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Scientia Horticulturae 126 (2010) 186–191

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

lassifying vegetable genetic resources—A case study with domesticatedapsicum spp.

odomiro Ortiza,∗, Francisco Delgado de la Flora, Gregorio Alvaradob, José Crossab

Programa de Investigaciones en Hortalizas, Universidad Nacional Agraria – La Molina (UNALM), Av. La Universidad s/n, La Molina, Lima 12, PeruCentro Internacional de Mejoramiento de Maíz y Trigo (CIMMYT), Apdo. Postal, México, D.F., Mexico

r t i c l e i n f o

rticle history:eceived 11 June 2010ccepted 6 July 2010

a b s t r a c t

Great variation for many traits seems to be available in Capsicum genetic resources but yet to be fully usedby plant breeders. Adequate characterization of genebank accessions is needed to facilitate the utiliza-tion of germplasm by end-users. The aim of this research was to use multivariate techniques with bothqualitative and quantitative descriptors in the five domesticated species of Capsicum for grouping them

eywords:hiliescriptorsenetic resourcesot pepperultivariate analysisodified location modelard

after assessing inter- and intra-specific variation. Key qualitative descriptors (seed color, corolla colorand spot, calyx constriction, numbers of flowers per node and filament color) served for assigning mostaccessions to their respective species, whereas intra-specific multivariate diversity was better assessedby quantitative descriptors such as fruit length/width ratio, numbers of days to flowering, leaf width,and anther, filament and pedicel length. The modified location model an adequate method for classifyingCapsicum accessions using quantitative descriptors.

© 2010 Elsevier B.V. All rights reserved.

. Introduction

Hot peppers (Capsicum spp.) are a major vegetable and the mostmportant spice crop in the world. They are eaten fresh, dehydratedr processed in several types of meals worldwide (Reifschneidert al., 2009). Their capsacinoid compounds, which are responsibleor the pungent flavor when eating the fruits, are widely used for

edicinal applications (Bosland and Votava, 2000). Matured redeppers are also an important source for natural red pigments, andther types are grown as ornamental plants in home gardens (Nagyt al., 2005).

Peppers were domesticated in the American continent wherearmers have grown the crop for several thousand years (Pickersgill,997). Perry et al. (2007) found a starch from hot peppers on ancientottery and stone tools at various archaeological sites (ranging fromahamas to southern Peru); being the oldest about 6100 years ago.heir findings suggest multiple domesticated hot pepper species by

bout 4000 years ago. The most important Capsicum species (out ofpproximately 30 in this genus) are Capsicum annuum, Capsicumaccatum, Capsicum chinense, Capsicum frutescens and Capsicumubescens, has attracted the attention of scientists for collection and

∗ Corresponding author. Current address: Martin Napanga 253, Apt. 101, Miraflo-es, Lima 18, Peru.

E-mail address: rodomiroortiz@gmail.com (R. Ortiz).

304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2010.07.007

conservation due to their economic importance and use (Heiser andSmith, 1953).

C. annuum is the most widely grown species, includes bothlarge-fruited bell peppers and smaller pungent types (e.g. Gua-jillo, Jalapeno, Morrón and Serrano from Mexico), and comprisesa species complex with C. chinense and C. frutescens, which areknown in Mexico as Habanero and Tabasco, respectively. C. bacca-tum (ají Amarillo, Mirasol or Pacae), C. chinense (ají Arnaucho, Limo,Mochero or Panca) and C. pubescens (Rocoto) are the predominantspecies for commercial hot peppers in Peru because of their originand adaptation to the horticultural systems of this country (Ortizand Delgado de la Flor, 1990). Local pungent types of C. annuumand C. frutescens are called ají Cerezo and Pipí de Mono, respec-tively. More recently Peruvian growers included paprika in theircoastal fields, and the country became one of the largest exportersof this crop, worldwide.

The variation in plant and fruit color, shape, length and pun-gency of Capsicum species seems to depend on human preferences,but yet to be fully exploited by plant breeders, because breed-ing programs concentrate on the non-pungent cultivars of C.annuum and mostly for host plant resistance to many biotic stresses

affecting its cultivation. Evaluation and clustering of Capsicumaccessions (mainly landraces) are helpful to establish evolution-ary relationships along with considering the history of the crop inthe region, and for their efficient utilization in crop breeding, e.g.by crossing apparently unrelated genotypes. The great intra- and

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nter-specific variability in Capsicum should be helpful for breedingrograms.

