Ardei 1
Ardei 1
Ardei 1
Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti
a r t i c l e
i n f o
Article history:
Received 4 May 2015
Received in revised form 18 January 2016
Accepted 4 March 2016
Available online 14 March 2016
Keywords:
Capsicum annuum
Pepper
Genetic diversity
Morphological characterization
SSR
a b s t r a c t
Pepper (Capsicum annuum L.) is one of the most valuable vegetables in the world. Over the last decades,
highly performing cultivars have progressively replaced the diversied and heterogeneous landraces
worldwide, causing wide genetic erosion in this crop. The recovery of these ancient landraces, which
might preserve alleles of agricultural interest and local adaptations, results of pivotal importance for the
development of new varieties and the maintenance of a sustainable agriculture. In the present work, a
collection of twenty-six landrace-derived inbred lines and three landraces from North-West Spain were
evaluated for their agronomic performance and genetic diversity based on a set of twenty-seven morphological descriptors and twenty microsatellite markers. The collection featured phenotypic variability
for all the studied traits, which were inuenced by the location, except for the yield. The principal component analysis divided the landraces in well-dened groups, with only Arnoia, Punxin and Blanco Rosal
showing some degree of overlapping. The greater part of the variance was accounted for traits such
as fruit weight, pericarp thickness and fruit shape and color. The molecular analyses suggested a high
level of genetic diversity within the collection and the presence of specic alleles, which were not previously detected in other Spanish pepper landraces. Multivariate and Bayesian clustering showed that
landraces were primarily grouped according to their geographical origin and secondarily in agreement
with the characteristics of their fruits. Six groups of landraces, with a great genetic differentiation, were
clearly identied. Only the landraces Mougan and Arnoia possessed an allele associated to the pungency
character.
2016 Elsevier B.V. All rights reserved.
1. Introduction
The genus Capsicum (family Solanaceae) originated in the tropical South American region centered in what is now Bolivia
(Eshbaugh, 1993). Nowadays, the number of recognized species
in the genus is twenty-seven, ve of which were domesticated
from distinct events at different primary diversication centers
(Andrews, 1995; Baral and Bosland, 2002). Among these ve,
Capsicum annuum L. is the most widespread and economically
important Capsicum species worldwide as well as the most used
in commercial cultivar breeding programs (Bosland and Votava,
2000). C. annuum was domesticated in Mexico from the wild bird
Corresponding author.
E-mail address: c.silvar@udc.es (C. Silvar).
http://dx.doi.org/10.1016/j.scienta.2016.03.006
0304-4238/ 2016 Elsevier B.V. All rights reserved.
pepper or Chiltepin (C. annuum var. glabriusculum) and subsequently introduced to Europe by Columbus in the 15th century
(Andrews, 1995; Perry et al., 2007; Kraft et al., 2014). Afterwards, it
was rapidly distributed to Africa, India and China, where it came
into wide cultivation giving way to a current crop of immense
cultural and economic importance because of its multiple uses
(Bosland and Votava, 2000; Kumar et al., 2006). Thousands of years
of human selections in multiple environments and cultural contexts led to the impressive phenotypic diversity of contemporary
C. annuum fruits (Nuez et al., 1996; Djian-Caporalino et al., 2007;
Nicola et al., 2013). In general, continued selection was driven to
obtain lines with non-deciduous, pendant, larger and non-pungent
fruits with greater shape variation and increased fruit mass (Paran
and van der Knaap, 2007). The main negative effect of migrations
and consequent articial selections in the secondary diversication
centers was the dramatic reduction in the genetic basis of pep-
Table 1
Common name and origin of the landraces from NW Spain used in the present work.
Landrace
Arnoia
Blanco Rosal
Couto
Couto Grande
Mougan
Oimbra
Padron
Pineira
Punxin
a
Linesa
AR330-05, AR55,
AR65, AR45,
AR274-05
BR97-04,
BR113-05
Co5A, Co10A,
Co12
CG-10-117
MG279-05,
MG77-04,
MG101-04,
MG335-05,
MG14-04,
MG318-05
OI27-05, OI30-04
ETH80-05,
ETH134-05,
ETH209-05,
PA145,
ETH211-05,
ETH166-05
PI309-05,
PI275-05
PX74-05,
PX118-04
Origin
Region
Province
Arnoia
Orense
O Rosal
Pontevedra
Narn
A Coruna
Narn
Guntin
A Coruna
Lugo
Oimbra
Herbn
Orense
A Coruna
Ribadeo
Lugo
Punxin
Orense
All lines, except AR45, AR55 and AR65, are landraces-derived inbred lines.
assessed with twenty-seven morphological traits (IPGR descriptors) in two different locations.
