Abstract
Citrus (Citrus sp.) germplasm collections are a valuable resource for citrus genetic breeding studies, and further utilization of the resource requires knowledge of their genotypic and phylogenetic relationships. Diverse citrus accessions, including citron (Citrus medica), mandarin (Citrus reticulata), pummelo (Citrus maxima), papeda (Papeda sp.), trifoliate orange (Poncirus trifoliata), kumquat (Fortunella sp.), and related species, have been housed at the Florida Citrus Arboretum, Winter Haven, FL, but the accessions in the collection have not been genotyped. In this study, a collection of 80 citrus accessions were genotyped using 1536 sweet orange–derived single nucleotide polymorphism (SNP) markers, to determine their SNP fingerprints and to assess genetic diversity, population structure, and phylogenetic relationships, and thereby to test the efficiency of using the single genotype-derived SNP chip with relatively low cost for these analyses. Phylogenetic relationships among the 80 accessions were determined by multivariate analysis. A model-based clustering program detected five basic groups and revealed that C. maxima introgressions varied among mandarin cultivars and segregated in mandarin F1 progeny. In addition, reciprocal differences in C. maxima contributions were observed among citranges (Citrus sinensis × P. trifoliata vs. P. trifoliata × C. sinensis) and may be caused by the influence of cytoplasmic DNA and its effect on selection of cultivars. Inferred admixture structures of many secondary citrus species and important cultivars were confirmed or revealed, including ‘Bergamot’ sour orange (Citrus aurantium), ‘Kinkoji’ (C. reticulata × Citrus paradisi), ‘Hyuganatsu’ orange (Citrus tamurana), and palestine sweet lime (Citrus aurantifolia). The relatively inexpensive SNP array used in this study generated informative genotyping data and led to good consensus and correlations with previously published observations based on whole genome sequencing (WGS) data. The genotyping data and the phylogenetic results may facilitate further exploitation of interesting genotypes in the collection and additional understanding of phylogenetic relationships in citrus.
Citrus is suggested to have originated from the tropical and subtropical areas of southeast Asia and was then spread to other regions in the world (Webber, 1967). The genus Citrus, including important commercial cultivars, has been grown in tropical and temperate areas over several thousands of years. Citrus taxonomy and phylogeny are complicated, partly because of the long history of cultivation, wide distribution, apomixis, high frequency of bud mutations, and sexual compatibility between Citrus and related genera. Two morphological characteristic-based citrus classifications, defined by Swingle (Swingle and Reece, 1967) and Tanaka (1977), have been widely used. However, the two classification systems differ greatly in species classification: 16 species were included in the Citrus genus in the classification of Swingle, whereas Tanaka described 162 species. The subsequent phylogenetic studies suggested that there were three true cultivated Citrus species, mandarin, citron, and pummelo, and that hybridization between them has generated other genotypes (Barrett and Rhodes, 1976; Scora, 1975, 1988). Molecular analyses have been widely used in Citrus taxonomy studies. The three cultivated Citrus species concept was supported by multiple subsequent studies using isozymes (Herrero et al., 1996; Torres et al., 1978).
