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Article

Phylogenetic Analysis and Molecular Diversity ofCapsicum Based on rDNA-ITS Region

1
Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
2
Education and Research Field, College of Life, Environment, and Advanced Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
3
Bioeconomy Research Institute, Research Center for the 21st Century, Osaka Prefecture University, Osaka 599-8531, Japan
*
Author to whom correspondence should be addressed.
Submission received: 16 October 2020 /Revised: 5 November 2020 /Accepted: 18 November 2020 /Published: 20 November 2020
(This article belongs to the SectionGenetics, Genomics, Breeding, and Biotechnology (G2B2))

Abstract

:
The genusCapsicum is comprised of 5 domesticated and more than 30 wild species. The region of nuclear ribosomal DNA internal transcribed spacers (rDNA-ITS) has widely been used for species identification, but has rarely been used inCapsicum. In this study, the evaluation of genetic diversity and a phylogenetic analysis were conducted using rDNA-ITS of 28Capsicum accessions, including five domesticated and two wild species. We surveyed six conventional keys of domesticated species and another five traits inCapsicum accessions. Specific morphological characteristics were found inC. annuum,C. baccatum, andC.pubescens. Three subclones of each accession were sequenced, and rDNA-ITS polymorphisms were detected in all accessions excludingC. annuum, suggesting that incomplete concerted evolution occurred in rDNA-ITS ofCapsicum. The genetic diversity was evaluated using nucleotide polymorphism and diversity.C. annuum had the lowest genetic diversity of all species in this study. The phylogenetic tree formed a species-specific clade forC. annuum,C. baccatum, andC. pubescens. TheC. chinense clade existed in theC. frutescens clade, implying that it was a cultivated variant ofC. frutescens.C. chacoense likely belonged to theC. baccatum complex according to its morphologic and genetic features. This study indicated that the rDNA-ITS region can be used for simple identification of domesticatedCapsicum species.

    1. Introduction

    The genusCapsicum has been cultivated since at least 6000 B.C. by Native Americans [1], and is now produced at over 40 million tons per year worldwide [2]. Fruits of the genus have good health properties such as stress relief and fat breakdown [3,4]. Capsaicin, which is the main pungent component ofCapsicum, has attracted much attention because of beneficial health properties [3,5].
    The genus has five domesticated species,C. annuum,C. baccatum,C. chinense,C. frutescens, andC. pubescens, and more than thirty wild species [6,7]. The origin of theCapsicum genus is postulated to be along the Andes of western to north-western South America [8]. The most commonly cultivatedCapsicum species isC. annuum, which is domesticated in northern Latin America [9,10].C. chinense andC. frutescens are domesticated in tropical northern Amazonia, whileC. baccatum andC. pubescens are more prevalent in Latin America and mid-elevation southern Andes, respectively [9]. The domesticated species can be classified by morphological traits: seed color, corolla yellow spot, number of flowers per axil, calyx annular constriction, and flower position [11].
    There are three genetic complexes based on the degree of genetic proximity and cross compatibility among the five domesticated species and closely related wild species. The complexes are theC. annuum complex (C. annuum,C. chinense, andC. frutescens),C. baccatum complex (C. baccatum,C. praetermissum, andC. tovarii), andC. pubescens complex (C. pubescens,C. cardenasii, andC. eximium) [12,13,14]. Additionally,C. chacoense is sometimes assigned to theC. annuum complex [15] orC. baccatum complex [8] depending on the phylogenic analysis method.
    In recent years, phylogenetic studies have been conducted to elucidate the relationship betweenCapsicum species based on molecular markers, including isozyme [16], random amplified polymorphic DNA [15], amplified fragment length polymorphism [17], simple sequence repeat [18,19], and single nucleotide polymorphism [20]. These studies analyzed the relationships between cultivated species, and indicated the species-specific clades formed; the species belonging to theC. annuum complex were genetically close to each other in phylogenetic trees.
    DNA sequencing of genes or specific regions is often performed in phylogenetic studies. The internal transcribed spacers from nuclear ribosomal DNA (rDNA-ITS) is the most commonly used region for DNA barcoding [21]. For plant DNA barcoding,rbcL,psbA-trnH spacer regions, andmatK on plastid DNA are proposed in addition to rDNA-ITS [21]. In the phylogenic analysis ofCapsicum species, thepsbA-trnH spacer region andmatK on plastid DNA, and the partial sequence ofwaxy on nuclear DNA have been used for DNA barcoding [8]. On the other hand, rDNA-ITS has been used for theCapsicum species identification of ‘Bhut Jolokia’ [22] and genetic diversity evaluation [23,24]. Besides, it has been used for phylogenetic analysis ofCapsicum species, although it is unclear whether rDNA-ITS can be used for the identification of domesticatedCapsicum species, as the number of lines surveyed was one for most species surveyed [25].
    The objectives of this study were to conduct phylogenetic analyses ofCapsicum species using rDNA-ITS, verify whether rDNA-ITS can identifyCapsicum species, especially domesticated ones, and describe their morphological characteristics. The rDNA-ITS sequences and morphological traits of domesticated and wildCapsicum species were examined. Moreover, genetic diversity within species was evaluated using rDNA-ITS sequences.