2. Materials and methods
2.1. Plant material
Twenty-six pepper (C. annuum) inbred lines derived from nine
different landraces, collected as seeds from farmers in the main
growing regions of Galicia (NW Spain) and three landraces (AR45,
AR55 and AR65), were used in this work (Table 1, Fig. S1).
2.2. Agro-morphological characterization
The twenty-nine lines were evaluated over three years
(20052007) in two experimental elds located in Northern and
Southern Galicia. The rst one was at the Centro de Investiga Spain) (43
ciones Agrarias de Mabegondo (Mabegondo, A Coruna,
15 N, 8 18 W). The second eld was located at Estacin Exper (Salceda de Caselas, Pontevedra,
imental Agrcola de Baixo Mino
Spain) (42 6 N, 8 33 W), about 10 Km far from the border with
Portugal. Both areas, approximately 200 Km distant, belong to the
same climate region (Mediterranean maritime) according to the
classication of Papadakis (1975) and they exhibit little differences
in their agro-climatic conditions. During the time of experiments
(20052007) slightly higher mean temperatures were observed in
Salceda (13.5 C) with respect to Mabegondo (11 C), with averages per year of 14 C, 13 C and 13.5 C for the rst and 11 C,
12 C and 11.5 C for the second site. On the contrary, the rainfall
accomplished better at the latter location (mean values of 85.4 L/m2
against 71.2 L/m2 ). Thus, average values per year (2005, 2006 and
2007) for Mabegondo were 70.1, 105.8 and 80.2 L/m2 , while 62.8,
82.4 and 68.5 L/m2 for Salceda. Seeds were sown under greenhouse conditions and the seedlings were transplanted to the eld
in the month of April. The experimental design was a complete
randomized block with three replications for a total of 84 plants
per plot. Agro-morphological data were collected from 10 plants
and 25 fruits per replicate. Twenty-seven traits associated to both
the plant and the fruit were evaluated on the basis of the Capsicum
descriptors developed by the International Plant Genetic Resources
Institute (IPGRI, 1995): plant height (cm), plant growth habit, plant
canopy width (cm), stem length (cm), stem diameter (cm), stem
color, stem shape, stem pubescence, nodal anthocyanin, fruit length
(cm), fruit width (cm), fruit weight (g), fruit pedicel length (cm),
fruit wall thickness (mm), placenta length (mm), number of locules,
fruit set, fruit color at intermediate stage, fruit color at mature
stage, presence of anthocyanin stripes, fruit shape, fruit shape at
pedicel attachment, neck at base of fruit, fruit shape at blossom
end, fruit blossom end appendage, fruit cross-sectional corrugation and fruit surface. Harvesting was performed from middle July
to middle September and the yield of each line was recorded in
kilograms per square meter (kg/m2 ) and number of fruits per plant
(n fruits/plant).
Quantitative data were examined with an analysis of variance (ANOVA) using a 99% condence interval followed by means
comparison with Waller-Duncans test or Least Signicant Differences (LSD) test ( = 0.05). Qualitative characters were expressed
as relative frequencies for each landrace at any specic location
(Mabegondo or Salceda). In addition, both sets of variables were
subjected to principal component analysis (PCA) to obtain a general
overview of the structure of variation within and among landraces.
All statistical analyses were performed with SPSS software version
17.0 (SPSS, 2008).
2.3. Molecular marker analysis
Genomic DNA was isolated from young leaves of each line following the CTAB method (Doyle and Doyle, 1987). DNA quality
was evaluated on agarose gels and DNA concentrations were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop
Technologies, Wilmington DE). Final DNA concentrations were
adjusted to 25 ng l1 and they were stored at 20 C until used.
