultural Materials Inspection Center (Japan), species (Transue et al., 1994; Ren et al., 2010; Mason 2015). Currently, the annual squash production in Japan et al., 2015). Incorrect species identification may reduce is 100,000 to 200,000 tons, half of which is from the utilization efficiency of genetic resources, leading to Hokkaido (northern region) (Food and Agricultural unexpected experimental results. DNA barcoding of
Materials Inspection Center (Japan), 2015). An F1 nuclear, chloroplast, and mitochondrial genes (or of all hybrid cultivar “Ebisu” (C. maxima, in some countries these genes combined) can help avoid this problem known as “Delica”) is the most widely cultivated (Ren et al., 2010; Pečnikar and Buzan, 2013; Wang (Agriculture & Livestock Industries Corporation et al., 2013). (Japan), 2019). Due to their wide adaptability and high In Japan, the National Agriculture and Food yield, Cucurbita cultivars bred in Japan have had a Research Organization (NARO) has collected a large worldwide influence. In Australia, China, Europe, number of Cucurbita genetic resources derived from North America, New Zealand, and other regions, Japan and other countries. However, the genetic diver‐ Cucurbita cultivars bred in Japan have been introduced sity of these resources remains to be explored. Many and are widely cultivated; they are not only loved by accessions have no information in terms of species local consumers, but are also used to improve local cul‐ identification or may be miss-identified. In this study, tivars (Morgan and Midmore, 2003; Cumarasamy et al., we aimed (1) to find a combination of SSR primers 2010; Liu et al., 2010; Formiga et al., 2019; Agricola suitable for the simultaneous analysis of accessions of Don Camillo, 2020; Takii Europe, 2020). various Cucurbita species preserved in NARO collec‐ Although a number of Cucurbita cultivars have been tions; (2) to confirm or correct species identification of developed around the world in the past few decades, the all accessions based on information on the polymor‐ demand for new cultivars is still urgent. One priority phism of their nuclear and organelle genes; and (3) to trait that should be improved is disease resistance. In analyze the genetic diversity of Cucurbita genetic Cucurbita production fields, many diseases such as resources preserved in Japan. powdery mildew, damping-off, and black root rot occur Materials and Methods from time to time, and new cultivars with high resis‐ tance are desired by farmers. In addition, because Materials Cucurbita plants with long vines and large leaves occu‐ We used 612 Cucurbita accessions from the Genetic py large areas in fields, managing and harvesting them Resources Center, Institute of Vegetable and Flori‐ is time-consuming. Therefore, cultivars with short culture Science, and Hokkaido Agricultural Research internodes are highly desirable (Hirai et al., 2004; Tsuji Center of NARO, and commercial cultivars owned by et al., 2011). New Japanese Cucurbita cultivars with the a seed company. They included 323 accessions of above-mentioned traits would increase the production C. moschata, 164 of C. maxima, 63 of C. pepo, 17 of of squash fruits. To develop new cultivars, it is neces‐ C. ficifolia, three of C. mixta, two of C. foetidissima, sary to identify species among the existing genetic and 40 of Cucurbita sp. (species unknown) (Table S1). resources and to clarify their characteristics and diversi‐ Among them, 307 were Japanese local accessions and ty at the phenotypic and genotypic levels. commercial cultivars, 286 were overseas accessions and In the last decade, the use of molecular markers to cultivars from other continents, and the origin of 19 study genetic diversity has developed