Phylogenetic analysis using nuclear gene sequence may be more informative for investigating the relationships between different Citrus genotypes. There are many types of molecular markers, among them are simple sequence repeat markers (SSRs) which are codominant, highly polymorphic, locus-specific, abundant, and distributed randomly throughout the genome (Tautz and Schlötterer, 1994). Thus, SSRs have been widely used in Citrus phylogeny studies (Barkley et al., 2006; Bretó et al., 2001; Fang and Roose, 1997; Fang et al., 1998; Garcia-Lor et al., 2012; Gulsen and Roose, 2001). In addition, random amplification of polymorphic DNA [RAPD (Bretó et al., 2001; Coletta et al., 1998; Federici et al., 1998; Nicolosi et al., 2000)], restriction fragment length polymorphism (Federici et al., 1998), and amplified fragment length polymorphism (Bretó et al., 2001) all have been used as markers in Citrus. Barkley et al. (2009) have noted that homoplasy in microsatellite alleles might cause incorrect conclusions in phylogenetic studies. Insertion/deletions (INDELs) were found to be more suitable than SSRs for tracing the genetic contributions of the basic taxa in cultivated Citrus (Garcia-Lor et al., 2012), but the low frequency of INDELs limits their usefulness. Although SNPs show lower polymorphism information content (PIC) relative to SSRs, high levels of reproducibility and high frequency of SNP occurrence throughout the genome make them ideal markers for population genetic studies (Helyar et al., 2011). High-throughput next-generation sequencing technology has resulted in the development of automated high-throughput SNP genotyping assays that are available for most crops and are useful tools to evaluate genome-wide allelic variation. In Citrus, SNPs were able to differentiate the basic taxa; therefore, several phylogenetic studies have been accomplished using SNPs, ranging in number from 67 to 1457 (Curk et al., 2014, 2015, 2016; Garcia-Lor et al., 2013, 2015; Ollitrault et al., 2012). In addition to molecular markers, primary and secondary metabolite analyses indicated that metabolite diversity was associated with phylogeny among tested basic taxa in cultivated Citrus (Fanciullino et al., 2006; Goldenberg et al., 2014, 2015; Luro et al., 2011).
It is now generally accepted that all cultivated Citrus are derived from interspecific hybridization between four ancestral taxa, mandarin, citron, pummelo, and Citrus micrantha (Nicolosi et al., 2000), but opinions differ on the origin of some specific species and cultivars. Most of the secondary species including sweet orange (C. sinensis), sour orange, grapefruit (C. paradisi), lemon (Citrus limon), and lime, generally, are propagated vegetatively and many come true to type from seed, thereby limiting further interspecific recombination and resulting in minimal intragroup genetic diversity (Ollitrault and Navarro, 2011). Natural biodiversity that exists in the citrus gene pool is essential for innovative citrus breeding. The understanding of phylogenetic origin and genomic structure of those secondary species and important cultivars can facilitate the utilization of the natural phenotypic variability (Krueger and Navarro, 2007). The recent WGS projects have made important contributions in elucidating the phylogenetic history of citrus domestication and deciphering the genetic relationships between C. sinensis, C. aurantium, and Citrus clementina with C. maxima and C. reticulata (Wu et al., 2014, 2018; Xu et al., 2013). The phylogenetic origin of some secondary species and important citrus cultivars resulting from sexual crosses remains unknown or controversial. This study aimed to genotype the citrus collection at the Florida Citrus Arboretum (maintained by the Bureau of Citrus Budwood Registration in the Division of Plant Industry, Florida Department of Agriculture and Consumer Services) in Winter Haven, FL, using the previously used 1536-SNP genotyping assay (Yu et al., 2016, 2017, 2018), and to determine their SNP fingerprints and to assess genetic diversity, population structure, and phylogenetic relationships. Use of this sweet orange–derived SNP assay may not be ideal for genotyping distantly related accessions but was much less expensive than genome sequencing, and the efficiency of using the relatively inexpensive approach for these analyses could be tested in this study. Our hypothesis was that genotyping using this SNP chip would nonetheless prove informative.
Materials and Methods
Plant materials, DNA extraction, and genotyping.
The 80 accessions were chosen from Citrus and Citrus relatives (Table 1) and included representatives of the major known cultivar groups. Leaves of the 80 accessions were sampled from the Florida Citrus Arboretum. The Swingle (1967) and Tanaka (1977) botanical classifications were used for scientific names. Genomic DNA extraction was performed using the CTAB method according to Aldrich and Cullis (1993), and all the individuals were genotyped using a GoldenGate 1536-SNP array platform in the University of Florida’s Interdisciplinary Center for Biotechnology Research. The 1536 SNPs were well characterized and evenly distributed based on sweet orange bacterial artificial chromosome (BAC) end sequences. Genotyping data were collected and analyzed by the software Genome Studio (Illumina, San Diego, CA). All SNPs were blasted against the clementine (C. clementina) genome V1.0 (Wu et al., 2014), and the physical locations of SNPs on corresponding chromosomes of the clementine genome were obtained.