Twenty publicly available microsatellite markers (thirteen genomic
and seven EST-based SSRs), distributed across the twelve pepper
chromosomes, were selected according to their observed polymorphism in Spanish pepper landraces (Gonzlez-Prez et al., 2014)
(Table 2). The sequences of primer pairs were obtained from Lee
et al. (2004), Minamiyama et al. (2006), Yi et al. (2006), Ben-Chaim
et al. (2006), Nagy et al. (2007). All 20 SSRs were unlinked, according to the genetic maps of Barchi et al. (2007), Wu et al. (2009),
Mimura et al. (2012) and Sugita et al. (2013). PCR amplication
and detection of microsatellite markers were performed according to the methodology reported by Schuelke (2000) based on the
uorescently labelling of universal primers. Briey, an M13 tail
(5 -CACGACGTTGTAAAACGAC-3 ) was added to the 5 end of each
publicly available forward primer cited above. A universal primer
with a complementary sequence to the M13 tail was labelled with
different uorophores (6-Fam, Hex or Ned). PCR was carried out in
a nal volume of 15 L, which contained 5075 ng of genomic DNA,
1X PCR Buffer (NZYTech), 2.5 mM MgCl2 (NZYTech), 0.02 M of the
forward primer, 0.2 M of reverse primer, 0.18 M of the universal primer, dNTPs (NZYTech) at 0.2 mM each, and 0.4 U of Taq DNA
Polymerase (NZYTech). All fragments were amplied using the following touchdown PCR prole: an initial denaturing step of 5 min at
94 C was followed by 35 cycles with denaturation at 94 C for 30 s
and extension at 72 C for 30 s, respectively. The annealing temperature was decreased in 0.5 C increments from 62 C in the rst cycle
to 56 C and was then kept constant for the remaining 35 cycles
(always 3050 s). A nal extension step was performed at 72 C for
10 min. DNA fragments were resolved in an Applied Biosystems
3130xl Genetic Analyzer. The allele sizes were assigned with the
GeneMapper 3.7 software (Applied Biosystems). The putative presence of the pungency trait in the C. annuum lines was assessed by
using the molecular marker MAP1, which was developed based on a
3. Results
3.1. Morphological characterization
A broad phenotypic variation was found for most of the parameters studied in the collection of pepper lines. The distribution
of the different qualitative morphological characters was represented as the percentage of relative frequencies for each group
of landraces at any different location (Fig. S2). Little discordance
was observed between the two experimental sites. Among the
agronomic traits, all the pepper plants showed a growth habit
erect, the stem colour was mostly green with sparse pubescence,
except for Mougan, which showed an intermediate-dense level of
Table 2
Microsatellite markers evaluated for the genotyping of the C. annuum collection. Name, chromosomal position, repeat sequence and source are indicated. For each locus:
number of alleles observed (Na), range of alleles sizes, observed heterozygosity (Ho ), Neis unbiased gene diversity (uHe ), Wrightxation index (Fis ) and Polymorphism
Information Content (PIC).
Marker
Chrom
Repeat
Source
Na
Range (bp)
Ho
uHe
Fis
PIC
Hpms1-148
Hpms1-139
Hpms1-143
HpmsE016
Hpms1-173
CAMS351
CA523558
Hpms1-5
EPMS426
HpmsCaSIG19
HpmsE145
EPMS342
Hpms2-24
HpmsE082
CAMS336
HpmsE031
Hpms2-2
CAMS405
HpmsE075
GPMS197
1
1
2
3
3
4
6
6
7
7
8
8
9
9
10
10
11
11
12
12
(GA)14
(CT)2 (AG)15
(AG)12
(TACA)3
(GA)16 (TG)2
(TG)3 . . .(AG)26
T20
(AT)11 (GT)17
(AT)15
(CT)6 (AT)8
(TTA)4
(CTT)7
(CT)17 (CA)5 A21
(ATC)5
(TC)16
(TC)6
(GT)9
(TC)18
(ACC)6
(GA)3 (TAT)16
2
3
4
4
4
3
2
3
3
3
2
2
2
2
3
3
2
4
2
7
211213
304320
240246
180210
176186
223227
194196
334338
118130
237241
233245
349360
204220
250254
178192
185189
167169
250256
218221
293351
0.000
0.069
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.037
0.000
0.069
0.000
0.000
0.000
0.000
0.000
0.000
0.034
0.103
0.508
0.550
0.498
0.709
0.745
0.547
0.373
0.653
0.547
0.662
0.382
0.460
0.373
0.195
0.668
0.249
0.334
0.587
0.354
0.849
1.000
0.872
1.000
1.000
1.000
1.000
1.000
1.000
1.000
0.943
1.000
0.847
1.000
1.000
1.000
1.000
1.000
1.000
0.901
0.876
0.375
0.440
0.456
0.641
0.684
0.476
0.299
0.563
0.476
0.575
0.305
0.350
0.299
0.173
0.582
0.227
0.274
0.523
0.287
0.814
pubescence. Stem shape was cylindrical and anthocyanin was visible in the internodes, varying from light to dark purple (Fig. S2).
