Morphological and Genetic Diversity Among and Within Common Bean

Scientia Horticulturae 180 (2014) 72–78

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Scientia Horticulturae

journal homepage: www.elsevier.com/locate/scihorti

Morphological and genetic diversity among and within common bean

(Phaseolus vulgaris L.) landraces from the Campania region

(Southern Italy)

a b b a a,∗

Daria Scarano , Fernando Rubio , Juan José Ruiz , Rosa Rao , Giandomenico Corrado

Dipartimento di Agraria, Università di Napoli Federico II, Portici, NA, Italy

Department of Applied Biology, Miguel Hernandez University of Orihuela, Orihuela, Spain

a r t a b

i s

c l e i n f o t r a c t

Article history: The common bean (Phaseolus vulgaris L.) is arguably the most important food legume and a fundamental

Received 3 May 2014

source of proteins especially for rural societies. In several countries, this species is characterized by a

Received in revised form 3 October 2014

number of locally adapted landraces and many of them are at risk of extinction. In Italy, common bean

Accepted 9 October 2014

cultivation has always been a typical element of rural economies especially in the Southern regions. We

Available online 28 October 2014

carried out an investigation of the morphological and genetic diversity in 25 common bean populations

cultivated in the Campania region (Southern Italy). We analyzed 26 qualitative and 11 quantitative

Keywords:

traits following the IPGRI descriptors. Furthermore, 10 SSRs were employed to examine genetic poly-

SSR

Diversity morphism, differentiation and population structure. Molecular and morphological data distinguished all

the landraces under investigation. A considerable phenotypic diversity among landraces was observed

Phenotypic variation

Genetic resources for many characters, including some related to agronomical performance. At molecular level, all the

SSRs were polymorphic, with an average of 8.5 alleles per locus. Moreover, the vast majority of the

landraces (92%) displayed intra-varietal differences. Our work indicated the presence of a wide-ranging

variation among and within cultivated common bean landraces. Moreover, it provided evidence that

the implementation of measures for their on-farm conservation, management and promotion should be

useful also to preserve genetic variability.

1. Introduction environments, the geographical isolation of several growing areas,

the cultivation technique (e.g. the frequent consociation with

Common bean (Phaseolus vulgaris L.) is a world-wide culti- local maize varieties) as well as aesthetical and organoleptic pre-

vated legume of agricultural interest in many countries (Lioi and ferences of speciﬁc areas. In Mediterranean countries, common

Piergiovanni, 2013). In Italy, it represents the main grain legume bean cultivation is present nearly in all the traditional agri-

for direct human consumption. Common bean was introduced cultural settings (Lioi and Piergiovanni, 2013; Negri and Tosti,

into Italy from the New World in the 16th century (Bitocchi 2002). The strong link between rural agriculture and common

et al., 2012; Piergiovanni and Lioi, 2010). The ﬁrst documenta- bean has given rise to landraces that are frequently associated

tion dates to 1532 and refers to a donation of seeds from the to restricted areas (Negri and Tosti, 2002; Piergiovanni and Lioi,

Spanish Emperor Charles V to the Pope Clemente VII (Lioi and 2010).

Piergiovanni, 2013). Since its introduction into Europe, this species During the last decades, in Italy as well as in other developed

has experienced an adaptive radiation being, for instance, cul- European countries, the cultivated area of P. vulgaris has declined

tivated for the production of dry seeds, shell seeds and green because of economic (e.g. low competitiveness, decreasing price on

pods (Angioi et al., 2010; Lioi and Piergiovanni, 2013). Bean diver- the international market), social (e.g. larger availability of animal

siﬁcation can be ascribed to different factors such as the need proteins, its strong identiﬁcation with a rural diet) and technical

for adaptation to the soil type and climatic conditions of new reasons (e.g. diffusion of fertilizers, mono-culture and mechanical

harvesting). This reduction has been particularly evident for elite

cultivars that replaced traditional varieties after WWII, while in

∗ several Italian cropping areas, farmers still maintain in cultivation

Corresponding author. Tel.: +39 0812539446.

common bean landraces (Lioi et al., 2005; Marotti et al., 2007; Negri

E-mail addresses: [email protected],

[email protected] (G. Corrado). and Tosti, 2002; Piergiovanni and Lioi, 2010).

http://dx.doi.org/10.1016/j.scienta.2014.10.013

D. Scarano et al. / Scientia Horticulturae 180 (2014) 72–78 73

Table 1

Currently, modern nutritional recommendations inspired by

List of the landraces investigated, their code and site of collection/growing area. AV:

the traditional dietary patterns of Greece, Spain, Portugal and

Avellino; BN: Benevento; NA: Naples; SA: Salerno.