List of 80 Citrus and Citrus relatives analyzed by a 1536-single nucleotide polymorphism genotyping assay.
Diversity and phylogenetic statistics.
The software PowerMarker 3.25 (Liu and Muse, 2005) was applied to calculate allele frequency, PIC, expected and observed heterozygosity (He and Ho), and inbreeding coefficient, and to test the Hardy–Weinberg equilibrium. Any SNP was excluded from the subsequent analysis if it had more than 10% missing calls or had less than 5% minor allele frequency (MAF). The software JMP Genomics (version 7; SAS Institute, Cary, NC) was used to calculate the genetic relationship matrix of the 80 accessions based on the identity by state (IBS) values from the genotypic data of SNPs. IBS computes the probability of two individuals sharing the same copy of an allele. The cluster analysis was performed using a matrix of IBS pairwise distances, and groups were determined by a permutation test. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were performed using the software GenAIEx 6.5 (Peakall and Smouse, 2006) based on allele frequencies.
Population structure.
The population structure of the 80 accessions was determined using the software STRUCTURE 2.3.4 (Pritchard et al., 2000). No prior population information was defined. The parameters were set for an admixture model and allele frequencies correlated. For estimating K, 10 independent STRUCTURE runs were performed at each of K = 1–10 with 10,000 steps of burn-in and 50,000 repetitions of Markov chain Monte Carlo. The results generated by STRUCTURE were entered into the website program Structure Harvester (Earl and Vonholdt, 2012) for visualizing likelihood values of multiple K and detecting the number of genetic groups that best fits the data.
Results and Discussion
Genetic diversity and allele frequencies.
A total of 1366 SNPs (missing calls ≤ 10% and MAF ≥ 5%) were selected to study the genetic diversity and population structure of the 80 accessions. The number of SNPs used for analysis of each accession ranged from 1088 for Microcitrus inodora to 1366 for 18 genotypes. Molecular marker properties were computed for each SNP, with PIC and Ho being 0.31 ± 0.07 and 0.34 ± 0.12, respectively (Supplemental Table 1). All the SNPs were evenly distributed throughout the chromosomes, varying from 105 on scaffold 7 to 236 on scaffold 3, and no significant differences were observed for PIC, Ho, and He between chromosomes (Table 2). Within the genus Citrus, the progenitor species mandarin, pummelo, and citron displayed low heterozygosity relative to other secondary species (Table 3; Supplemental Table 2). Ho ranged from 0.09 in citron to 0.89 in sweet orange, whereas PIC values varied from 0.06 in sour orange to 0.16 in sweet orange and 0.18 in pummelo. For Citrus accessions, Ho varied from 0.04 in Buddha’s hand citron to 0.94 in Cara Cara navel orange (C. sinensis), and PIC ranged from 0.04 in Nules clementine to 0.35 in Sha Tian You pummelo. With respect to the accessions not in Citrus, kumquat displayed a low Ho value of 0.05, and trifoliate orange exhibited a high PIC value of 0.27.
The number, polymorphism information content (PIC), observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (IC) of 1366 single nucleotide polymorphisms (SNPs) on different scaffolds of the clementine genome assembly.
Polymorphism information content (PIC) and observed heterozygosity (Ho) of citrus genotype groups analyzed by 1366 genome-wide single nucleotide polymorphism (SNP) markers.
Phylogenetic analysis.
The 80 accessions were grouped into five main clusters according to the IBS values calculated from the genotypic data of 1366 SNPs (Fig. 1). The first main cluster included all kumquats, all citrons, all trifoliate oranges, all Microcitrus, two papedas, and two limes. The next major cluster consisted of all pummelos, which were clustered together. The third main group comprised all mandarin-type accessions. The mandarin group was divided into four subclusters. The calamandarin (C. reticulata × C. madurensis) and Iyokan (C. reticulata × C. sinensis) were clustered closely together in the subcluster formed with Sunki and Shekwasha mandarins. The fourth major group included most of the sour oranges, one papeda, one lime, four lemons, and all citranges. Eureka and Lisbon lemons were grouped closely together with palestine sweet lime. ‘Bergamot’ and all other C. aurantium were clustered together. ‘Kinkoji’ and ‘Hyuganatsu’ were clustered closely together in the subcluster formed with all citranges. The last main cluster comprised all sweet oranges.
