Euphytica 109: 71–77, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

71

Capsicum tovarii, a new member of the Capsicum baccatum complex

Nankui Tong & Paul W. Bosland

Department of Agronomy and Horticulture, New Mexico State University, Las Cruces, New Mexico, 88003, U.S.A.

Received 6 March 1998; accepted 2 September 1998

Key words: Capsicum tovarii, chile, interspecific hybrid, meiotic chromosomes, pepper, random amplified
polymorphic DNA marker

Summary
Interspecific hybrid performance and meiotic chromosome behavior of F1 hybrids were studied to elucidate the
genetic relationship between C. tovarii and the other Capsicum species. C. tovarii was hybridized, as a female
and a male parent, to C. annuum, C. chinense, C. frutescens, C. chacoense, C. galapogense, C. baccatum, C.
praetermissum, C. cardenasii, C. eximium and C. pubescens. When the hybridization of C. baccatum × C. tovarii
was performed, F1 , F2 and backcross progenies were successfully obtained. In addition, a successful hybridization
of C. praetermissum × C. tovarii was also obtained. A cytological investigation of F1 hybrids of C. baccatum × C.
tovarii revealed that most meiotic chromosomes paired as bivalents. However, multivalents, chromosome bridges,
and chromosome lags were observed. These results suggest that C. baccatum differs from C. tovarii by at least
a chromosomal reciprocal translocation. Crosses of C. tovarii to C. chinense and C. frutescens produced plump
seeds, but none of the seeds germinated. Hybridizations of C. tovarii to C. pubescens, C. eximium and C. cardenasii
did not produce seed. These hybridization results indicate that C. tovarii is genetically more closely related to the
C. baccatum complex than to the C. annuum complex or the C. pubescens complex.

Introduction cies is of great benefit to plant breeders. Not only are

wild species useful in breeding for disease resistance,
but they can be used to increase nutritional quality,
The Capsicum genus represents a diverse plant group
yield, and adaptation to stress (Bosland, 1993).
and includes twenty-seven species, five domestic-
Capsicum tovarii Eshbaugh, Smith & Nickrent is
ated and twenty-two undomesticated species (DeWitt
a wild species native to Peru (Eshbaugh et al., 1983).
& Bosland, 1993). The Capsicum species with
Primary morphological description of the species was
n = 12 chromosomes belong to one of three species-
provided by Eshbaugh et al. (1983). They stated that
complexes: C. annuum complex, C. baccatum com-
the corolla color of the flower for this species varied
plex, and C. pubescens complex. A species complex
from violet to cream, marked with two green spots
includes species that can hybridize albeit sometimes
at the base of each corolla lobe. The fruit is pungent
with difficulty. The C. annuum complex includes C.
with the mature fruit color being red (Eshbaugh et
annuum L., C. frutescens L., C. chinense Jacq., C. cha-
al., 1983). The mature fruit easily separates from the
coense Hunz. and C. galapagoense Hunz (Pickersgill,
calyx, which is typical of wild species of Capsicum.
1991; Zijlstra et al., 1991). The C. baccatum com-
Successful crosses of C. tovarii to other Capsicum
plex consists of C. baccatum L. and C. praetermissum
species had not been obtained (McLeod et al., 1979,
Heiser & Smith. The C. pubescens complex contains
1983; Pickersgill, 1991). McLeod et al. (1983) men-
C. pubescens Ruiz & Pav., C. cardenasii Heiser &
tioned that this species was not closely related to any
Smith and C. eximium Hunz.
other Capsicum species based on electrophoretic ana-
As genetic resources, wild Capsicum species are
lysis of enzymatic and non-enzymatic proteins. Flow
important assets for breeding improved chile peppers.
cytometric estimation of nuclear DNA content in dif-
Knowing the relationship among the Capsicum spe-

Article: euph 4835 Pips nr.190851 (euphkap:bio2fam) v.1.1

euph4835.tex; 16/08/1999; 14:49; p.1
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Table 1. Interspecific hybridizations between several Capsicum species and C. tovarii

Species No. of flowers No. of fruits Avg. seeds/fruit Germination ∗ Seedling

pollinated (%) survival(%)

C. annuum complex
C. annuum
PI 338490 11 0
NuMex
Joe E. Parker 34 1 39∗∗ 0
NuMex
Big Jim 35 0
C. chacoense
NMCA 50027 40 4 11∗∗ 0
C. chinense
NMCA 30031 32 2 5 0
PI 439483 13 4 10 0
C. frutescens
PI 355808 13 0
NMCA 40011 86 16 4 30 0
C. galapagoense
Grif 1567 26 5 2∗∗ 0
C. baccatum complex
C. baccatum
NMCA 20061 80 16 17 24 100
C. praetermissum
NMCA 90015 42 10 10 N/A
NMCA 90022 31 14 3 18 100
C. pubescens complex
C. cardenasii
PI 590507 34 0
C. eximium
NMCA 90006 39 0
C. pubescens
NMCA 80026 46 0
PI 590503 24 0
∗ For the hybridization of C. tovarii to C. galapagoense, 10 seeds were tested for germination and for the
hybridization of C. tovarii to NMCA 90015, the seed germination data is not available. A minimum 30 seeds
were tested for germination for each of the other hybridizations.
∗∗ Only a seed coat was formed in these hybridizations.