rapidly. Research accessions was unknown. methods based on RAPD, AFLP, ISSR, SRAP, SSR, SNP, and other markers are well established, and meth‐ Tissue collection and DNA extraction ods based on next-generation sequencing analysis are Seeds were germinated in 72-cell trays filled with also developing rapidly (Schulman, 2007; Varshney Nippi Horticultural Soil No. 1 (Nihon Hiryo Co., Ltd., et al., 2007; Liu et al., 2008; El-Esawi, 2017). SSR Tokyo, Japan). The first true leaf was collected and if markers are widely used for genotyping plants because the seeds did not germinate, cotyledons were collected of their wide, codominant distribution in the genome, from them. DNA was extracted using a DNeasy Plant Hort. J. Preview 3
Mini Kit (Qiagen GmbH, Hilden, Germany) and diluted Organelle marker analysis in TE to a final concentration of 10 ng·μL−1. For accessions that may have been misidentified in the phylogenetic tree based on SSR marker genotyping SSR marker analysis and accessions without identification information, re- A total of 77 Cucurbita nuclear SSR markers identification was performed based on polymorphism of (Table S2) were prescreened against eight accessions their organelle DNA sequences. Polymorphism of four (C001, C002, C003, C019, C117, C429, C431, C436) chloroplast genome regions (matK, rbcL, trnL–trnF, chosen based on their origins (Gong et al., 2012). Thirty rpl16–rpl14) and one mitochondrial genome region SSR markers with a single peak and clear, reproducible, (atp4–ccmC) was tested by amplification and sequenc‐ polymorphic amplification products were then applied ing in four accessions from different species (C077, to all other accessions (Table S2). PCR mixtures C243, C268, and C435) (Nakamura et al., 1997; Zheng, (10 μL) contained template DNA (10 ng), 1× KAPA 2G 2011). Because the number of C. mixta accessions was buffer A (KAPA Biosystems Inc., Woburn, MA, USA), very small and its importance in agriculture is low, no
200 nM dNTPs, 0.5 mM MgCl2, 0.1 U KAPA 2G Fast accessions of this species were included. The primer DNA polymerase, 2 pmol reverse primer, and 0.5 pmol sequences for amplification and sequencing are listed in forward primer. The forward primers were 5′-labeled Table S3. PCR cycling conditions and sequencing were with the fluorescent dyes 6-FAM, VIC, NED, or PET as described by Nguyen et al. (2019). The sequences (Shimizu and Yano, 2011). PCR was performed in a obtained were aligned and assembled in GeneStudio C1000 Thermal Cycler (Bio-Rad Laboratories, Inc., (GeneStudio, Inc. Suwanee, GA, USA). UPGMA trees Hercules, CA, USA) with the cycling program were constructed in MEGA X (Kumar et al., 2018) described by Chen et al. (2020). PCR amplicons were using the sequence data. analyzed using an automated DNA analyzer (model Based on the polymorphism of these regions, the 3130xl) with a GeneScan-500LIZ size standard and atp4–ccmC region was selected because it could distin‐ GeneMapper v. 4.0 software (all from Thermo Fisher guish all four different Cucurbita species. Then, 57 Scientific Inc., Waltham, MA, USA). accessions with possible identification errors, 40 acces‐ The number of alleles, major allele frequency, poly‐ sions without identification information, and 38 acces‐ morphism information content (PIC), and F statistics sions (C. moschata, C. maxima, C. pepo, C. ficifolia, indices were calculated in PowerMarker v. 3.25 soft‐ and C. mixta) that were considered to have no identifi‐ ware (Liu and Muse, 2005). Observed heterozygosity cation errors in the SSR marker analysis were analyzed (Ho) and expected heterozygosity (He) were calculated as above (Table S1). in GenAlEx v. 6.502 software (Peakall and Smouse, Results 2012). Cluster analysis among