Dendrogram of 80 Citrus and Citrus relatives based on identity by state (IBS) calculated from the data of 1366 genome-wide single nucleotide polymorphism markers. IBS is the allelic similarity between two individuals at the given loci regardless of their common ancestry, via computing the probability of two individuals sharing the same copy of an allele. The cluster analysis is performed using a matrix of IBS pairwise distances, and groups are determined by a permutation test.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04394-18
Multivariate analysis.
PCA was performed for the 80 accessions with genotypic data of the 1366 SNPs. The first three components (46.7%, 25.2%, and 5.4%) explained 77.3% of the total diversity, revealing a strong structuration. The principal component 1 (PC1) mainly distinguished mandarins and their hybrids from other species (Fig. 2A). The PC2 separated sour oranges and their hybrids from C. sinensis. The PC3 differentiated C. maxima from C. medica (Fig. 2B). The calamandarin was located in the mandarin cluster, whereas the calamondin was positioned between the mandarin and Fortunella clusters however, closer to the mandarin cluster (Fig. 2B; Supplemental Fig. 1A). ‘Kinkoji’ was close to the sour orange cluster (Fig. 2; Supplemental Fig. 1A). Multivariate PCoA was used to investigate the genetic distances among the 80 accessions by using the 1366 SNPs. The first three axes (48.69%, 19.21%, and 7.88%) explained 75.78% of the total diversity. The first axis mainly differentiated mandarins and their hybrids and C. maxima from other species (Supplemental Fig. 1A). The second axis separated C. sinensis from sour oranges and their hybrids. The third axis distinguished C. limon from sour oranges and their hybrids (Supplemental Fig. 1B).
Principal component analysis differentiating the 80 accessions of Citrus and Citrus relatives into different groups using the 1366 genome-wide single nucleotide polymorphisms. The percentage of total variance explained by each principal component (PC) is reported. (A) PC1/PC2, (B) PC1/PC3.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04394-18
Population structure.
Five groups (K = 5) including pummelos, mandarins, citrons, trifoliate oranges, and kumquats were indicated by the structure analysis, but no K was defined for the papeda group which probably resulted from the absence of pure C. micrantha being included in the dataset (Fig. 3). A similar observation has been previously reported and even sweet oranges did not show most C. maxima contribution (Barkley et al., 2006). It might be difficult to infer a correct value for K in a dataset that consists of a large number of individuals with admixture and a small number of individuals with no admixture, as the whole dataset is likely treated as a panmictic population (Ramadugu et al., 2013). In addition, small introgressions of Fortunella and Poncirus were found in some secondary species, lower than 5%, and therefore were not considered significant. Based on previous publications, <5% genetic contribution could be an error in STRUCTURE run from limited markers such as 1536 SNPs (Barkley et al., 2009; Garcia-Lor et al., 2015).