Southern Italy have increased the value of legumes as healthy

Code Name Site of collection/growing area Province

source of proteins. Moreover, consumers are more attracted by

agricultural and food products that are distinguished from oth- BM Bianco Montefalcone BN

ers by certain characteristics, qualities or reputations that derive C di Controne Controne SA

DM Dente di Morto Marigliano NA

from their geographical origin. For all these reasons, traditional

EU Dente di Morto Acerra NA

common bean varieties are receiving a growing attention from

F a Formella Visciano NA

both consumers and policy makers (Piergiovanni and Lioi, 2010).

MI Mustacciello Ischia NA

The primary policy goal for conservation and exploitation of MP Mustacciello Pimonte NA

OC Occhio nero Oliveto Citra SA

agricultural biodiversity should focus on the assessment of the

OS Occhio nero alta valle del Sele SA

existing diversity not only between but also within landraces

P1 Sel Pirolo 1 Acerra NA

(Esquinas-Alcazar, 2005). This effort is essential to prospect strate-

P2 Sel Pirolo 2 Acerra NA

gies for the implementation of adequate on-farm conservation P3 Sel Pirolo 3 Acerra NA

schemes and it is as a prerequisite for the possible develop- P4 Sel Pirolo 4 Acerra NA

P5 Sel Pirolo 5 Acerra NA

ment of breeding programs (Esquinas-Alcazar, 2005; Huang et al.,

R della Regina Casalbuono SA

2010).

RG della Regina (di Gorga) Stio SA

The evaluation of morphological traits is a traditional, important

RL della Regina San Lupo BN

method for the description and the determination of relationship RO Cannellino Romano Agro-acerrano-nolano NA

SI Screziato impalato Castellabate SA

among common bean landraces (Skroch and Nienhuis, 1995). In

TBC Tondino bianco Caposele SA

addition, molecular analysis provides additional information that

TC Tondo Caposele SA

is independent of environmental effects and in P. vulgaris, it has

TV Tondino Villaricca NA

proved to be a valuable tool for association mapping and the study V Cannellino Viscardi Agro-acerrano-nolano NA

of genetic diversity, population structure and phylogenetic rela- ZI Zampognaro d’Ischia Ischia NA

ZO Zolfariello Visciano NA

tionships (Beebe et al., 1995; Bitocchi et al., 2012; Blair et al.,

2009; Metais et al., 2000; Shi et al., 2011). Moreover, DNA anal-

yses of plant genetic resources are valuable to identify duplicate

2.2. Analysis of morphological data

accessions in core collections and possible cases of homonymy and

synonymy in cultivated landraces (Corrado et al., 2014; Rao et al.,

2009). Qualitative characters were transformed into dummy binary

attributes as reported (Romesburg, 2004). Pairwise similarities

In Italy, the common bean cultivation has always been a

between landraces were calculated by using the Jaccard similarity

typical element of rural economies especially in the Southern

coefﬁcient (Sneath and Sokal, 1973). Variability of the quantita-

regions (Piergiovanni and Lioi, 2010). In those areas, common

tive traits between and within landraces was evaluated calculating

bean is probably the horticultural crop with the highest number of

the coefﬁcient of variation (i.e. the absolute value of the standard

landraces (Perrino et al., 1984). The germplasm of different Italian

deviation of the mean/mean). This coefﬁcient ranges from 0 to 1.