    2. Materials and Methods

    2.1. Plant Materials

    Twenty-sixCapsicum accessions, including five domesticated and two wild species, were provided by the National Agriculture and Food Research Organization Genebank (Tsukuba, Japan) or the USDA/ARSCapsicum germplasm collection (Griffin, GA, USA) (Table 1). After seeds were germinated on moistened filter papers in petri dishes, all seedlings were transplanted to pots (9 cm diameter, 10 cm depth) filled with culture soil (Sakata Super Mix A, Sakata Seed Co., Yokohama, Japan). The seedlings were cultivated in constant conditions (25 °C, 12/12 h light/dark, 85 µmol m−2 s−1). At 30 days after germination, plants were transferred to bigger pots (21 cm diameter, 15 cm depth) and placed in a greenhouse (natural day length; Osaka Prefecture University, Sakai, Osaka, Japan). OnlyC. pubescens was kept at room temperature because it is not suitable for cultivation at a high temperature.
    For morphologic investigations, five key traits (seed color, corolla yellow spot, number of flowers per axil, calyx annular constriction, and flower position) were described for the classification of domesticatedCapsicum species according to the International Board for Plant Genetic Resources [11], in addition to other five traits (plant growth habit, anther color, calyx margin, fruit shape, and fruit pungency) (Figure 1 andFigure 2). Morphologic surveys, excluding plant growth habit, seed color, fruit shape, and pungency, were conducted when each plant flowered. Plant growth habits were evaluated 5 months after germination when plant growth habits no longer change. Seed color was evaluated before sowing, and fruit shape and pungency were evaluated when each plant’s fruits ripened. Fruit shapes were visually classified based on the illustration described by the International Plant Genetic Resources Institute (IPGRI) [26]. Fruit pungency was evaluated as presence or absence of pungency by sensory test. Three plants per accession were investigated to determine each trait and whether the traits were uniform within each accession.