The qualitative fruit characters showed broader variation. Fruit set
ranged from intermediate to high and fruit shape varied from triangular (Blanco Rosal, Oimbra and Punxin) to blocky (Couto Grande,
Pineira,
Mougan and Padron) (Fig. S2). Fruit color at intermediate
stage was white for Blanco Rosal, Oimbra and Punxin, and green
for the others, whereas fruit color at mature stage mostly varied
between red and dark red. All fruits had a smooth surface and only
few showed anthocyanin stripes (Fig. S2).
The analysis of variance for quantitative descriptors was rstly
performed independently at each experimental location, in order
to check the potential effect of variable climatic conditions over the
evaluated periods. Signicant differences among years were found
at each site for all traits, except for fruit length (Mabegondo), fruit
wall thickness and number of locules (Salceda). Differences in yield
traits were detected only in Mabegondo (Table S1). Nevertheless,
the percentage of variance explained by the year together with
the interaction landrace year was small compared to that arising from the genotypic effects (Table S1), and it mostly resulted
from divergent means observed in 2006 (data not shown). ANOVA
analysis considering the two different locations showed signicant
(p < 0.001) differences for all of the quantitative characters under
study. In general, differences were detected among landraces, environments and for the interaction landrace location (Table 3). The
yield, expressed as n fruits/plant and kg/m2 was slightly inuenced
by the experimental site. Thus, landrace location interaction was
not signicant for the trait kg/m2 , but the location showed a signicant effect on the n fruits/plant (Table 3). Couto and Padron
exhibited the highest yield for both places (141 and 146.6 kg/m2 ,
Salceda (Table S3). The landraces that displayed a greater differentiation depending of the environment were Blanco Rosal, Mougan
and Punxin, especially for the attributes plant height, plant width
and fruit weight (Table S3). Only the total number of harvested
kilograms per square meter were not signicantly different at both
locations, the mean value for this character at Mabegondo being of
53.2 kg/m2 , while 58.7 kg/m2 for Salceda (Table S3).
A principal component analysis was performed separately with
both sets of descriptors in order to more accurately determine the
most effective attributes in discriminating among landraces, and
to avoid a potential reduction in the variance due to excessive
number of variables or the presence of scale-based traits. Those
traits related to yield were considered as quantitative characters.
PCA based on quantitative data resulted in three principal components with eigenvalues > 1, cumulatively accounting for 85.61%
of the total variance (Table S4). The rst component explained
47.21% of the total variance and it was positively and strongly correlated with fruit weight, width and wall thickness, and negatively
correlated in a robust manner with kg/m2 (Table S4). The second
component, which explained 29.98% of the total variance, had plant
height, width and stem length with the highest coefcients. Finally,
the third component contributed only for 8.42% of the total variance and the n fruits/plant was the main trait responsible for the
observed variability (Table S4). These three components were used
to obtain the diagram of dispersion for all the twenty-nine lines,
giving the picture of the differences among the nine groups of lan
draces (Fig. 1). Couto Grande, Pineira,
Oimbra, Padron, Couto and
Mougan, were represented as well-dened clusters, while Arnoia
and Punxin, and Blanco Rosal to a lesser extent, showed some
degree of overlapping (Fig. 1). The rst axis, mainly related to variation in fruit characters, clearly separated the landraces with large
Table 3
Analysis of variance (sum of squares) for test of signicance of differences among landraces, locations and their interaction for thirteen quantitative traits in a collection of
twenty-nine Galician lines.
Source of variation
df
Plant width
Landrace (L)
Location (Lo)
L Lo
Error
8
1
8
2807
174043.95
54212.37
22096.56
418739.13
Source of variation
Landrace (L)
Location (Lo)
L Lo
Error
df
8
1
8
2807
*,**,***
Stem length
***
***
***
90959.47
450.07
1172.62
54655.31
78.60
127.34
17.47
252.48
***
***
***
N of locules
262.41
***
16.32
***
24.48
***
1128.51
854375.17
213559.21
81402.63
1120461.95
Fruit lenght
***
***
***
Placenta lenght
18712.88
***
170.93
**
1009.33
***
86345.38
14384.94
85.89
354.20
6992.66
Fruit width
***
***
***
N fruits/plant
34.54
***
15.40
***
45.95
***
587.51
5666.77
45.39
22.02
1655.45
kg/m2
1.14E + 06
1.76E + 03
8.09E + 03
2.16E + 05
Fruit weight
***
***
***
7.70E + 06
5.22E + 04
5.33E + 04
2.62E + 06
***
ns
ns
***
***
***
Fig. 1. Scatter plot of the PCA analysis based on thirteen quantitative characters. (A) PC1 vs PC2, (B) PC3 vs PC2.