regions has been characterized (Lioi et al., 2012; Mercati et al.,

For continuous variables, pairwise dissimilarity coefﬁcients were

2013; Piergiovanni and Lioi, 2010; Raggi et al., 2013), but despite

computed using squared Euclidean distances after standardization

the long history of cultivation, the number of accessions (Hammer

of mean values (Sneath and Sokal, 1973). To calculate the stan-

et al., 1989) and the presence of some well-known landraces that

dardized value Zij for the ith attribute and jth object, we subtracted

excel for product quality (e.g. “di Controne”, “Dente di Morto”),

the mean of the values of the ith attribute from the corresponding

the germplasm of the Campania region (Southern Italy) has been

value Xij in the data matrix and divided the result by the standard

poorly investigated.

deviation of the values of the ith attribute. On the basis of each

The aim of this study was to collect, characterize and evalu-

resemblance matrix, landraces were clustered by the unweighted

ate the diversity of traditional common beans that are cultivated

pair-group method with arithmetic averages (UPGMA) (Sneath and

in the Campania region (Southern Italy) using morphological and

Sokal, 1973). A cophenetic value matrix (Sneath and Sokal, 1973) of

molecular data.

the UPGMA clustering was also used to test for the goodness-of-ﬁt

of the clustering to the resemblance matrix on which it was based,

by computing the product-moment correlation coefﬁcient (r) with

2. Materials and methods

1000 permutations (Rohlf and Fisher, 1968). These analyses were

conducted using the NTSYSpc v. 2.1 package (Rohlf, 1998).

2.1. Plant material and its description

The study took place at the Genetics’ Experimental Station of 2.3. SSR analysis

the Faculty of Agricultural Science, University of Naples Federico

II (Portici, Italy), during the 2012–2013 period. The investiga- Total DNA was isolated from leaves using a previously reported

tion was performed on 25 common bean landraces (P. vulgaris procedure (Caramante et al., 2009). We analyzed three plants per

L.) from different geographic areas of the Campania region landrace. Ten SSR loci were used for PCR ampliﬁcation (Buso et al.,

(Table 1). Collected seeds were not multiplied before trials. 2006; Gaitan-Solis et al., 2002; Grisi et al., 2007; Hanai et al.,

The morphological characterization was done according to the 2010; Yu et al., 2000). SSR features, primer sequences and anneal-

IPGRI recommendations, including the number of replications ing temperature (Ta) are reported in Supplementary Table 1. PCR

(IPGRI, 1982). Brieﬂy, we recorded a number of highly hered- was performed in a 25 ␮l volume containing 20 ng genomic DNA,

itable characters selected as being easily seen and expressed 1.5 mM MgCl2, 100 mM dNTPs, 0.2 mM ﬂuorescently labeled for-

in all environments (IPGRI, 1982). In total, we analyzed 26 ward primer and unlabeled reverse primer, and 0.5 U Taq DNA

qualitative and 11 quantitative traits. Traits under investiga- polymerase (Promega) in 1× PCR buffer. The ampliﬁcation condi-

◦

tion, along with their IPGRI code, are reported in Supplementary tions were: one denaturing step at 94 C for 5 min, followed by 35

◦ ◦ ◦

Tables 2 and 3. cycles of 94 C for 45 s, Ta C for 45 s and 72 C for 1 min 30 s. After

74 D. Scarano et al. / Scientia Horticulturae 180 (2014) 72–78

◦

the ﬁnal cycle, a 5 min step at 72 C was added. Ampliﬁcation prod- closest match possible and then averaged across replicates, using

ucts were separated by agarose gel-electrophoresis to verify the the Greedy algorithm of the software CLUMMP (Jakobsson and

presence of ampliﬁed fragments. For allelic discrimination, the ﬂu- Rosenberg, 2007). To validate the estimated population structure,

orescent fragments were resolved by capillary electrophoresis and we calculated pairwise Fst between populations using MicroSatel-

detected in an ABI Prism 3130 Genetic Analyzer system (Applied lite Analyzer (Dieringer and Schlotterer, 2003). Null distribution

Biosystems). Signal peak height and allele sizes were scored using for p-value estimation was obtained from 10,000 permutations.

the Gene Mapper 4.0 software (Applied Biosystems) on the basis of

the GeneScan 500Liz molecular weight standard (Applied Biosys- 3. Results

tems).