accessions was per‐ formed using the unweighted pair-group method with Genotyping with SSR markers an arithmetic mean (UPGMA) based on Nei’s genetic The 30 selected SSR markers amplified a total of similarity (Nei et al., 1983) estimated using the 30 378 alleles in the 612 accessions, with an average of markers in PowerMarker. Hierarchical molecular vari‐ 12.6 alleles per marker (Table 1), ranging from 4 ance analysis (AMOVA) of accessions of the three (CMTp248) to 24 (CMTp193). The mean genetic diver‐ major species was calculated with Arlequin v. 3.5.2.2 sity indices of these accessions were 0.53 for major software (Excoffier and Lischer, 2010). Genotype data allele frequency (frequency of the allele with the high‐ for the SSR markers were also analyzed in the model- est frequency), 0.62 for He (indicating moderate to high based STRUCTURE v. 2.3.4 software (Pritchard et al., levels of polymorphism), 0.10 for Ho (indicating that 2000) to determine the most probable number of clus‐ almost all individuals were highly homozygous), and ters (K value). These analyses were performed separate‐ 0.58 for PIC (indicating that these markers were infor‐ ly for all 612 accessions and for accessions that mative). belonged to C. moschata, C. maxima, and C. pepo. The K value was determined by running an admixture and Cluster analysis by SSR markers related frequency model with K = 1 to 10 (20 replica‐ A phylogenetic tree of the 612 accessions revealed tions per K value); the burn-in period of each run was four major groups, each composed mainly of accessions set to 100,000 and the Monte Carlo Markov Chain of one species (C. ficifolia, C. pepo, C. maxima, and length was set to 1,000,000. The web-based program C. moschata; Fig. S1A). A considerable number (63) of STRUCTURE HARVESTER was used to estimate the accessions were found to have incorrect identification optimal K value (Earl and vonHoldt, 2012); this pro‐ information. Forty accessions of unknown species were gram follows the ΔK method of Evanno et al. (2005). classified into all groups except that dominated by Principal coordinate analysis (PCoA) was carried out C. ficifolia. using GenAlEx software separately for the 612 acces‐ sions and for the C. moschata, C. maxima, and C. pepo Cluster analysis by organelle marker accessions. Phylogenetic trees based on organelle markers are Table 1. Diversity indices of the 30 SSR markers used for genotyping of 612 Cucurbita accessions. 4 R. Chen, K. Takamura, K. Sugiyama, D. Kami, K. Shimomura and Y. Yoshioka
Table 1. Diversity indices of the 30 SSR markers used for genotyping of 612 Cucurbita accessions.
Major allele Marker name Naz Hez Hoz PICz frequency CMTp39 16 0.51 0.67 0.09 0.64 CMTp245 10 0.79 0.37 0.06 0.36 CMTp88 16 0.50 0.63 0.09 0.57 CMTp224 10 0.32 0.74 0.12 0.69 CMTm68 12 0.52 0.64 0.05 0.59 CMTp248 4 0.67 0.45 0.02 0.36 CMTp125 12 0.30 0.79 0.14 0.76 CMTm29 13 0.58 0.62 0.17 0.59 CMTm111 6 0.67 0.50 0.09 0.45 CMTp183 13 0.60 0.60 0.34 0.58 CMTm65 15 0.33 0.73 0.15 0.68 CMTp106 13 0.48 0.70 0.12 0.67 CMTm168 6 0.58 0.56 0.02 0.49 CMTmC14 13 0.54 0.62 0.03 0.56 CMTp36 5 0.45 0.64 0.10 0.57 CMTm232 16 0.33 0.77 0.11 0.74 CMTm131 11 0.50 0.65 0.15 0.60 CMTm261 19 0.36 0.81 0.33 0.79 CMTp187 14 0.59 0.60 0.07 0.57 CMTp193 24 0.61 0.58 0.04 0.55 CMTm259 12 0.54 0.61 0.04 0.56 CMTmC11 5 0.57 0.60 0.05 0.55 CMTm54 13 0.51 0.62 0.07 0.55 CMTm219 9 0.46 0.64 0.13 0.57 CMTmC15 13 0.63 0.57 0.07 0.55 CMTmC61 12 0.64 0.56 0.12 0.53 CMTp68 17 0.41 0.73 0.11 0.69 CMTp62 20 0.54 0.61 0.02 0.56 CMTm98 17 0.61 0.58 0.11 0.54 CMTm130 12 0.68 0.47 0.13 0.41 Average 12.6 0.53 0.62 0.10 0.58 z Na, number of alleles; He, expected heterozygosity; Ho, observed heterozygosity; PIC, polymorphism informa- tion content.