    2.2. DNA Sequence of rDNA-ITS Region

    Total DNA was extracted from individual leaves using the cetyltrimethylammonium bromide (CTAB) method [30] with minor modifications. Each leaf was ground in a mortar with liquid nitrogen. The ground leaf was mixed with CTAB isolation buffer (2% w/v CTAB, 1.4 M NaCl, 0.2% v/v β-mercaptoethanol, 20 mM ethylenediaminetetraacetic acid (EDTA), 100 mM Tris-HCl, pH 8.0) preheated at 60 °C, and the mixture was incubated at 60 °C for 60 min. The suspension was extracted twice with chloroform/isoamyl alcohol (24:1) and centrifuged for 15 min at 500 g. The aqueous phase was transferred to a new tube; nucleic acids were precipitated by the addition of isopropanol (2/3 volume) and centrifuged for 20 min at 1,000 g. The pellet was washed with 70% ethanol and dissolved in 40 µL of Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
    To enhance the specificity, the rDNA-ITS region was amplified by touchdown PCR using forward primer (5′-CTGCGGAAGGATCATTGTCG-3′) and reverse primer (5′-TAAACTCAGCGGGTAATCCC-3′), which were designed forCapsicum [31]. Touchdown PCR was performed in 40 μL reaction mixture containing 0.2 mM of dNTP, 0.2 μM primers, 1 U of KAPATaq EXtra DNA Polymerase (Kapa Biosystems Inc., Wilmington, MA, USA), 5×KAPATaq EXtra Buffer, and approximately 50 ng of DNA as template. The first step started with 94 °C for 3 min, followed by the touchdown phase, and PCR phase. The touchdown phase started with 94 °C for 30 s, annealing for 50 s, followed by elongation at 68 °C for 15 s. The annealing step of the touchdown phase had a temperature ramp from 67 to 64 °C in seven cycles (0.5 °C per cycle). The PCR phase had 33 thermal cycles, and each cycle had melting at 94 °C for 30 s, annealing at 64 °C for 50 s, and elongation at 72 °C for 15 s. The amplicons were verified on a 2% agarose gel in single band pattern. The amplicons were purified with Plus Gel Elution Kit (GMbiolab Co., Ltd., Taichung, Taiwan) following the manufacturer’s protocol.
    The preliminary attempt to use direct nucleotide sequencing for rDNA-ITS amplicons frequently failed to obtain good electropherograms, suggesting the possibility that each band contained different sequences derived from rDNA-ITS paralogs within individuals. Therefore, PCR products were cloned using the pGEM®-T Easy Vector System I cloning kit (Promega Co. Ltd., Madison, WI, USA) with competent cells ofEscherichia coli strain DH5α. The sequences of three cloned amplicons from one plant perCapsicum accession were determined using BigDye Terminator (version 3.1) cycle sequencing kit (Applied Biosystems Co. Ltd., Waltham, MA, USA), M13 forward (5′-GTAAAACGACGGCCAGT-3′) and reverse (5′-CAGGAAACAGCTATGAC-3′) primers, and an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Nucleotide sequences of rDNA-ITS determined are available in DDBJ/EMBL/GenBank (accession No. LC510550– 510603).

    2.3. Sequence Alignment and Phylogenetic Analysis

    The obtained rDNA-ITS sequences were aligned, using MUSCLE in the MEGA6 program [32], with other rDNA-ITS sequences downloaded from GenBank, including three sequences fromC. eximium,C. lycianthoides, andSolanum nigrum (Table 1). The sequence ofS. nigrum was used as the outgroup. The evolutionary history was inferred using MEGA6, with the maximum-likelihood method based on the general time reversible model [33]. Branch support was assessed by bootstrap resampling with 1000 replications.

    2.4. Evaluation of Genetic Diversity

    Based on the rDNA-ITS sequences, the number of nucleotide mutations, haplotype number and diversity, average number of nucleotide differences within population, and nucleotide polymorphism (θw) and diversity (π) were calculated using the DnaSP package ver. 6.0 [34].

    3. Results

    3.1. Morphology Characters of Each Capsicum Species

    The morphological traits of 26Capsicum accessions, including 11C. annuum, 4C. chinense, 3C. frutescens, 3C. baccatum, 1C. pubescens, 1C. chacoense, and 1C. exmium, were studied (Table 1,Supplemental Table S1). In addition to six key traits used for domesticated species classification, five traits were also surveyed (Figure 1 andFigure 2,Table S1). The results showed that seed color was black inC. pubescens, while straw in others. The corolla color was yellow inC. baccatum, white or purple inC. annuum, white or greenish inC. chinense, greenish inC. frutescens andC. eximium, purple inC. pubescens, and white inC. baccatum andC. chacoense. Some accessions inC. annuum andC. chinense had two or more flowers per axil, whereas others only had one. Two out of fourC. chinense accessions had calyx annular constriction. Flower position was erect inC. frutescens,C. chacoense, andC. eximium, erect or intermediate inC. annuum, erect, intermediate, or pendant inC. chinense, intermediate or pendant inC. baccatum, and intermediate inC. pubescens. All accessions of five domesticated species had been correctly classified according to morphological traits. However, theC. eximium accession was likely to be misclassified because it is characterized by a purple corolla [35]; this misclassification was also inferred from the following phylogenetic analysis based on rDNA-ITS in the present study. The morphological traits of theC. chacoense accession were in line with those described in literature [36].
    Plant growth habit was compact inC. annuum andC. baccatum, erect or compact inC. chinense, erect inC. frutescens andC. eximium, and prostrate inC. pubescens andC. chacoense. Anther color was yellow inC. baccatum andC. chacoense, while black in others. Calyx margin was dentate inC. annuum,C. pubescens andC. chacoense, smooth inC. chinense, and smooth or intermediate inC. frutescens andC. baccatum. Fruit shape was elongated or blocky inC. annuum, elongated, campanulate, or almost round inC. chinense, elongated inC. frutescens,C. eximium, andC. chacoense, elongated or campanulate inC. baccatum, and almost round inC. pubescens. Fruit pungency was present or absent inC. annuum, while present in others.