3.1. Morphological characterization

2.4. Molecular data analysis

Twenty-six binary or multistate characters were examined in 25

For each SSR locus, we calculated the number of different common bean landraces. Six traits were not polymorphic, while the

alleles, the Shannon’s information index (−1 × Sum (pi × ln(pi)), remaining showed a considerable level of variation (Supplemen-

the observed heterozygosity (Ho; number of heterozygotes/N), the tary Table 2). All the landraces could be distinguished by at least one

polymorphic information content (PIC; 1 − Sum pi , equivalent to character. Taking into account the polymorphic characters, forty-

the expected heterozygosity) and the ﬁxation index, where N is one different IPGRI morphological phenotypes (out of a total of 65)

number of individuals, pi the frequency of the ith allele for the pop- were present. Sixteen landraces possessed at least one unique phe-

ulation and (Sum pi ) represent the sum of the squared population notype (i.e. not present in the other landraces). Considering the

allele frequencies. The detected intra-landraces variability (DILV) number of IPGRI-phenotypic classes that were covered, a trait that

per SSR represents the percentage of within-landraces polymor- displayed a remarkable variability was the dry pod colour. Large

phic loci. Analysis of Molecular Variance (AMOVA) was carried out differences were also present for the dry seed colour and shape

using a distance matrix and suppressing within individual analysis. (Lioi and Piergiovanni, 2013) and the most frequent bean colour

Permutations (999) were used to test for signiﬁcance. All these cal- was white (Fig. 1). Polymorphic qualitative data were used to clus-

culations were performed using the Genalex 6.5 software (Peakall ter landraces. The average (±s.d.) of Jaccard’s pair-wise similarity

and Smouse, 2012). Population structure was estimated using a coefﬁcients was 0.37 (±0.12) and ranged from a minimum of 0.11

model-based Bayesian procedure implemented in the software to 0.81. Similarity coefﬁcients were used to build a dendrogram

Structure v2.3 (Pritchard et al., 2000) with the aid of Structure using the UPGMA algorithm (Fig. 2A). The cophenetic correlation

Harvester (Earl and Vonholdt, 2012), using previously reported coefﬁcient was good and signiﬁcant (r = 0.79; p < 0.01). This analy-

settings (Corrado et al., 2013). The most informative number of sis indicated that the nine landraces with large white kidney shape

subpopulations was identiﬁed using the K method (Evanno et al., beans clustered together. Despite having a different seed colours,

2005). The estimated cluster membership coefﬁcient matrices the two landraces originating in the Ischia Island (MI and ZI) clus-

of the 20 runs were permuted so that all replicates have the tered together.

Fig. 1. Dry beans of the 25 landraces under investigation. For landrace codes see Table 1. Scale bar: 1 cm.

D. Scarano et al. / Scientia Horticulturae 180 (2014) 72–78 75

Fig. 2. Hierarchical cluster analysis of the 25 landraces based on qualitative (A) or quantitative (B) traits. (A) UPGMA cluster based on similarities calculated with the

Jaccard coefﬁcient. (B) UPGMA dendrogram based on Squared Eculidean Distances calculated on normalized quantitative data (Z-scores). For landrace codes see Table 1. See

Supplementary Tables 2 and 3 for data.

The analysis of 11 quantitative traits is reported in Supplemen- 3.2. Molecular characterization

tary Table 3. The data indicated a widespread variability among

landraces (the average coefﬁcient of variation was 0.25; Table 2), An analysis of the genetic diversity was carried out using 10

including variables that are considered the main components of SSRs selected from the literature. All these loci were polymorphic

been productivity (pods per plant, seeds per pod and weight of in our germplasm collection. Main genetic parameters of the pop-

100 seeds) (Nienhuis and Singh, 1986). The traits “pods per plants” ulation under investigation are presented in Table 3. We detected

and “racemes per plants” had the higher coefﬁcient of variation 85 alleles, whose length ranged from 100 to 314 bp. Large differ-

between landraces and, as average, also within each landrace. The ences were present in the number of alleles per locus, which ranged

landraces with the higher average coefﬁcient of variation of the from 2 (PVBR5) to 15 (PVBR163). We did not observe a signiﬁcant

traits analyzed were TO and TBC. To group landraces, dissimilar- difference (p > 0.05; Mann-Whitney test) between the number of

ities were calculated based on the squared Euclidean distances. alleles of transcribed vs untranscribed SSRs (Supplementary Table