Multivariate data on continuous and categorical traits areollected for genetic resources conservation and plant breedingCrossa and Franco, 2004). Individual genotypes can be conceptual-zed as being located in a multidimensional space in which there isne dimension for each trait. The shape and structure of the groupsf genotypes in this multidimensional space in unknown, but thessociation (correlation) between traits influences the shape of theroups, and the structure is affected by the true composition ofhe groups. Hierarchical, non-hierarchical, and statistical classifi-ation methods attempt to recover, as much as possible, the truehape and structure of the underlying groups. In genetic resourcesonservation and the formation of core subsets, the main objec-ive is to select accessions that best represent the entire collectionith the minimum loss of genetic diversity (Ortiz et al., 2008a,b).

herefore, the best numerical classification strategy is the onehat produces the most compact and well-separated groups (i.e.,

inimum variability within each group and maximum variabilitymong groups).

Two type of techniques, geometric and statistical, can be usedequentially in order to create what is known as a ‘classificationtrategy’ in two stages, which are expected to display the charac-eristics of both types of techniques. The underlying idea is thathe initial groups formed by the geometric technique are based onhe principle that rules such a technique; for example, a minimumariance within groups (Ward, 1963). Then the mixture distribu-ion approach named the Modified Location Method (MLM) actspon the previous clusters, particularly regarding shape, direction,nd volume of the clouds of points that make up the groups in thedimensional space. The maximization of the likelihood functionegins at a point that has been reached using the geometric tech-ique and it will then reach a peak (which could be local) nearhe starting point that contains the characteristics of the geomet-ic technique. This two-stage classification approach is called the

ard-MLM method (Franco et al., 1998).The objective of this research was to assess the value of the two-

tage multivariate technique for classifying Capsicum accessionssing available quantitative and qualitative descriptors.

. Materials and methods

About 90 Capsicum accessions from the genebank of the Pro-rama de Investigaciones en Hortalizas (PIH) of the Universidadacional Agraria La Molina (UNALM, Lima, Peru) were included for

his research (Table 1). The experiments were planted in the fieldsf PIH-UNALM located about 5 km from Lima (12◦15′S, 76◦57′W,38 m above sea level) Eight week old seedlings were transplanted

n the field at a spacing of 0.9 m between rows × 0.5 among plants.he field experiments started during the spring (October) or sum-er seasons (January) and the harvest was in the autumn (either

ate March or May). The temperatures ranged from 14.5 to 28 ◦Cwith an average of 19.7 ◦C), whereas the relative humidity variedetween 53.5 and 98% (with an average of 83.2%). The sandy loamoil had a pH 7.7 and low N, P and K levels. At planting 70 kg ha−1 of, 100 kg ha−1 of P2O5 and 80 kg ha−1of K2O were therefore used,

able 1ccession and descriptor numbers used for assessing variation and classifying Capsicum g

Set Descriptors C. annuum C. baccatum

1 12a 3 112 11a 17 163 47b 6 7

a All qualitative traits.b 26 qualitative and 21 quantitative traits, respectively

urae 126 (2010) 186–191 187

and 3 months after planting 80 kg ha−1 of N were added. The fieldswere irrigated frequently.

The experimental field layout was a completely randomizeddesign with a minimum of five replicates per accession. Sets of qual-itative and quantitative descriptors showing continuous variationwere selected from the available literature on the crop (IPGRI etal., 1995). Descriptors were recorded in five samples per plant andusing a minimum of five plants per accession. Quality descriptorswere evaluated in the most representative plant of the accessionand compared with the other plants to observe if there was traitsegregation.