    3.2. Variations of Sequence Length and GC Content, and Genetic Diversity in rDNA-ITS

    We obtained the rDNA-ITS sequences from three subclones for each of the 26Capsicum accessions. In all accessions ofC. annuum, each had the same sequence among three subclones.C. chinense ‘Habanero’ and PI 441609 had the same sequence in two of the three subclones. In other accessions, three subclones from the same individual were different from one another. Additionally, rDNA-ITS sequences ofC. eximium,C. lycianthoides, andSolanum nigrum were obtained from the NCBI database. Lengths of ITS1 were from 140 to 245, 138 to 161 for 5.8S rDNA, and 200 to 233 for ITS2 (Table S2). The GC percentages of ITS1 were from 51.9% to 72.0%, 43.8% to 55.0% for 5.8S rDNA, and 52.7% to 70.0% for ITS2 (Table S2).
    The genetic diversity of rDNA-ITS in each species was evaluated (Table 2). InC. annuum, 7 of 11 sequences were the same haplotype, and 2 of the 10 sequences were the same haplotype inC. chinense, whereas sequences of other species were all different haplotypes. Therefore, haplotype diversity was lower inC. annuum andC. chinense than in others. We used two indicators of genetic diversity: nucleotide polymorphism (θw) [37], which reflects the number of mutation nucleotides in a population, and nucleotide diversity (π) [38], which reflects the average number of nucleotide differences between two sequences selected at random. The genetic diversity of rDNA-ITS inC. annuum was lowest among all species used in this study according to nucleotide polymorphism and diversity (Table 2).

    3.3. Phylogenetic Relationship Between Capsicum Species Based on rDNA-ITS

    We constructed a phylogenetic tree using the maximum-likelihood method based on the rDNA-ITS sequences (Figure 3,Figure S1). The phylogenetic tree showed that in-group species were divided into two clades (C. pubescens clade vs. the others) with 50% support value (Figure 3). TheC. annuum clade then formed a monophyletic group with 71% support value. In addition to theC. annuum clade and theC. pubescens clade, the phylogenetic tree formed theC. chinense andC. frutescens clade and theC. baccatum clade, although the bootstrap value was less than 50%. TheC. chinense clade was derived from theC. chinense andC. frutescens clade.C. eximium PI 645681 existed in theC. chinense andC. frutescens clade, whileC. chacoense PI 273419 existed in theC. baccatum clade.