These are insensitive to additive and proportional translation. A 1). As anticipated for a selﬁng species, the observed heterozygosity

dendrogram was built using the UPGMA algorithm (Fig. 2B). The was very low (on average, 6%), except for the BM188 and PVBR163

correlation between Euclidean dissimilarities and ultrametric dis- loci. The latter is one of the three loci that have been described as

tances was high and signiﬁcant (r = 0.89; p < 0.01). Although the associated to QTLs (the others being PVBR5 and PVBR14) (Wright

correlation between qualitative and standardized quantitative data and Kelly, 2011). The PVBR163 locus had the higher diversity,

was very low and not signiﬁcant (r = 0.11; p > 0.05), landraces with number of alleles and PIC. A signiﬁcant correlation between the

white kidney shaped beans formed one cluster, with the exception number of alleles per locus and its observed heterozygosity was

of the P1 and P3. not present (p > 0.05; Spearman’s Rho). For instance, two (PVat007

and PvM115) of the tree loci that were homozygous in all samples

had a number of allele well above the average. Allele frequency

spanned from a maximum value of 0.813 (PVBR5) to a minimum

of 0.007 for two alleles of the BM143 locus. These two rare alleles

Table 2

are present only in one plant of the RG landrace. The locus with the

Variation in quantitative traits among the 25 landraces. For each trait, the table

reports the coefﬁcient of variation (CV) and the maximum (max), average (mean) lower minor allele frequency (MAF) was PVBR163. Considerable

and minimum (min) value of the 11 quantitative traits analyzed. differences among SSRs were also present in the PIC, which ranged

from 0.30 (PVBR5) to 0.89 (PVBR163). The former has the lowest

Trait CV Max Mean Min

number of diversity, number of alleles, and PIC. PIC signiﬁcantly

Leaﬂet lenght (cm) 0.24 7.8 5.49 3.4

correlated with the number of alleles per SSR (p < 0.01; Spearman’s

Racemes per plant 0.40 6.4 2.59 0.8

Rho). The Fixation index showed substantial positive values and

Pods per plant 0.34 5.9 3.08 1.6

Pod lenght 0.23 163.4 100.12 67.4 limited differences among SSRs.

Pod beak lenght (mm) 0.29 41.0 25.53 14.0

All the employed SSRs were able to detect intra-landraces vari-

Locules per pod 0.23 7.6 4.14 2.6

ability, although with a different efﬁciency (Table 3). The locus

Seeds per pod 0.22 6.6 3.87 2.8

with the higher (resp. lower) number of alleles was PVBR163 (resp.

Seed lenght (mm) 0.22 15.9 12.40 7.2

Seed height (mm) 0.12 9.0 6.85 5.0 PVBR5). On average, intra-landraces variability was detected at 3.12

Seed widht (mm) 0.20 5.9 4.36 2.8 loci but varied largely among landraces (s.e.: 0.48). Two landraces

Seed weight (g) 0.26 67.9 46.09 19.3

(MI and OS) did not display genetic variability at the tested loci. On

76 D. Scarano et al. / Scientia Horticulturae 180 (2014) 72–78

Table 3

Main genetic indices of the common bean landraces obtained by SSR analysis. Na: number of different alleles; I: Shannon’s information index; MAF: major allele frequency;

Ho: observed heterozygosity; PIC: polymorphic information content; F: ﬁxation index; DILV: detected intra-landrace variability.

Locus Na Size range (bp) I MAF Ho PIC F DILV (%)

PVBR5 2 171–179 0.48 0.81 0.00 0.30 1.00 8

PVBR14 3 164–180 0.61 0.78 0.04 0.35 0.89 16

PVBR20 3 163–181 0.59 0.78 0.04 0.35 0.89 16

PVBR25 11 136–172 1.80 0.40 0.00 0.77 1.00 24

BM188 5 163–184 1.56 0.31 0.26 0.78 0.67 48

GATS91 8 226–258 1.75 0.31 0.03 0.79 0.97 44

BM143 11 116–184 1.78 0.36 0.01 0.77 0.98 28

PVat007 13 100–224 2.14 0.31 0.00 0.84 1.00 40

PVBR163 15 192–314 2.41 0.17 0.25 0.89 0.72 80

PVM115 14 112–188 2.03 0.37 0.00 0.80 1.00 40

Average 8.5 1.52 0.46 0.06 0.67 0.91 34

Standard error 1.6 0.22 0.07 0.03 0.07 0.04 6.6

Table 4

The groups deﬁned by the Structure’s analysis represent statisti-

Analysis of molecular variance among and within landraces. Df: degree of free-

cally different subpopulations, as indicated by the evaluation of

dom; SS: sum of squares calculated from a square genetic distance matrix; Est. Var.:

genetic differentiation (Supplementary Table 5). One subpopula-

estimated variance.

tion (Fig. 4, blue) comprised landraces with large white beans, such

Source df SS Est. Var. %

as the Cannellino, Dente di Morto and also the MP. The latter has

Among landraces 24 365.68 2.36 68 white-dark broadly stripped beans. For the other two subpopula-

Within landraces 125 135.83 1.09 32

tions, a clear distinction according to the area of origin or seed traits

was not evident. Nonetheless, 95% of the samples had a member-

Total 149 501.51 3.44 100

ship coefﬁcient higher than 0.8 (Supplementary Table 6), indicating

that the vast majority of the genotypes were strongly assigned to

subpopulations. At K = 3, the three analyzed plants per landrace

the contrary, the three plants of the TBC landrace had at least one

were attributed to the same subpopulation (Supplementary Table

different allelic proﬁle at each of the 10 SSR loci.

6), suggesting that the genetic differences within landraces rep-

To hierarchical partition genetic variation among landraces, we

resent true landrace variability. The only exception was the TBC.

used the AMOVA statistical procedure. The results indicated that a

For this landrace, the plants were assigned to three different sub-

considerable fraction of genetic variation (32%) was present within

populations, implying possible mixture of seeds or very strong

landraces (Table 4). Furthermore, Fst analysis was used to ascertain

genetic admixture.

the degree of genetic differentiation among landraces (Supplemen-

tary Table 4). Out of the 300 possible pairwise combinations, 13

were not significant (p > 0.05). Briefly, the non-significant Fst val- 4. Discussion

ues were obtained for comparisons that involved landraces with

the same seed type (e.g. kidney-shaped white beans). Different regions of Southern Italy are considered rich of tra-

ditional varieties of horticultural species, including common bean

3.3. Genetic structure analysis (Hammer et al., 1989; Perrino et al., 1984). In the Campania region,

there are several common bean types frequently reported in grey

The identiﬁcation of genetically similar groups of plants was literature and archival materials but a systematic evaluation has

performed using an admixture model-based clustering analysis not yet been carried out. Common bean types are traditionally clas-

implemented in the software Structure. The Evanno’s test indi- siﬁed according to their origin, use and features of the seed but

cated that the most informative number of subpopulations (K) is the local denomination does not always express true genetic dis-

3 (Fig. 3). The inferred population structure is presented in Fig. 4. tinction (Negri and Tosti, 2002). According to our morphological

analysis, local names were in many instances correctly related to

seed colour or shape, such as Dente di Morto (dead man’s tooth),

Solfariello (sulphurous, yellowish), Formella (in Neapolitan lan-

guage the “formella” is the button) and dell’occhio (eye-shaped).

The evaluation of phenotypic variation in common bean is cru-

cial in determining adaptation, agronomic potential and breeding

value of landraces. Many of the characters analyzed here pre-

sented a considerable phenotypic diversity among the genotypes.

All the traditional varieties could be distinguished by at least

one character, suggesting a signiﬁcant diversiﬁcation of the crop

in the Campania region. Considering the bean cultivars that are

commonly available on the market, many landraces were easily

distinguishable especially considering seed morphology. Landraces

displayed a potentially interesting variation also in traits that are

related to productivity such as pods per plants. Although a spec-

trum of bean phenotypes was present (Lioi and Piergiovanni, 2013),

the most frequent seed colour was white. It is likely that its diffu-

sion is because Italian consumers associate this colour with a thin

tegument and higher digestibility. In addition, the qualitative and

Fig. 3. Estimation of the optimum number of clusters for the common bean geno-

quantitative phenotypic measurements were consistent in indi-

types according to the Evanno’s method. The graph displays the DeltaK [mean (lL”