Fig. 2. Scatter plot of the PCA analysis based on eight qualitative traits. (A) PC1 vs PC2, (B) PC3 vs PC2.
Table 4
Diversity parameters calculated for the different groups of landraces. N = number of
genotypes, Ho = observed heterozigosity, uHe = Neis unbiased gene diversity (uHe ),
Fis = Wrightxation index. Numbers in brackets in the last column indicate the
frequencies for each private allele.
Landrace
Ho
uHe
Fis
N private alleles
Arnoia
Blanco Rosal
Couto
Couto Grande
Padron
Mougan
Oimbra
Pineira
Puxin
5
2
3
1
6
6
2
2
2
0.000
0.000
0.033
na
0.000
0.042
0.025
0.025
0.000
0.21
0.00
0.05
na
0.00
0.16
0.09
0.13
0.00
1.00
na
0.20
na
na
0.72
0.56
0.67
na
1 (60%)
1 (100%)
4 (100%), 1 (50%)
1 (na)
1 (na)
1 (83%), 1 (75%)
1 (na)
Total
29
0.016
0.51
0.97
12
Fig. 3. (A) Dendrogram of twenty-nine landraces-derived inbred lines revealed by UPGMA cluster analysis based on Dice distance derived from twenty SSRs. Numbers in
brackets indicate bootstrap values expressed as percentages. (B) Structure bar plots at K = 6 and K = 8 (C) The twenty-nine genotypes are ordered according to the UPGMA
tree. Each colour represents a different group and the proportion of a given genotypes color represents the proportion that genotype belongs to the corresponding group.
Table 5
AMOVA partition of molecular variance for three and six groups suggested by the
UPGMA derived dendrogram and the STRUCTURE analysis.
Source of variations df
3 groups
Among groups
Within groups
Total
2 63.37
55 138.52
57 201.89
1.56
2.52
4.08
38.35
61.65
100
0.0001
6 groups
Among groups
Within groups
5 141.58
52 60.32
2.9
1.16
71.46
28.54
0.0001
Total
57 201.89
4.06
100
locally by farmers across many years on the basis of a recognizable performance, are still cultivated nowadays because of
their high quality and good acceptance in the national market.
On a global scale, this collection represents a valuable source of
unexploited variability in a cultivated background that might be
easily employed with breeding purposes. For that goal, the accurate assessment and description of trait variation is of crucial
signicance in the success of any program aimed at the selection of genotypes harboring those features mainly demanded by
consumers. Similarly, the evaluation of genetic variability among
genotypes results essential for the conservation and protection of
any genetic resource, but also for broadening the genetic basis of
cultivated varieties, promoting a sustainable agriculture (FAO, 2010
Ortiz et al., 2010; Sparato and Negri, 2013). Both approaches are
addressed in the present work in order to gain a better knowledge
on Galician pepper landraces and design suitable strategies for their
future exploitation.
Agronomic results of eld trials pointed out a notable phenotypic variability among landraces. Qualitative traits were not
highly affected by the location and only a few discrepancies arose
among and within landraces, which could be also attributed to
the ambiguity of any visual classication based on pre-established
morphological categories. On the contrary, the year and location of
eld trials exerted a certain inuence on the majority of quantitative descriptors, although the latter possessed a more remarkable
effect. Productivity, expressed as kg/m2 , was signicantly different
over the three assayed years in Mabegondo, but it was not signicantly affected by the regional conditions, performances of all
distinct landraces being comparable between both experimental
sites. Indeed, differences in yield among lines within each landrace were neither detected for this parameter, suggesting a high
adaptability of the different genotypes. Other parameters associate
to yield, such as the number of fruits per plant and fruit weight
were signicantly affected by the location, although in those cases
the landrace location interaction represented a small percentage of the total variance. Indeed, a low correlation (20%) was found
between the number of fruits per plant and the kg/m2 . In oppo-
10
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