    4. Discussion

    All accessions of five domesticated species had been correctly classified by key morphological characteristics for domesticatedCapsicum species classification [11].C.chacoense PI 273419 would be correctly classified because the morphological traits were not in conflict with those described in the literature [36]. However,C. eximium PI 645681 may be misclassified, because the corolla color of PI 645681 was greenish, while it is purple in general [35]. According to the key morphological characteristics of domesticatedCapsicum species [11],C. eximium PI 645681 should be classified asC. frutescens. AlthoughC. eximium PI 645681 belonged to theC. chinense andC. frutescens clade, theC. eximium (AY665841) obtained from NCBI did not belong to any clades in the phylogenetic tree on rDNA-ITS (Figure 3). The deviation ofC. eximium (AY665841) in the tree seemed to reflect thatC. eximium is not very close to domesticatedCapsicum species [8]. Therefore,C. eximium PI 645681 seemed to beC. frutescens considering its morphological and molecular traits.
    The rDNA-ITS is a tandem repeat unit of hundreds or thousands of copies [39]. Individual copies of rDNA-ITS are homogenized in the same sequence type via concerted evolution, which is thought to be induced by unequal crossing over and high frequency of gene conversion [40]. However, in this study, rDNA-ITS polymorphisms within individuals were detected inCapsicum species, except inC. annuum, suggesting that concerted evolution was incomplete. The polymorphism of rDNA-ITS paralogs within individuals is also observed in other plants [41,42]. Although the mechanism of incomplete concerted evolution has not been elucidated thoroughly, some reports have shown that it occurs in cases where hybridization is involved [43], or when paralogous rDNA-ITS sequences are present in a non-homologous locus [44]. Although further analysis is needed to determine the factors of incomplete concerted evolution, this study revealed that it occurs in rDNA-ITS ofCapsicum, except forC. annuum.
    The genetic diversity inCapsicum species was investigated using rDNA-ITS (Table 2). The results suggested that the genetic diversity inC. annuum was much lower than otherCapsicum species. Moreover, the genetic differences between lines ofC. annuum were small in the phylogenetic tree (Figure S1). Low diversity ofC. annuum was also observed in the analysis ofC. annuum lines worldwide using 746k polymorphic sites [45]. Only Japanese cultivars of theC. annuum accessions were used in this study. It is possible that the genetic diversity ofC. annuum, especially in Japanese cultivars, has decreased due to factors like intensive selective breeding.
    The phylogenetic tree based on rDNA-ITS formed theC. annuum clade, theC. chinense andC. frutescens clade, theC. baccatum clade, and theC. pubescens clade. Therefore, rDNA-ITS can distinguish domesticatedCapsicum species, but this was difficult forC. chinense andC. frutescens (Figure 3). However, the clades supported by bootstrap values above 50% were just theC. annuum clade and theC. pubescens clade. Therefore, rDNA-ITS should be used only for a rough identification of domesticatedCapsicum. TheC. chinense clade might be divided into two groups: one consisting of mainlyC. frutescens and another consisting ofC. chinense, suggesting thatC. chinense may have evolved fromC. frutescens. Consequently,C. chinense may be a cultivated variant ofC. frutescens [46]. It was unclear to which complexC. chacoense belongs [8,15], but according to the phylogenetic tree based on rDNA-ITS, it belonged to theC. baccatum complex (Figure 3); it also had yellow anther, the same morphological trait asC. baccatum.
    The phylogenetic analysis using rDNA-ITS almost agreed with previous studies using molecular markers [15,16,18,19,20] and DNA barcoding [8] with sequences except rDNA-ITS in terms of the formation of species clades and phylogenetic relations, although rDNA-ITS could not differentiate betweenC. chinense andC. frutescens. The analysis using molecular markers or DNA barcoding except rDNA-ITS requires manyCapsicum species for species identification, and thus takes a lot of effort. On the other hand, many sequences of the rDNA-ITS region inCapsicum species have been accumulated in the NCBI database. Therefore, phylogenetic analysis using the rDNA-ITS region would be a low-effort method forCapsicum species identification.
    The evolution of the morphological traits of each species on a phylogenetic tree was also studied (Figure 3). In the key morphological traits for domesticated species classification, black seed and purple corolla were the characteristic traits of theC. pubescens clade [46]. Most accessions in theC. chinense andC. frutescens clade had a greenish corolla (Figure 3,Table S1) [46,47], although it was not a characteristic trait ofC. chinense andC. frutescens clades. In the traits not used for domesticated species classification, common traits were found in each clade. In theC. pubescens clade, prostrate growth habit and almost-round fruit were the characteristic traits [46]. A dentate margin of the calyx was aC. pubescens characteristic trait (Figure 3,Table S1), although a large-scale survey is needed to confirm that. The phylogenetic tree inFigure 3 suggested that yellow anther in theC. baccatum clade seemed to be a trait obtained after differentiating from a common ancestor ofC. annuum,C. chinense, andC. frutescens. It was necessary to investigate the anther color of other species in theC. baccatum complex to verify yellow anther as the characteristic trait of theC. baccatum complex.C. tovarii andC. praetermissum, belonging to theC. baccatum complex, had blue and purple anthers, respectively [13,48]; therefore, yellow anther was not a characteristic trait of theC. baccatum complex, but it may be useful for the identification of some species belonging to theC. baccatum complex. In theC. annuum clade, dentate calyx margin was a trait distinct from theC. chinense andC. frutescens clades (Figure 3,Table S1). However, dentate calyx margin cannot be used as a characteristic trait because there were some lines inC. annuum that had smooth or intermediate calyx margin [49]. AllC. frutescens accessions had erect flower position and growth habit, but these traits cannot distinguishC. frutescens fromC. annuum andC. chinense (Figure 3,Table S1). No new traits were found to distinguishC. annuum,C. chinense, andC. frutescens through the key traits of domesticated species, but based on their morphological traits, they were closely related.
    This study revealed the phylogenetic relationships betweenCapsicum species based on 11 morphological traits and rDNA-ITS sequences, and the low genetic diversity ofC. annuum cultivars based on rDNA-ITS sequences. These results may be used for future studies on species differentiation and genetic resources inCapsicum.

