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Scientia Horticulturae 111 (2007) 197–202

Genetics and distribution of fertility restoration associated RAPD

markers in inbreds of pepper (Capsicum annuum L.)

Sanjeet Kumar a,*, Vineeta Singh b, Major Singh a, Shubha Rai a, Sanjeev Kumar a,Sunil Kumar Rai a, Mathura Rai a

a Indian Institute of Vegetable Research, P.O. Box 5002, P.O. BHU, Varanasi 221005, Indiab Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, BHU, Varanasi 221005, India

Received 26 May 2006; received in revised form 21 August 2006; accepted 23 October 2006

Abstract

Experiments were conducted to study genetics of fertility restoration and to examine distribution of RAPD markers (OPW19800 and OPP131400)

linked with fertility restoration gene (Rf) in pepper (Capsicum annuum L.) inbreds. Forty-two hot and five sweet pepper inbreds were crossed on a

cytoplasmic male sterile (cms) line CCA-4261 and F1s were evaluated for fertility restoration under open field conditions. DNA of 5 plants of CCA-

4261 and individual plants of 47 inbreds was isolated and PCR reaction was performed using OPW19 and OPP13 primers. The results revealed that

most of the hot pepper lines posses Rf gene. The Rf gene associated two markers, viz., OPW19800 and OPP131400 were not frequently distributed in

the restorer inbred lines because presence of marker bands often does not coincide with the presence of Rf gene identified in many restorer inbreds.

The case specific applications of both the RAPD markers have been described.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Capsicum; cms; Fertility restoration; Markers; Pepper; RAPD

1. Introduction

Among the five domesticated Capsicum species, pungent and

non-pungent forms of Capsicum annuum L. (pepper) are most

popular and have a worldwide commercial distribution (Bosland

and Votava, 2000). In India, hot pepper (syn. chilli) is an

important commercial crop, cultivated for vegetable, spice, and

value-added processed product (Kumar and Rai, 2005).

Although a majority of Indian farmers cultivate locally adapted

populations, hybrid cultivars have recently become popular

(Kalloo et al., 2001). Many Indian farmers produce hybrid seed

of hot pepper using a nuclear male sterile line (Dash et al., 2001).

Nevertheless, hybrid seed production based on cytoplasmic–

nuclear male sterility (cms) would be more cost effective (Kumar

et al., 2002a). The first male sterile Capsicum plant was identified

in an Indian C. annuum population (PI-164835), and fertility

restoration was found to be under the control of one major

fertility restoration gene (Rf) (Peterson, 1958). Complementary

genes and a major gene affected by minor genes controlling

* Corresponding author. Tel.: +91 542 2635247; fax: +91 544 3229007.

E-mail address: sanjeetk1@gmail.com (S. Kumar).

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2006.10.021

fertility restoration have also been reported (Novak et al., 1971;

Shifriss, 1997). Two types of cms plants have been described in

pepper based on anther morphology (Kaul, 1988). However, all

of these independently isolated cms lines were genetically

identical and had temperature sensitive male sterility expression

(Shifriss, 1997). In the recent past, however, stable cms hot

pepper lines have been developed and utilized for commercial

hybrid seed production in South Korea, China and India (Kumar

and Rai, 2005; Liu and Gniffke, 2004; Shifriss, 1997; Zhang

et al., 2000).

The limited availability of maintainer allele (rf) in hot

pepper has been a handicap in rapid transfer of male sterile

cytoplasm (nuclear diversification of cms), leading to

restriction in the choice of the parents (Shifriss, 1997;

Zhang et al., 2000). Screening of inbred lines for Rf /rf

allele’s constitution has been conventionally performed by

developing and evaluating fertility restoration in the testcross

progenies (rfrf � RfRf or rfrf). Molecular marker-assisted

selection (MAS) utilizing marker(s) tightly linked to a Rf

gene would facilitate: (i) rapid identification of inbred lines

for Rf /rf gene constitution without testcross progeny

evaluation, (ii) transfer of the rf gene to female hot pepper

parental stocks (maintainer breeding), and (iii) transfer of the

S. Kumar et al. / Scientia Horticulturae 111 (2007) 197–202198

Rf gene to male sweet pepper parental stocks (restorer

breeding) without testing progeny of the selected plant(s)

after each backcross. The effective deployment of Rf marker

technology for MAS involves the identification of markers

tightly linked with the Rf gene, testing validity of such

marker(s) in an array of restorers and maintainers and the

utilization of such marker–trait association(s) for the indirect

selection of restorer/maintainer plants. Although linkages

between an Rf gene and Random Amplified Polymorphic

DNA (RAPD) markers (Pakozdi et al., 2002; Zhang et al.,

2000) and QTLs for fertility restoration (Wang et al., 2004)

in pepper have been demonstrated, it is imperative to test

their utility in elite advanced generation restorer and

maintainer lines. Therefore, the present study was conducted

to examine genetics of fertility restoration and distribution

of two previously reported Rf gene associated markers

in an array of newly identified restorer and maintainer

lines.