ferent Capsicum species indicated that C. tovarii was the other Capsicum species. Random amplified poly-
closely related to the C. annuum complex (Belletti, et morphic DNA (RAPD) analysis was used to confirm
al., 1995). To date, it is still unknown to what species the interspecific hybrids.
complex C. tovarii belongs. In addition, the analysis
of meiotic chromosomes of C. tovarii has not been
documented. Materials and methods
This study explored the biological relationship of
C. tovarii to the three species-complexes by invest-
The seeds of C. tovarii (Grif 14033) used in this study
igating hybridization of C. tovarii to the other Cap-
were supplied by Paul G. Smith, at the University of
sicum species. Interspecific hybrid performance and
California-Davis. The C. tovarii distribution is in the
meiotic chromosome behavior of F1 hybrids elucid-
Rio Mantaro basin of Peru. C. tovarii was hybridized,
ated the genetic relationship between C. tovarii and
as a female and a male parent, to three accessions of C.

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annuum (PI 338490, ‘NuMex Joe E. Parker’ and ‘Nu- steps were repeated for 45 cycles: 1 minute of denat-
Mex Big Jim’), one accession of C. baccatum (NMCA uration at 94 ◦ C, 1 minute annealing at 35 ◦ C, and 2
20061), two accessions of C. chinense (NMCA 30031 minutes of elongation at 72 ◦ C and then held at 4 ◦ C.
and PI 439483), two accessions of C. frutescens (PI PCR products were run on 3% agarose gel, stained
355808 and NMCA 40011), one accession of C. cha- with ethidium bromide, and photographed on a UV
coense (NMCA 50027), one accession of C. galapa- transilluminator.
goense (Grif 1567), one accession of C. cardenasii
(PI 590507), one accession of C. eximium (NMCA
90006), two accessions of C. praetermissum (NMCA Results
90015 and NMCA 90022) and two accessions of C.
pubescens (NMCA 80026 and PI 590503). A total of Interspecific hybridization
586 pollinations of C. tovarii to Capsicum species was
In order to obtain interspecific progeny, all hybridiza-
accomplished (Table 1). The pollinations were done in
tions were done by controlled pollination and included
a wind and insect proof greenhouse at the Fabian Gar-
reciprocal hybridizations. In addition, for hybridiza-
cia Science Center, New Mexico State University, Las
tions of C. tovarii to species from the C. pubescens
Cruces, NM, U.S.A. To obtain interspecific progeny,
complex, bulk-pollen pollination, repeated pollination
methods including controlled pollination, reciprocal
at different flower bud stages and a shortened style
hybridization, bulk-pollen pollination, repeated pol-
were also employed. Interspecific hybridization results
lination at different flower bud stages (3 pollinations
are listed in Table 1. The data in Table 1 reveal that hy-
of the same flower of a female parent from emascula-
bridizations of C. tovarii to some species (C. chinense
tion day to the third day after the emasculation), and a
and C. frutescens) from the C. annuum complex yiel-
shortened style were used. To produce F2 generations,
ded true seeds with an embryo and endosperm, while
F1 hybrids were self-pollinated and pollinated to the
hybridizations to C. annuum, C. chacoense and C.
parental plants to produce backcross generations.
galapagoense produced only a seed coat. None of the
For the meiotic chromosome investigation, young
seeds from hybridizations to the species within the
flower buds were fixed in an ethanol-acetic acid (3:1)
C. annuum complex had normal germination. Seeds
mixture for 24 h and stored in 70% ethanol for at
of C. frutescens × C. tovarii germinated producing a
least 24 h. A chromosome squash was made from
radicle, however, no cotyledons emerged. When the
the anthers of the flower bud. Chromosome pairing
hybridizations of C. tovarii to the species within the C.
and behavior were observed at diakinesis, metaphase-
pubescens complex (C. pubescens, C. cardenasii and
I and anaphase-I of pollen mother cells (PMCs) using
C. eximium) were accomplished, none of the flowers
a normal aceto-carmine squash method with modific-
set fruit. However, hybridizations of C. tovarii to the
ation (Tong and Bosland, 1997). Pollen viability was
C. baccatum species-complex (C. baccatum and C.
determined by aceto-carmine stainability.
praetermissum) produced seed that contained an em-
For random amplified polymorphic DNA (RAPD)
bryo and an endosperm. The successful interspecific
analysis, leaf samples from C. baccatum, C. tovarii
F1 plants were obtained by usingC. baccatum as the
and the putative F1 hybrids of C. baccatum × C. to-
female parent and C. tovarii as the male parent. The
varii were taken and DNA was isolated using a CTAB
backcross and F2 progenies of C. baccatum × C. to-
DNA isolation technique with modification (Votava et
varii were also obtained. The rate of seedling survival
al., 1996). Samples were rehydrated in 100 µl TE buf-
was 27% for the F2 generation and 15% for the back-
fer. DNA concentration was measured using a Hoefer
cross generation. In addition, plants of F1 hybrid of
fluorometer, and diluted to 10 ng/µl. For PCR amp-
C. praetermissum × C. tovarii have been successfully
lification, a random oligonucleotide primer OPA-11
obtained.
(50 -CAATCGCCGT-30 ) (Operon Technologies) was
selected for this study after screening 7 random oligo-
Morphological characteristics
nucleotide primers. Amplification was carried out in
a Perkin Elmer 2400 Thermocycler in 20 µl volumes To confirm that the F1 plants were truly of an interspe-
that contained 1X Stoffel Buffer, 3.5 mM MgCl2 , cific origin, several traits were examined. The plant of
0.1 mM dNTPs, 2 units Stoffel fragment, 0.22 µM C. tovarii is woody and has a round stem. There are
primer, and 20 ng DNA template. After an initial DNA 2 to 4 flowers per node. The corolla color is cream,
denaturation step of 94 ◦ C for 4 minutes, the following marked with two green spots at the base of each lobe.