    Supplementary Materials

    The following are available online athttps://www.mdpi.com/2311-7524/6/4/87/s1, Figure S1: Molecular phylogenetic tree based on a maximum-likelihood analysis of rDNA-ITS sequences with evolutionary distances, Table S1: Morphological traits of Capsicum investigated in the present study, Table S2: Length range (bp) and GC content (%) analysis of ITS sequences.

    Author Contributions

    Conceptualization, T.T.; methodology, T.T.; validation, K.S., S.Y., and T.T.; investigation, K.S.; data curation, K.S.; writing—original draft preparation, K.S.; writing—review and editing, K.S., S.Y., and T.T.; visualization, K.S.; supervision, S.Y.; project administration, T.T.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

    Funding

    This research was funded by JSPS KAKENHI (Grant Number JP17K15224 and JP20K05988) from the Japan Society for the Promotion of Science.

    Acknowledgments

    We are grateful to the National Agriculture and Food Research Organization Genebank (Tsukuba, Japan) and the USDA/ARSCapsicum germplasm collection (Griffin, GA, USA) for providingCapsicum seeds.

    Conflicts of Interest

    The authors declare no conflict of interest.

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    Horticulturae 06 00087 g001 550
    Figure 1. Traits for classification of domesticated species.
    Figure 1. Traits for classification of domesticated species.
    Horticulturae 06 00087 g001
    Horticulturae 06 00087 g002 550
    Figure 2. Other traits surveyed in this study.
    Figure 2. Other traits surveyed in this study.
    Horticulturae 06 00087 g002
    Horticulturae 06 00087 g003 550
    Figure 3. Molecular phylogenetic tree based on a maximum-likelihood analysis of rDNA ITS sequences, and evolution of morphological traits inCapsicum. Bootstrap = 1000 replicates (any clade with a hyphen has a bootstrap < 50). Trait state changes are indicated by black rectangles with the number of the trait on the phylogenetic tree. Traits 1 to 6 (written in red) are the key morphological traits of domesticated species.
    Figure 3. Molecular phylogenetic tree based on a maximum-likelihood analysis of rDNA ITS sequences, and evolution of morphological traits inCapsicum. Bootstrap = 1000 replicates (any clade with a hyphen has a bootstrap < 50). Trait state changes are indicated by black rectangles with the number of the trait on the phylogenetic tree. Traits 1 to 6 (written in red) are the key morphological traits of domesticated species.
    Horticulturae 06 00087 g003
    Table
    Table 1.Capsicum and rDNA-ITS accessions used in this study.
    Table 1.Capsicum and rDNA-ITS accessions used in this study.
    SpeciesAccession No.Cultivar and Line NameOriginSourcesrDNA-ITS Accession No.Reference of rDNA-ITS
    C. annuumJP 32511Sapporo Onaga NanbanJapanNAROaLC510550This study
    JP 32520Mie MidoriJapanNAROaLC510551This study
    JP 32523AkashiJapanNAROaLC510552This study
    JP 32549YatsubusaJapanNAROaLC510553This study
    JP 32555ZairaiJapanNAROaLC510554This study
    JP 32562NikkoJapanNAROaLC510555This study
    JP 32566FushimiamanagaJapanNAROaLC510556This study
    JP 82498TakanotsumeJapanNAROaLC510557This study
    JP 123787ShosukeJapanNAROaLC510558This study