shown in Figure S2. Only the sequence of the mito‐ SSR markers was repainted, and the Japanese and over‐ chondrial atp4–ccmC region could distinguish the four seas accessions are marked with different colors in species used in this analysis. After multiple attempts, Figure S1B. we were unable to obtain sequence information for five accessions for which DNA was directly extracted from Genetic diversity of Cucurbita accessions seeds; these accessions were therefore excluded from The genetic diversity indices for each Cucurbita the final analysis (Table S1). All accessions that were species are summarized in Table 2 (C594 is not includ‐ considered to be identified correctly by SSR marker ed). Among the seven species, the Na ranged from 1.27 analysis were grouped into a cluster according to their (C. ficifolia) to 5.30 (C. pepo). The ranges for He, Ho, species (Fig. 1). For almost all of the accessions that and PIC were 0.06–0.51, 0.004–0.22, and 0.08–0.46, had no identification information or with incorrect iden‐ respectively. Among the species, C. ficifolia had the tification, analysis based on the atp4–ccmC region con‐ lowest values and C. pepo had the highest. For firmed the results of SSR marker analysis. The only C. moschata, C. maxima and C. pepo, the diversity exception was C594: cluster analysis using SSR mark‐ indices of Japan and overseas accessions were calculat‐ ers suggested that it belongs to C. moschata, whereas ed separately. The Japanese accessions had the lowest analysis of the atp4–ccmC region indicated that it values for He, Ho, and PIC, even though there were belongs to C. maxima. Based on the combined results of more Japanese accessions than overseas accessions both analyses, the phylogenetic tree established using (C. moschata and C. maxima). Hort. J. Preview 5
C506
C505 14 17 C077 C020 C439 C136 C1 C383 C1 C435
C368 C449 C366 C518 C547 C365 0.012 C553 C364 C560 C363 C580 C336 0.011 C590 C039 C333 C121 C326 0.01 C130 C318 C143 C277 0.009 C144 C248 C145
C247 C146 0.008 C238 C147
C234 C148 0.007 C229 C154 C198 C225 0.006 C251 C224 C265 C223 0.005 C266 C222 0.004 C300 C221 C309 C210 0.003 C313 C205 C341 C188 0.002 C343 C153 0.001 C352 C150 C355 C149 C360
C125 C361
C124 C367 C540 C441 Colored ranges C538 C499 C517 C500 C. ficifolia C482 C510 C5 C480 11 C. ficifolia ? C424 C512
C308 C513 C519 C. maxima C268 C594 C209 C457 C103 C. maxima ? C219 C082 C220 C052 C242 C. mixta C036 C508
C014 C200
C004 C243 C. mixta ? C555 C284
C378 C501 C502 C377 C. pepo C503 C359 C504
C327 C516 C290 C522 C539 C283 C119 C. pepo ? C189 C006 C024 C152 C132 C133
C134 C151
5
C141 3
C140
1
C139
C138
C137 C C. moschata
C. moschata ?
Fig. 1. Phylogenetic tree of 130 Cucurbita accessions based on atp4–ccmC region sequences. “?” means the accession is probably belong to the Table 2.species Genetic inferred diversity by SSR estimates marker in analysis. Cucurbita accessions for each species.
Table 2. Genetic diversity estimates in Cucurbita accessions for each species.
Number of Species Naz Hez Hoz PICz accessions C. moschata 339 4.50 0.27 0.10 0.23 Japan 169 3.33 0.22 0.08 0.19 Overseas 164 3.50 0.27 0.11 0.23 C. maxima 179 3.77 0.26 0.07 0.22 Japan 124 3.07 0.19 0.07 0.16 Overseas 50 2.87 0.27 0.07 0.27 C. pepo 66 5.30 0.51 0.22 0.46 Japan 7 3.00 0.43 0.13 0.38 Overseas 54 4.87 0.51 0.22 0.46 C. ficifolia 18 1.27 0.06 0.00y 0.08 C. mixta 7 2.27 0.33 0.16 0.29 C. foetidissima 2 1.40 0.22 0.18 0.38 z Na, number of alleles; He, expected heterozygosity; Ho, observed heterozygosity; PIC, polymorphism informa- tion content. y 0.004.