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Figure 1. Morphological characteristics of C. tovarii.

Table 2. Meiotic chromosome pairing in pollen mother cells of C.

baccatum, C. tovarii, and F1 hybrids of C. baccatum × C. tovarii
tovarii exhibited normal growth. The F1 plants had an
intermediate leaf size, flower size and fruit size, and a
Sources Chromosome pairing No. of cells examined round stem shape. There were 1 to 3 flowers at each
I II (ring, rod) IV X node and the flower had a white corolla with green
spots and blue anthers. The red mature fruit easily
C. baccatum 12 (2–6, 6–10) 20 (100%)
separated from the calyx. These traits, except corolla
C. tovarii 12 (0–4, 8–12) 11 (100%)
F1 hybrid 5 (4, 1) 1 1 1 (6.7%)
color, resemble those of the male parent, C. tovarii.
8 (4, 4) 2 4 (26.7%)
The fruit was conic with a pointed blossom end. More
2 9 (4, 5) 1 1 (6.7%) than 100 fruits set on each F1 plant.
10 (0–4, 6–10) 1 5 (33.3%)
12 (1–4, 8–11) 4 (26.7%)
Pollen viability
I, II, IV and V denote univalent, bivalent, quadrivalent, and
quinquevalent, respectively.
A total of 2282, 1690, and 1620 pollen grains were
stained for testing the pollen viability of C. tovarii,
The anthers are blue. The fruit is small, round and C. baccatum, and the F1 progeny, respectively. The
red when ripe (Figure 1). The mature fruit easily sep- percentage of stainable pollen of the F1 plants was
arates from the calyx. In contrast, the C. baccatum 32.2%, significantly lower than the 84.7% for C. to-
var. pendulum has yellow spots on a white corolla, varii and 96.5% for C. baccatum. Even though the
yellow anthers, a square stem, one flower per node, pollen stainability was low in the F1 plants, the flowers
large elongated and persistent fruit. The mature fruit were highly fertile. The average number of seeds per
color is orange. The F1 plants of C. baccatum × C. fruit of the F1 plants was 1.2 from self-pollination,

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Figure 2. Meiotic chromosome pairing and behavior in F1 hybrids of C. baccatum × C. tovarii. Chromosome bridge (arrow) and chromosome
lag (arrow) at meiotic anaphase-I.

Figure 3. RAPD profiles by primer OPA-11 in F1 hybrids of C. baccatum × C. tovarii and parental plants. Lanes 1–5 from left to right: female
parental plant C. baccatum, male parental plant C. tovarii, F1 hybrids of C. baccatum × C. tovarii (lane 3–4) and 100 bp DNA ladder (lane 5).