The average clustering method was used in the first twoexperiments with qualitative descriptors for grouping Capsicumaccessions. The data were standardized to get a mean = 0 andvariance = 1. Clusters were merged sequentially based on anEuclidean distance (Ed) using an algorithm that initially usedeach accession as a cluster. The Euclidean distance (Ed) betweentwo accessions P1 and P2 was a straight line defined as Ed (P1,P2) = [(Y11 − Y12)2 + (Y21 − Y22)2 + · · · + (Yp1 − Yp2)2]1/2, where Yij wasthe frequency of a descriptor state “i” in individual “j”, which couldhave the values of 0 (if they match) or 1 (for a mismatch). Thedendrogram showed the diagramatic depiction of the Euclideandistance coefficient. Accessions with close genetic distances wereplaced in close proximity in this hierarchical analysis.

The initial groups in the third experiment first defined usinga hierarchical clustering method based on the minimum vari-ance within groups’ principle (Ward, 1963). The MLM was thenused with those groups (Ward-MLM) because improves the initialgroupings (Franco et al., 1998). It assumes that the mean vectorsof the ysj random vectors do not depend on the multinomial cellin which they appear, but they only depend on the subpopulation.The number of groups was defined using pseudo-F and pseudo-t2

criteria (SAS, 1999) combined with the likelihood profile associatedwith the likelihood-ratio test (Mardia et al., 1979). The sequentialclustering strategy allows modifying the form of the initial groupsobtained by the Ward strategy (spherical clusters) to a form (ellip-tical clusters) that permits the formation of more homogeneousgroups.

3. Results

Wide variability was note in all the characters (except for thelanceolate leaf shape shared by all accessions) included as descrip-tors in this research. Key traits used by taxonomy for discriminatingamong Capsicum species were helpful individually for assigningmost accessions to each of the species. They were seed color, corollacolor and spot, calyx constriction, numbers of flowers per nodeand filament color. For example, accessions of C. chinense showedup to 3 white flowers per node and calyx constriction, whereas C.pubescens had distinctive purple flowers and black seeds, and a spotin the corolla was seen only in C. baccatum accessions. Accessions

of C. annuum exhibited a solitary white flower per node with non-purple filaments while C. frutescens showed a greenish corolla andpurple filaments.

The dendrograms ensuing from the average linkage clusteringof qualitative data (Figs. 1 and 2) did not allow a clear cut grouping

rown in Peru.

C. chinense C. frutescens C. pubescens

3 – –9 – –6 1 1

188 R. Ortiz et al. / Scientia Horticulturae 126 (2010) 186–191

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ig. 1. Dendrogram of 18 Capsicum accessions using the average linkage clusteringevel of cut.

f the most popular domesticated Capsicum species: C. annuum,. baccatum and C. chinense. In the first set of accessions (Fig. 1),

he larger cluster (below the level of cut) includes all three specieshile the other four clusters were defined by one or a couple of

ccessions of the same species. Similar results were found for theargest cluster in the second set of accessions (Fig. 2). However,

ig. 2. Dendrogram of 42 Capsicum accessions using the average linkage clustering methoevel of cut.

od after using 12 qualitative descriptors. The perpendicular red line indicates the

there were other well defined clusters for C. baccatum (3) or the C.annuum–C. chinense complex but not single feature could explain

any of these multivariate groupings.

Pseudo-F and pseudo-t2 criteria indicated that the optimumnumber of groups when using quantitative descriptors for group-ing Capsicum germplasm was four because the log-likelihood

d method after 11 qualitative descriptors. The perpendicular red line indicates the

R. Ortiz et al. / Scientia Horticult

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ig. 3. Profile of the log-likelihood function (log L) versus number of groups (nroups). The highest increase in the log-likelihood function from 3 groups to 4 groupss marked with an arrow.

rofile, which is associated with the likelihood-ratio test, showedhat the highest increase of the likelihood function was from the- to the 4-group level (Fig. 3). Fig. 4 shows the distribution ofhe accessions of the five domesticated Capsicum species andhe groups along the first two canonical variables for the fourlusters formed by the Ward-MLM method. The first canonicalariable (CAN 1 in the X-axis) was correlated mostly with numbersf days to flowering (0.6271), filament length (0.5576) and leaf

idth (0.5468), whereas fruit length/with ratio (0.7001), pedicel

ength (0.5465) and anther length (0.5359) loaded the secondanonical variable (CAN 2 in the Y-axis) (Table 2). There werehree clear species clusters, one each for the bell peppers of C.nnuum, other for the long-fruited ají Panca of C. chinense, and

Fig. 4. Bi-plot of the first two canonical variables and the distribution o

urae 126 (2010) 186–191 189

the last for the late flowering ají Amarillo of C. baccatum. Thefourth cluster included mostly the pungent types of C. annuum,one accession each for early maturing, almost round or triangulateaccessions of C. baccatum and C. chinense, and the sole accessions ofC. pubescens and C. frutescens, which showed the smallest fruit. Thesequential Ward-MLM classification strategy reveals that sometypes within each Capsicum species (i.e., intra-specific variability)are well defined by quantitative descriptors, which are often usedin natural systems of germplasm classification.