Population genetic structure among 80 Citrus and Citrus relatives using the 1366 genome-wide single nucleotide polymorphisms. Each genotype is represented by a vertical bar partitioned into K = 5 segments representing the estimated membership fraction in five population groups. The five groups are depicted using the following color codes: red, Citrus maxima; green, Citrus reticulata; blue, Citrus medica; yellow, Poncirus trifoliata; and purple, Fortunella.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04394-18
Interspecific introgressions were found in several mandarins and their hybrids, and pummelo was a common genetic contributor to mandarin, but contribution varied greatly. Three of the mandarin hybrids (‘King’, ‘Nules’, and ‘Fortune’) showed genetic contributions from C. maxima (12% to 13%). In addition, Sunki mandarin, mostly used as a rootstock, was detected without any interspecific introgression. Curk et al. (2015) observed three mandarins (‘Sunki’, ‘Cleopatra’, and Citrus daoxianensis) with 100% C. reticulata, and they all shared the acidic mandarin mitotype (Froelicher et al., 2011). The proportions of C. maxima were found in sour orange (38%), sweet orange (35%), ‘Clementine’ (12%), and ‘Murcott’ (9%), which is well correlated (r = 0.997) with the previous study using the WGS data that inferred values of 49%, 44%, 21%, and 15% for the same cultivars, respectively (Wu et al., 2014). The good agreement between our observations and the WGS data-based findings reveals the relatively high efficiency of using such an inexpensive SNP array for a genetic diversity study. ‘Fortune’ and ‘Murcott’ are heterozygote admixed, and the proportion of C. maxima in their hybrids is inherited in a segregating but not an additive manner. Because of inheritance of large pieces of parental linkage groups, the C. maxima introgression ranged from 3% to 11% in the three ‘Fortune’ × ‘Murcott’ hybrids, when compared with their parents ‘Fortune’ (13%) and ‘Murcott’ (9%).
‘Kinkoji’ was considered to result from hybridization between a mandarin and a grapefruit (Hodgson, 1967), or a pummelo and a mandarin (Bayer et al., 2009). In the IBS-based phylogenetic tree (Fig. 1), ‘Kinkoji’ and citranges were clustered together with another subgroup mainly including C. aurantium. In PCA (Fig. 2B) and PCoA (Supplemental Fig. 1A), ‘Kinkoji’ was close to the sour orange cluster including the other three genotypes. ‘Kinkoji’ displayed an admixture of C. maxima (50%) and C. reticulata (39%), which supports the hypothesis of a pummelo–mandarin origin for ‘Kinkoji’. ‘Hyuganatsu’ contained higher contribution from C. maxima (60%), compared with the contribution to sweet oranges, sour oranges, and their hybrids from C. maxima (33% to 41%). ‘Natsumikan’, ‘Sanbokan’, and ‘Nansho Daidai’ showed a similar pattern to ‘Kinkoji’ as reported by Barkley et al. (2006). ‘Hyuganatsu’ was believed to be either a mutated ‘Yuzu’ or a hybrid of ‘Yuzu’ and a pummelo (Hodgson, 1967). Here, ‘Hyuganatsu’ exhibited a similar pattern to ‘Kinkoji’, but contained high contribution from C. maxima (60%). However, nearly no C. maxima contribution was observed in ‘Yuzu’. In both PCA and PCoA, ‘Hyuganatsu’ was clustered distantly from ‘Yuzu’. Therefore, we suggest that ‘Hyuganatsu’ cannot be a mutated ‘Yuzu’.
The hybrids of C. sinensis × P. trifoliata displayed higher introgression (27% to 42%) from C. maxima when compared with the seedlings of the reciprocal cross (C. maxima introgression: 14% to 23%). The difference may arise from preferential selection of traits associated with the sweet orange cytoplasmic DNA, even though no cytoplasmic SNPs were included here. Reciprocal differences persist in plants because of the unequal contributions of cytoplasmic determinants from the female and male gametes. Cytoplasmic and maternal effects are the major components of extranuclear effects for plant seeds (Mosjidis and Yermanos, 1984), and the characterization of extranuclear effects on quantitative traits is important for plant breeding and seed quality improvement. In addition, the citranges contained low contribution (22% to 30%) from one of its parents, P. trifoliata, which might be caused by the absence of trifoliate orange–derived SNPs in the genotyping assay used here; nonetheless, the citrange group was properly placed (Fig. 2).
Hodgson (1967) considered ‘Bergamot’ as a sour orange hybrid based on morphological characteristics, and C. aurantium has been widely suggested as one of its parents, by hybridization with a lime, lemon, or citron (Federici et al., 1998). Nicolosi et al. (2000) clustered ‘Bergamot’ within the citron group using RAPD, sequence-characterized amplified region, and cpDNA markers, and this observation was supported by a later study using SSRs (Barkley et al., 2006). In the phylogenetic analysis presented here, ‘Bergamot’ and all other C. aurantium accessions were clustered together, and PCA and PCoA were not able to distinguish ‘Bergamot’ from other C. aurantium. The structure analysis results are consistent with the three specific origins (C. medica, C. maxima, and C. reticulata) provided by Curk et al. (2016), but here C. reticulata (49%) predominates.