Genetic relationships among Cucurbita accessions allelic frequencies. In the STRUCTURE analysis of all AMOVA of the three major Cucurbita species 612 accessions, the computation of Evanno’s ΔK indi‐ showed that for each species the genetic variation cated K = 8 as the most likely model (Fig. S3), suggest‐ between Japanese and overseas accessions was much ing the presence of eight main clusters (1 to 8, Fig. 2). smaller than that within each of these groups (Table 3). With 0.8 as the likelihood (Q-value) of classifying each We used STRUCTURE analysis and PCoA to obtain accession into one of the eight clusters, each of 556 information about the population structure of all the accessions (90.8%) was grouped into one of the eight Cucurbita accessions and the three major species from clusters. Almost all accessions belonging to C. ficifolia, Table 3. Analysis of molecular variance (AMOVA) of genetic diversity of 3 major Cucurbita species. 6 R. Chen, K. Takamura, K. Sugiyama, D. Kami, K. Shimomura and Y. Yoshioka
Table 3. Analysis of molecular variance (AMOVA) of genetic diversity of 3 major Cucurbita species.
Variance of Percentage of Source of variation d.f. Sum of squares components variation (%) C. moschata Among Japan/Overseas 1 233.901 0.692 16.4 Within Japan/Overseas 664 2342.003 3.527 83.6 C. maxima Among Japan/Overseas 1 36.056 0.024 14.7 Within Japan/Overseas 346 489.671 1.415 85.4 C. pepo Among Japan/Overseas 1 25.429 0.798 12.4 Within Japan/Overseas 120 676.890 5.641 87.6
C. foetidissima, C. maxima, and C. mixta were grouped clusters (Fig. 4B). In the STRUCTURE analysis of into species-specific clusters. Accessions of C. maxima accessions, the ΔK was highest at K = 2 C. foetidissima and C. mixta were grouped into the (Fig. S4B), suggesting the presence of two main clus‐ same cluster, whereas those of C. moschata and C. pepo ters, which differed from the results of the analysis with were grouped into multiple clusters. Most of the all accessions. Most Japanese accessions were grouped C. moschata accessions were grouped into cluster 4 or 5 into one cluster and most overseas accessions into depending on their origin (Japan or overseas, respec‐ another cluster (Fig. 4C). In Figure 4D, we marked the tively), whereas the rest were mainly accessions from commercial C. maxima cultivars sold in Japan; they Nigeria grouped into cluster 6. The accessions of have a certain degree of centralized distribution and C. pepo were grouped into clusters 7 and 8. Cluster 8 show a relatively close genetic relationship. In the contained mini-pumpkins (C167, C502, C555), indicat‐ STRUCTURE analysis of the C. pepo accessions, the ing a certain genetic difference between mini-pumpkins ΔK was highest at K = 2 (Figs. 4E and S4C), suggesting and other C. pepo. For C594 (“Tetsukabuto”), which the presence of two main clusters, consistent with the could not be unambiguously assigned to a species, the results of analysis of all 612 accessions together. How‐ Q-values of clusters 3 and 4 were relatively high, but ever, PCoA analysis of the C. pepo accessions did not neither was greater than 0.8, indicating that it has a show two obvious clusters (Fig. 4F). The Q-values of genetic background from both C. moschata and the STRUCTURE analysis and the PCoA coordinates C. maxima. are listed in Table S4. The ΔK value was also relatively high when K = 4 Discussion (Fig. S3), indicating K = 4 as the next most-likely model. When K = 4, the accessions of C. ficifolia, In this study, the genetic diversity of worldwide C. foetidissima, and C. mixta were grouped into the Cucurbita genetic resources preserved in Japan was same cluster (cluster 1, Fig. 2), showing a close genetic analyzed for the first time. A total of 30 single-locus relationship between them. The C. moschata accessions SSR markers with clear amplification products were from Nigeria were also grouped in this cluster. Most of selected and used to analyze the genetic diversity of the remaining accessions of C. moschata, C. maxima, accessions belonging to six species of Cucurbita and C. pepo