euph4835.tex; 16/08/1999; 14:49; p.5

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4.6 from a backcross to C. baccatum, and 2.6 from
a backcross to C. tovarii.
Cytological features
Meiotic chromosome pairing patterns in pollen mother
cells of the two parental species, C. tovarii and C. bac-
catum, and of the F1 hybrid were observed. Because
of the ‘stickiness’ of meiotic chromosomes, it is often
difficult to observe chromosome pairing at prophase
or metaphase (Lanteri & Pickersgill, 1993). Fortu-
nately, in this study clear preparations were obtained,
and the meiotic chromosome associations of C. to-
varii, C. baccatum, and the F1 hybrids could be clearly
observed. The cytological investigation revealed that
chromosomes of the two parental species paired nor-
mally as bivalents (Table 2). When the meiotic cells
at diakinesis and metaphase-I of both C. tovarii and
C. baccatum were respectively analyzed, 100% of the
cells had 12 bivalents and had regular meiotic chro-
mosome behavior. The number of chromosomes that
paired as a ring varied from 0 to 4 per cell for C. tovarii
and 2 to 6 per cell for C. baccatum. No chromosome
bridges or chromosome lagging were observed.
When the cells at diakinesis and metaphase-I of the
F1 plants of C. baccatum × C. tovarii were observed,
complex chromosome associations were found. Most
chromosomes paired as bivalents, while univalents,
quadrivalents and a quinquevalent were also seen
(Table 2). Chromosome bridges and chromosome lag-
ging were observed in the F1 plants (Figure 2). Both
types had low frequencies of chromosome lagging
(4.7%) and for chromosome bridge formation (6.9%).
DNA analysis
The random amplified polymorphic DNA (RAPD)
analysis of C. baccatum, C. tovarii and the F1 hybrids
of C. baccatum × C. tovarii revealed that RAPDs
could be used to distinguish the two parents and the
F1 progeny. The F1 plants had a molecular marker at
600 bp that was unique to the male parent (Figure 3).
The RAPD results and the morphological traits con-
firmed that the F1 plants were true hybrids between C.
baccatum and C. tovarii.
Discussion
The complex chromosome associations observed in
the F1 hybrids of C. baccatum × C. tovarii are in-
dicative of chromosome structural repatterning in the
divergence of C. baccatum and C. tovarii. As clearly
shown in Figure 2, the irregular meiotic chromosomal
pairing with multivalents such as quadrivalents and
a quinquevalent, along with the chromosome bridges
and lagging at meiotic anaphase-I indicated that C.
tovarii could differ from C. baccatum by at least
the occurrence of a reciprocal translocation (Egawa
et al., 1986; Swanson et al., 1981). The occurrence
of univalents was very rare. The result suggests that
C. tovarii and C. baccatum share a structurally dif-
ferentiated but basically homologous genome (Egawa
et al., 1986). Theoretically, a translocation ring has
three possible arrangements in the meiotic metaphase
plate, which are two types of adjacent arrangement
and one type of alternate arrangement. If the three
types of orientation occurred at random, it would be
expected that a translocation would lead to inviability
in about two-thirds of the gametes (Swanson et al.,
1981). In this study, lower pollen stainability (32.2%)
of F1 progeny could have resulted from chromosomal
structural differentiation through at least a reciprocal
translocation.
The domesticated species of Capsicum have been
grouped into three species-complexes. These group-
ings have been based on studies of morphology,
isozyme analysis, cytology, and hybridization compat-
ibility (Eshbaugh, 1993; Pickersgill, 1988; Jensen et
al., 1979; McLeod et al., 1983; Moscone et al., 1993,
1996). Moscone et al. (1996) suggested that at least
three independent ancestral lines lead to the cultivated
Capsicum taxa. Their investigations on fluorochrome
karyotypes of cultivated species of Capsicum indic-
ated that the C. baccatum complex belonged to a dis-
tinct lineage that was between the C. annuum complex
and the C. pubescens complex. This study demon-
strated that C. tovarii can not successfully hybridize
with the C. annuum complex or the C. pubescens com-
plex, but is able to successfully hybridize with the
C. baccatum complex. Successful hybridizations to C.
baccatum and C. praetermissum establish that C. to-
varii is a member of the C. baccatum complex. The
fruitful F1 , F2 and backcross generations of C. bac-
catum × C. tovarii also provide good evidence of the
genetic relatedness of C. tovarii to the species within
the C. baccatum complex. Thus, the C. baccatum com-
plex consists of at least three species, C. baccatum, C.
praetermissum, and now, C. tovarii.
euph4835.tex; 16/08/1999; 14:49; p.6

Acknowledgements
We are grateful to Dr. P.G. Smith, Department of Ve-
getable Crops, University of California, Davis, for
supplying the original C. tovarii seed, Mr. Eric Vo-
tava, Department of Agronomy and Horticulture, New
Mexico State University, for his assistance in photo-
graphs and DNA analysis, Dr. Marisa M. Wall and
Dr. Roy G. Cantrell, Department of Agronomy and
Horticulture, New Mexico State University, for their
helpful suggestions and critical reading of the manu-
script. This article is a contribution of the New Mexico
Agricultural Experiment Station, New Mexico State
University, Las Cruces.
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