(K)l/SD(L(K))] for each K value. cating a cluster of landraces with white large seeds. Overall, the

D. Scarano et al. / Scientia Horticulturae 180 (2014) 72–78 77

Fig. 4. Estimated population structure of the the common bean landraces for K = 3 (see also Fig. 3). Each genotype is represented by a vertical line, which is partitioned into

colored segments that represent the estimated membership fractions in the three clusters. (For interpretation of the references to color in this ﬁgure legend, the reader is

referred to the web version of this article.)

plant and seed phenotypes provided an identity to each landrace, common name, were clearly different. It is worth to add that accord-

although this could be achieved by scoring several descriptors. ing to the legendary origin of these landraces, the three Regina’s

The number of alleles and a locus-speciﬁc allelic richness beans under investigation refer to different queens, indicating that

revealed the presence of a diffuse genetic variation. An estimated not only folk taxonomy but also historical information is useful to

outcrossing of 6% can be inferred considering the average observed facilitate the understanding of traditional varieties (Teshome et al.,

heterozygosity. This value is similar to that reported by Free 1997).

(1970) (8%). Cross-fertilization is expected to occur more frequently In conclusion, the combined use of molecular and morphologi-

between accessions with overlapping ﬂowering periods and there- cal markers allowed a detailed description of the variability present

fore, our estimate may be affected by the fact that (genetically) in our collection. As some of the traits are linked to agronomic per-

similar accessions are more likely to inter-cross. Of the two loci formance, our data allow an informed choice of the varieties to be

that displayed the highest Observed Heterozigosity (PVBR163 and further investigated for nutritional and breeding value. Despite a

BM188), the former was associated to a QTL related to plant height low estimated rate of outcrossing and admixture, common beans

(Wright and Kelly, 2011). from Campania comprise an appreciable level of inter- and intra-

Almost all landraces (23 out of 25) displayed genetic variability. landrace diversity both at morphological and molecular level, a

It is expected that garden-forms of common bean (i.e.: very roughly, result that needs to be considered when planning conservation

those cultivated in few hundreds plants) tend to reach rapidly strategies or breeding programmes. These traditional varieties still

homogeneity, because fewer than a dozen plants per year will be have a market share and many are receiving increasing support

sufﬁcient to produce seeds (Maxted et al., 2011). We analyzed lan- from local institutions as vital component of food tourism in rural

draces that are still cultivated for local markets, thus making more areas (e.g. food festivals). For that reason, the here presented char-

likely the presence of some genetic variability. The AMOVA showed acterization is also an essential step to (re-)establish quality brands

that a signiﬁcant part of the genetic variation is present within lan- associated with traditional common bean varieties.

draces, indicating that, despite autogamy, the analysis of genetic

variability on a single plant per landrace may provide limited infor-

Acknowledgments

mation. Furthermore, the DNA analysis implies that measures to

support on-farm conservation of common bean would be effective

This work was supported by the “Salvaguardia della biodiver-

to preserve genetic variability (Corrado et al., 2014; Negri and Tosti,

sità agroalimentare in Campania” (SALVE) project, Programma di

2002).

Sviluppo Rurale per la Campania 2007–2013, misura 214 az. f2

The observed subpopulation structure indicated that our

(Regolamento CE no. 1698/2005 del Consiglio del 20 settembre

germplasm collection could be clearly subdivided in three sub-

2005).

populations. The membership coefﬁcient of the genotypes to

speciﬁc sub-populations was very high and possible admixture

Appendix A. Supplementary data

was detected in a reduced number of landraces. It is likely that

the high self-pollination rate of bean, along with the fact that

Supplementary data associated with this article can be

the cultivation of traditional variety is usually restricted to spe-

found, in the online version, at http://dx.doi.org/10.1016/j.scienta.

ciﬁc agricultural settings, contribute signiﬁcantly to these features.

2014.10.013.

Moreover, local farmers do not usually select beans considering

yield (Zeven, 1997), but their distinctive visible features, making

more likely a maintenance breeding. Of the three subpopulations References

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