were grouped into their respective clusters (C. moschata, C. maxima, C. pepo, C. ficifolia, C. mixta, (Fig. 2). The results of PCoA for the 612 accessions are and C. foetidissima). Amplification products were shown in Figure 3. The first two axes explained 28.12% detected in all six species; most of the 30 markers had of the variation in the genetic distance matrix. The high PIC values (> 0.5) (Table 1) and were thus a suit‐ results of the PCoA and phylogenetic analysis were in able marker set for Cucurbita genetic diversity analysis. good agreement. A triangle-like distribution was found, Cluster analysis using these markers revealed that with the three vertices being C. moschata, C. maxima, about 10% of the 612 accessions may have been and C. pepo; the accessions belonging to the remaining misidentified (Fig. S1). This situation could lead to species were distributed in the middle of the triangle. missing some accessions that may be interesting or mis‐ Accessions of the same species were grouped together, takenly selecting accessions that turn out to be different with no mixing between groups. from what is expected. This issue does not happen only In the STRUCTURE analysis of C. moschata acces‐ in the gene banks in Japan (Cao et al., 1999; Mason sions, the ΔK was highest at K = 3 (Fig. S4A), suggest‐ et al., 2015). Forty accessions that had no identification ing the presence of three main clusters, consistent with information were classified into certain clusters, sug‐ the results of analysis of all 612 accessions together: the gesting that they may belong to the same species as Japanese and overseas accessions tended to be grouped other accessions in the cluster. If their classification is into different clusters (Fig. 4A). PCoA also showed a confirmed, the barrier to the utilization of these genetic difference between Japanese and overseas accessions, resources due to lack of identification will be eliminat‐ but they were not clearly divided into three different ed. Currently, morphological traits have been used to Hort. J. Preview 7
0.00 0.20 0.40 0.60 0.80 1.00 0.00 0.20 0.40 0.60 0.80 1.00 distinguish different species in the Cucubita genus, but C. ficifolia this method sometimes results in miss-identification. In C. foetidissima the study of other species, molecular markers such as RAPD, SSR, chloroplast and mitochondrial markers have been used for species identification (Singh et al., 2004; Mugue et al., 2008; Tuler et al., 2015; Poovitha et al., 2016). The set of SSR markers we selected could meet the need for species identification of the Cucurbita species in this study. C. maxima We screened five organelle markers, and found that all six Cucurbita species could be identified with just one mitochondrial marker. The results of the analyses with SSR markers and with the mitochondrial marker were almost identical, confirming the accuracy of both DNA markers in species identification. A good marker set, if widely used as a standard marker set, can be used to compare the results of differ‐ C. mixta ent research groups around the world (Fukuda et al., 2013). The SSR marker set used in this study has high PIC values and can distinguish six Cucurbita species, so it has the potential to become a generic marker set for species identification of new accessions and genetic diversity analysis in Cucurbita collections. Accession C594 requires a special mention. It was identified as C. moschata with SSR markers, but as C. maxima with the mitochondrial marker (Fig. 1). According to the breeding history of this cultivar, C594 is an interspecific hybrid between these species, so it is reasonable that it has genetic characteristics of two dif‐ ferent species. Its nuclear genome is closer to that of C. moschata C. moschata, whereas its mitochondria are derived from C. maxima. Some studies use only nuclear markers such as SSR markers to identify interspecific hybrids (Kimball et al., 2013; Pathirana et al., 2016; Saha et al., 2017), but this example shows that when it is impossi‐ ble to find nuclear markers polymorphic