    JP 124339MurasakiJapanNAROaLC510559This study
    PI 640723ShishitoJapanNAROaLC510560This study
    C. chinensePI 159236USAUSDAbLC510561-510563This study
    PI 315008Scarlet LanternPeruUSDAbLC510564-510566This study
    PI 438614HabaneroMexicoUSDAbLC510567-510568This study
    PI 441609BrazilUSDAbLC510569-510570This study
    C. frutescensPI 439512Rat chiliMexicoUSDAbLC510571-510573This study
    PI 586675TabascoUSAUSDAbLC510574-510576This study
    PI 634826Greenleaf TabascoUSAUSDAbLC510577-510579This study
    C. baccatumPI 640882PeruUSDAbLC510580-510582This study
    PI 640885IndiaUSDAbLC510583-510585This study
    PI 653669ColombiaUSDAbLC510586-510588This study
    C. pubescensPI 593624Chile de caballoGuatemalaUSDAbLC510589-510591This study
    Grif 1613UnknownUSDAbLC510592-510594This study
    Grif 1614MexicoUSDAbLC510595-510597This study
    C. chacoensePI 273419ArgentinaUSDAbLC510598-510600This study
    C. eximiumPI 645681AustraliaUSDAbLC510601-510603This study
    MexicoAY665841[27]
    C. lycianthoidesUSADQ314158[28]
    Solanum nigrumChinaFJ980391[29]
    a National Agriculture and Food Research Organization Genebank (Tsukuba, Japan),b USDA/ARSCapsicum germplasm collection (Griffin, GA, USA).
    Table
    Table 2. Evaluation of genetic diversity within species based on rDNA-ITS.
    Table 2. Evaluation of genetic diversity within species based on rDNA-ITS.
    SpeciesNumber of Plant MaterialsNumber of SequencesaNumber of HaplotypesHaplotype DiversityNumber of Mutation NucleotidesNucleotide Polymorphism (θw)Average Number of Nucleotide DifferencesNucleotide Diversity (π)
    C. annuum111140.4909190.008081.781820.00476
    C. chinense41090.95556460.0366114.555560.03239
    C. frutescens39911500.1338540.472220.09922
    C. baccatum39911100.1079534.111110.09187
    C. pubescens3991740.0618818.527780.04449
    C. chacoense133184n.d.56n.d.
    C. eximium133149n.d.32.66667n.d.
    Total2657480.9812790.1443934.3230.08192
    a Number of sequences indicates the number of subclones, excluding overlapped subclones in each accession. “n.d.” indicates that the calculations could not be performed due to insufficient numbers of sequences.
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    Shiragaki, K.; Yokoi, S.; Tezuka, T. Phylogenetic Analysis and Molecular Diversity ofCapsicum Based on rDNA-ITS Region.Horticulturae2020,6, 87. https://doi.org/10.3390/horticulturae6040087

    AMA Style

    Shiragaki K, Yokoi S, Tezuka T. Phylogenetic Analysis and Molecular Diversity ofCapsicum Based on rDNA-ITS Region.Horticulturae. 2020; 6(4):87. https://doi.org/10.3390/horticulturae6040087

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    Shiragaki, Kumpei, Shuji Yokoi, and Takahiro Tezuka. 2020. "Phylogenetic Analysis and Molecular Diversity ofCapsicum Based on rDNA-ITS Region"Horticulturae 6, no. 4: 87. https://doi.org/10.3390/horticulturae6040087

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    Shiragaki, K., Yokoi, S., & Tezuka, T. (2020). Phylogenetic Analysis and Molecular Diversity ofCapsicum Based on rDNA-ITS Region.Horticulturae,6(4), 87. https://doi.org/10.3390/horticulturae6040087

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