4. Discussion

Germplasm characterization provides an important link for con-servation through use of the genetic endowment of a species. Thesystematic description of each accession should lead to classifica-tion in small and well-organized groups of genebank accessionsthat will facilitate their enhanced utilization. Most of the descrip-tors, which are identifiable and measurable traits, for germplasmcharacterization and evaluation are species-specific. Corolla colorand spots, filament and seed color, number of flowers per nodeand calyx constriction are the key qualitative descriptors for dis-criminating among the five domesticated species. The lack ofsound clustering using the average linkage methods and qualitativedescriptors in the first two experiments suggests that multivari-ate analysis of qualitative traits that are not key for classifyingaccessions to their respective species may mislead grouping of thisCapsicum germplasm. Likewise, a limited number of inappropri-ate descriptors may lead to misleadingly simplistic interpretations

of dendrograms ensuing from multivariate analysis. The non-keyspecies-clustering traits used in both experiments with C. annuum,C. baccatum and C. chinense were growth habit, stigma exertion,pubescence, ovary position, sepal spot, anther color, and fruit colorand shape.

f 22 Capsicum accessions after using 23 quantitative descriptors.

190 R. Ortiz et al. / Scientia Horticulturae 126 (2010) 186–191

Table 2Correlations among quantitative descriptors and the first three canonical axes (CAN1, CAN2, and CAN3) from the Ward-MLM method considering four groups of Capsicumaccessions.

Descriptor CAN1 CAN2 CAN3

Plant height 0.002790 0.294066 −0.287231Plant canopy width ratio −0.293171 0.277486 −0.519547Flowering days 0.627108 −0.022383 0.008510Stem width −0.385425 0.217475 0.215843Leaf length 0.349985 0.518996 0.141038Leaf width 0.546783 0.405390 0.128679Leaf length/width ratio −0.566541 0.006090 −0.216241Pedicel length −0.119806 0.546482 −0.076654Filament length 0.557617 −0.162155 0.120135Corolla length 0.118932 0.346879 0.369388Corolla width 0.040844 −0.065755 0.627894Corolla length/width ratio 0.135800 0.204531 −0.215224Anther length 0.002168 0.535930 −0.002049Fruit weight −0.009978 −0.350438 0.890263Fruit length 0.283972 0.385926 0.427690Fruit width −0.051149 −0.453603 0.792866Fruit length/width ratio 0.291829 0.700120 −0.213309Fruit wall thickness −0.084483 −0.426213 0.676157

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Seed width 0.076214Seed number 0.257235Maturity days 0.448489

In recent years, molecular markers are also assisting to assessiodiversity, identify duplicates in genebanks, and estimate geneticelationships. For example, diagnostic random amplified polymor-hic DNA (RAPD) markers discriminate among Capsicum speciesRodriguez et al., 1999). They also allowed reassigning a fewccessions to the most appropriate species, which were early mis-lassified based on qualitative descriptors. The findings of ourlustering with quantitative descriptors for C. annuum are similaro those by Paran et al. (1998) using RAPD and amplified frag-

ent length polymorphisms (AFLP); i.e., the large-fruited sweetultivars from the small-fruited pungent peppers, and the for-er group showed less divergence than the latter. Quantitative

rait variation among bell peppers of C. annuum is indeed limitedompared with that among pungent types or other domesticatedpecies, though restriction fragment length polymorphisms (RFLP)evealed similar patterns of variation (Prince et al., 1992, 1995).ery recently, Ince et al. (2009, 2010) used a polymerase chaineaction (PCR) based approach involving the directed amplifica-ion of minisatellite DNA region (DAMD-PCR), and RAPD markersased on touch-down PCR (Td-RAPD-PCR) to establish genetic rela-ionships within and between Capsicum species. They divided theccessions into four major groups, three of which corresponded tohe three distinct complexes: C. annuum–C. chinense–C. frutescens,. baccatum and C. pubescens. The remaining group belongs to theild C. chacoense. The Capsicum complexes contain species that