between par‐ ents from different species, using organelle markers could help identify parents of interspecific hybrids. After the identification information was corrected, C. pepo had the highest He and PIC values and showed higher genetic diversity than the other species (Table 2). The average number of alleles in C. pepo was 5.3, higher than the average of 3.0 (determined with 134 SSR markers) for 104 C. pepo accessions stored in the United States (Gong et al., 2012). The PIC value of C. pepo was 0.46, which is close to the 0.42 reported in a Spanish study (Formisano et al., 2012). Although the C. pepo difference in the markers and accessions used allows only a rough comparison between studies, it still shows that the diversity of C. pepo accessions preserved in Cucurbita sp. Japan is no lower than that in other countries, and that K=8 K=4 C. pepo could be an ideal resource for future breeding. Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 3 Cluster 4 In contrast, although C. moschata had the largest num‐ Cluster 5 Cluster 6 Cluster 7 Cluster 8 ber of accessions, its diversity indices were almost as low as those of C. maxima. A study of the worldwide Fig. 2. Population structure of 612 Cucurbita accessions based on 30 SSR markers. At K = 8, all accessions were classified into 8 (mainly European) C. maxima accessions found a PIC groups by STRUCTURE analysis. value of 0.51 (Kaźmińska et al., 2017), which was 8 R. Chen, K. Takamura, K. Sugiyama, D. Kami, K. Shimomura and Y. Yoshioka
0.5
0.3
0.1
-0.1
-0.3 C. moschata
-0.5 C. maxima Coord .2 (10.64%) C. pepo -0.7 C. ficifolia -0.9 C. mixta
-1.1 C. foetidissma
Cucurbita sp. -1.3 -1.0 -0.5 0.0 0.5 1.0 Coord.1 (17.48%) Fig. 3. Scatter diagram from principal coordinate analysis of the 612 Cucurbita accessions.
higher than the 0.22 in this study, indicating lower C. mixta. These results show that the nuclear and cyto‐ genetic diversity of the C. maxima accessions preserved plasmic genetic differentiation of the Cucurbita genus in Japan. Cucurbita ficifolia had the lowest diversity may be not consistent. Since the results of these studies indices, and many accessions were very closely related were based on partial genome information and a limited to each other in the phylogenetic tree, although they number of germplasms, further studies based on com‐ came from different countries (Fig. 2; Table 2). A study plete genome sequences using a greater number of on C. ficifolia in Mexico also revealed low diversity germplasms are needed to confirm this phenomena. index values (Moya-Hernández et al., 2018). Among In AMOVA, the genetic variation of the accessions the three major species, the diversity indices of acces‐ of the three major Cucurbita species was mainly among sions from Japan were lower than those of overseas individuals, and the correlation with their origin (Japan accessions, indicating that the overseas accessions pre‐ or overseas) was relatively low (Table 3). This indicates served in Japan are more genetically diverse than the that Japanese accessions have no overall characteristics accessions bred in Japan. Therefore, the overseas acces‐ that distinguish them from overseas accessions. The sions preserved in Japan can be used as a material STRUCTURE and PCoA analyses of C. moschata library for the development of new cultivars. In fact, revealed that, among Japanese accessions, some are overseas Cucurbita accessions have already been used traditional cultivars similar to overseas accessions, but in Japanese squash and pumpkin breeding (Kami, most are different from overseas accessions (Fig. 4). 