All lime and lemon accessions show partial apomixis, and this results in conflicting classifications (Scora, 1975). Several lime and lemon cultivars were suggested to have originated from a single clonal parent through sporadic mutations, transposable elements, or deletion of genomic fragments (Carvalho et al., 2005; Curk et al., 2015, 2016; Gulsen and Roose, 2001; Snoussi et al., 2012). Tanaka (1977) classified key lime and palestine sweet lime into C. aurantifolia and Citrus limettioides, respectively, whereas Swingle (1967) considered both of them as C. aurantifolia. Here, palestine sweet lime always showed a close relationship to C. limon accessions. Citrus aurantifolia has been considered as one of palestine sweet lime’s parents, by hybridization with C. medica or C. limetta Tan (Webber, 1967), C. sinensis (Barrett and Rhodes, 1976), and C. medica (Carvalho et al., 2005). Curk et al. (2016) excluded C. aurantifolia as one of its parents and proposed a (C. maxima × C. reticulata) × C. medica model by cytogenetic and nuclear analysis. Our structure analysis agrees with the model, as palestine sweet lime mainly contained genetic contributions from C. maxima, C. reticulata, and C. medica, which was a very similar pattern to ‘Eureka’ and ‘Lisbon’.
It was reported that the SNP chip data may be affected by ascertainment biases, which were caused by the procedure used to develop SNPs, and the degree of the ascertainment bias was dependent on the number of individuals selected as SNP discovery panels (Helyar et al., 2011; Nielsen et al., 2004). Here, the SNPs used for genetic diversity analysis of the Citrus and Citrus relatives were mined from sweet orange BAC end sequences, and as a result, the inferred admixture of the genotyped germplasm may suffer from the ascertainment biases due to the size of the ascertainment panel. In this article, some observations were different from the common knowledge, such as the inferred admixture for citranges, and the subsequent study with WGS data may support our results. Interestingly, good consensus and correlations were found between our population structure analysis with the limited and inexpensive set of SNPs and the published observations based on the SNPs, SSRs, and WGS data. We consider that the estimations of the inferred admixture structure based on the single genotype-derived SNP chip, which is much less expensive than genome sequencing, may reveal the true phylogenomic structures of some genotyped germplasms and be capable of conducting such genetic diversity studies on more distantly related accessions.
In summary, we present a detailed phylogeny of some Citrus and Citrus relatives using a 1536-SNP genotyping assay with much lower cost compared with genome sequencing. The SNPs solely mined from sweet orange BAC end sequences were used for analysis of the admixture structure of 80 genotypes including actual cultivars and rootstocks. The contribution of the ancestral species inferred with the set of SNP markers and the published results from WGS data were in good agreement. Citrus maxima contribution was detected in several recently selected mandarin cultivars, which indicates that C. maxima introgressions play an important role in mandarin domestication. Many secondary citrus species and important cultivars were studied and their inferred admixture structures were confirmed or revealed. Further study on interspecific admixture and the inferred phylogenetic origins of the main subgroup in Citrus will be critical for a better utilization of citrus biodiversity to breed new citrus cultivars and rootstocks.
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Principal coordinate analysis (PCoA) distribution of 80 Citrus and Citrus relative accessions from the 1366-single nucleotide polymorphism marker genotyping. The percentage of total variance explained by each coordinate (Coord) is reported. (A) Coord.1/Coord.2, (B) Coord.1/Coord.3.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 143, 6; 10.21273/JASHS04394-18
Summary statistics of 1366 genome-wide single nucleotide polymorphisms (SNPs) for genotyping 80 Citrus and Citrus relatives.
Polymorphism information content (PIC) and observed heterozygosity (Ho) of citrus genotypes analyzed by 1366 genome-wide single nucleotide polymorphism (SNP) markers.