llow for genetic exchanges, albeit with some difficulty, betweenpecies (Bosland and Votava, 2000). Pickersgill (1997) indicate thatarriers to inter-specific gene transfer are similar to those found

n other genera of Solanaceae, e.g. unilateral incompatibility, post-ertilisation abortion, and nucleo-cytoplasmic interactions leadingo male sterility or other abnormalities.

DNA markers along with morphological traits have been alsosed for cultivar protection (Lefebvre et al., 2001), or for calculatingenetic distances and establishing their relationships with hetero-is (Geleta et al., 2004). Accessions of C. annuum to were assigned toeterotic groups and early maturing could be predicted using theenetic distances among parents (Geleta et al., 2004). Quantitative

raits have been also used for calculating distances among parentsor further hybrid breeding between hot and sweet peppers, thoughesults showed a lack of correlation between phenotypic diversitymong parents and heterosis for yield (Shifriss and Sacks, 1980).urther research by Thul et al. (2009) showed that that taxonomic

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characteristics are not always related to agronomic traits, therebysuggesting the need for characterizing Capsicum germplasm withquantitative descriptors of economic use or affecting crop field per-formance (as we did in our third experiment).

Our results of the third experiment indicated that the Ward-MLM strategy is an adequate method for classification usingquantitative descriptors, particularly when the relation betweenthe number of observations to be classified and the total numberof cells is such that any initial cluster solution will produce emptycells. Franco et al. (1998) found that using all the information (con-tinuous and categorical variables) produces better clusters than theuse of some information (only continuous variables). The use ofdifferent forms of categorical variables does not limit the properfunctioning of the strategy. The two-stage strategy begins with acluster built to minimize the sum of squares within a group; forthis reason, it is expected that in the Ward-MLM strategy, the max-imum reached by the likelihood be nearer to the initial likelihood,that a smaller number of observations be reclassified, and hence,the reduction of the generalized variance.

This research also confirms Pickersgill’s view (1997) that thereare significant amounts of genetic diversity within as well asbetween Capsicum species, which can be a useful source for fur-ther genetic betterment of the crop. In this regard, there are severalassessments of molecular, qualitative and quantitative variationof Capsicum accessions from Brazil (Sudré et al., 2010), Colom-bia (Medina et al., 2006; Pardey et al., 2006; Toquica et al., 2003),China (Chen et al., 2009), Guatemala (Guzmán et al., 2005), India(Choudhary and Samadia, 2004), Italy (Lanteri et al., 2003), Mexico(Loaiza-Figueroa et al., 1989; Votaba et al., 2005), Nigeria (Adetula,2006), Peru (Ortiz and Delgado de la Flor, 1990), Taiwan (Rodriguezet al., 1999, Turkey (Bozokalfa et al., 2009), southern USA (Votabaet al., 2005) and former Yugoslavia (Zewdie and Zeven, 1990); anda core collection was established with 137 accessions that cap-tured 100% of the known morphological variability of the speciesincluded, namely C. annuum, C. baccatum, and C. chinense (Zewdieet al., 2004). Although the extent of variation and their clusteringtraits (many of them fruit descriptors) and methods vary, all of them

provide entry points for utilizing this variation for Capsicum breed-ing. A note by Pickersgill (1997) information on the occurrenceand effects of barriers to gene transfer among species are availableto guide Capsicum breeders to use inter-specific hybridization forgermplasm enhancement. Likewise, more saturated genetic link-

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ge maps (Barchi et al., 2007a; Kang et al., 2001; Livingstone et al.,999, Paran et al., 2004) and advances in quantitative trait loci map-ing (Barchi et al., 2007b; Ben Chaim et al., 2001, 2003a,b; Lefebvrend Palloix, 1996; Ogundiwin et al., 2005; Rao et al., 2003, Zygiert al., 2005) in Capsicum will pave the way for tapping the geneticesources for breeding-by-design this crop.

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