2015). Given the low genetic diversity of C. moschata Japanese pumpkins originated overseas, so there should and C. maxima accessions, to meet needs such as off- be some old cultivars that are similar to overseas acces‐ season supply, bush-type plants, and seedless fruits, sions. The difference between Japanese and overseas introducing more exotic germplasms is an effective accessions revealed that some native cultivars have option (Kikuchi and Iizuka, 2014; Kami, 2015). been grown and bred over a long period. Crops intro‐ The phylogenetic tree in this study also reflects the duced into Japan, such as loquat, rapeseed, and tea, relativeness among the investigated species. As can be often produce cultivars with Japanese characteristics seen in Figure 1, C. pepo is closer to C. moschata, and (Fukuda et al., 2013; Taniguchi et al., 2014; Chen et al., C. ficifolia is closer to C. maxima on the mitochondrial 2017). Although these Japanese cultivars have different atp4–ccmC region. In contrast, the phylogenetic tree levels of genetic diversity, their uniqueness shows that drawn based on nuclear SSR markers shows that they constitute an indispensable part of the global crop C. pepo is closer to C. maxima, and C. moschata is genetic resource. closer to C. mixta (Fig. S1). In other studies based on In the PCoA of C. maxima, we analyzed the commer‐ mitochondrial and chloroplast regions, both showed cial cultivars sold in Japan separately (Fig. 4D). that C. moschata and C. mixta were closer, while the Although they came from different companies, they relationship between C. pepo and C. maxima varied were not widely distributed in the PCoA chart, showing (Sanjur et al., 2001; Zheng, 2011), while all these stud‐ high genetic similarity. The development of new culti‐ ies showed that C. foetidissima is distant from C. pepo, vars often involves selecting the best available cultivar, C. maxima, C. moschata, and C. mixta. However, the so genetically similar parents are repeatedly used for phylogenetic tree based on SSR markers in this study breeding, which may be the cause of this genetic simi‐ showed that C. foetidissima is closer to C. moschata and larity. The use of genetically similar parents for breed‐ Hort. J. Preview 9
A 0.0 0.2 0.4 0.6 0.8 1.0 B 0.5 Japan 0.3
0.1
-0.1
Coord.1(10.65%) -0.3
Overseas Japan Overseas -0.5 Unknown Unknown -0.7 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 Coord.1(17.48%) C 0.0 0.2 0.4 0.6 0.8 1.0 D 0.8 0.6
Japan 0.4 0.2 0.0 -0.2 Coord.1(10.64%) -0.4 Overseas -0.6 Japan Overseas -0.8 Commerical F1 Unknown Unknown -1.0 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Coord.1(15.85%)
E Japan 0.0 0.2 0.4 0.6 0.8 1.0 F 1.6
1.1
Overseas 0.6
Coord.1(8.15%) 0.1
-0.4 Japan Unknown Overseas Unknown -0.9 -2.4 -1.9 -1.4 -0.9 -0.4 0.1 0.6 Coord.1(25.44%) Cluster 1 Cluster 2 Cluster 3 Fig. 4. Population structure and scatter diagram from principal coordinate analysis of C. moschata, C. maxima, and C. pepo accessions based on 30 SSR markers. A, B: C. moschata; C, D: C. maxima; E, F: C. pepo. ing may not be able to meet new requirements such as other. This misclassification may result in breeders large-scale mechanized cultivation. Therefore, it is rea‐ choosing accessions that do not match their expecta‐ sonable to consider using genetic resources that are tions. Therefore, we suggest that accessions with the genetically very distant from existing commercial culti‐ same name in the Japanese Genebank should be con‐ vars (Kami, 2015). firmed using Table S4 of this paper to understand the We also found that some accessions with the same genetic relationship between them before selection for name are genetically very different. For example, C064, breeding. C195, and C373 are all named BGH4138, but C373 and In this study, we selected 30 markers suitable for the the other two are not closely related according to analysis of six different species of Cucurbita crops, STRUCTURE analysis and PCoA (Fig. 4; Table S4). used them to study the genetic diversity of the Similarly, accessions C019, C122, C126, C129, C209, Cucurbita accessions in Japan, and corrected errors in C252, C374, C490, and C598 are all named “Hyuuga the identification of these genetic resources. These 14”, but some of them are not closely related to each markers could help researchers better understand the 10 R. Chen, K. Takamura, K. Sugiyama, D. Kami, K. Shimomura and Y. Yoshioka genetic relationships within these species, and help Domains; 2020.
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