2. Materials and methods

2.1. Plant materials

Forty-two hot and five sweet pepper (all C. annuum L.)

inbred lines (Table 1) were utilized along with a stable hot

pepper cms line (CCA-4261). CCA-4261 and its maintainer

were introduced from the Asian Vegetable Research and

Development Center, Taiwan. Kaala (hot pepper) and Waialua

(sweet pepper) inbred lines derived from the same cross

(Takeda et al., 1996) were included to examine consequences of

restorer/maintainer genes (Rf /rf) during segregation of fruit

size and pungency.

2.2. Development and evaluation of F1s, F2 and testcross

2.2.1. Seasons 1999–2000 and 2000–2001

During the winter of 1999–2000, a cross between cms line

CCA-4261 and Pant C-1 (already known strong restorer), was

made and its F1 progeny were raised to develop testcross

progeny [CCA-4261 � (CCA-4261 � Pant C-1)] and F2 seeds

during 2000–2001 under open field conditions. During 2000–

2001, a F1 between CCA-4261 and Pant C-1 was produced

again.

2.2.2. Seasons 2001–2002 and 2002–2003

In 2001–2002, F1 (CCA-4261 � Pant C-1), F2 and

testcross progenies were raised along with parental lines,

and fertility restoration ability was examined. The F1 progeny

were also utilized to produce F2 and testcross seed. During

2001–2002, 47 cms (CCA-4261) based crosses derived

from 42 hot and 5 sweet pepper inbred lines (Table 1)

were developed and evaluated for fertility restoration in the

next season. During 2002–2003, CCA-4261 � Pant C-1

derived F1, F2 and testcross progeny were again for the

fertility restoration. The Chi-square goodness-of-fit test was

applied in F2 (3:1) and testcross (1:1) segregation

progeny.

2.3. Male fertility determination

From the F2 and testcross generations, all plants and from

parents and F1s, 5–10 plants were examined for fertility

restoration expression. Male fertility was determined by three

methods through: (i) visual inspection for the presence (male

fertile) versus absence (male sterile) of pollen from 5 to 10 fully

developed flowers of each plant during three stages (45, 65 and

95 days after transplanting), (ii) selfing of one branch (bagging

from muslin cloth bags) of plant and then examining seed set

ability (male fertile) versus non-ability (male sterile), and (iii)

staining of pollen in 2% carmine prepared in 45% acetic acid

and counting of stained (male fertile) versus non-stained pollen

(male sterile).

2.4. DNA extraction

Total DNA from the leaves of 5 plants of CCA-4261 (cms

line) and individual plants of 47 inbreds was isolated using the

DNeasy Plant Mini Kit (Qiagen, Germany) following a

previously developed protocol (Kumar et al., 2002b). Quanti-

fication of DNA for PCR reactions was made using a Hoefer

DyNA Quant 200 Fluorometer (Amersham Biosci., New

Territories, Hong Kong).

2.5. RAPD analysis

Two operon primers OPP13 and OPW19 (Sigma, Louis,

US) were selected based on their association with a major

fertility restorer gene (Rf) in hot pepper (Zhang et al., 2000). A

tight linkage (0.37 cM) was reported between the OPP131400

and Rf , while OPW19800 was reported to be 8.12 cM away

from the Rf (Zhang et al., 2000). For PCR reaction, master mix

consisted of 0.4 mM of each dNTPs, 2.5 mM MgCl2, 0.4 unit

Taq polymerase (5 units/ml) with the supplied polymerase

buffer (Bangalore Genei Pvt. Ltd., Bangalore, India), 0.02 mM

primer and 50 ng genomic DNA. The amplification profile

consisted of 42 cycles of 20 s at 94 8C for denaturation, 40 s at

36 8C for primer annealing, and 1 min and 20 s at 72 8C for

primer extension and DNA synthesis. At the beginning of

cycling profile, the reaction was held for 3 min at 94 8C, and

the final cycle was extended to 5 min at 72 8C. The

amplification products were electrophoresed in a 1.2% agarose

gel and also in a 4% native polyacrylamide gel and then stained

with ethidium bromide (Sambrook and Russell, 2001; Zhang

et al., 2000). The RAPD fragments were then analyzed using

Alpha ImagerTM 3400 Gel Documentation System (Alpha

Innotech, US).

3. Results

3.1. Fertility restoration in F1s

Based on the results of fertility restoration in 47 cms-based

F1s, 47 pepper inbred plants (male parents) with various fruit

size and shape (Fig. 1) were classified in three categories: (i)

inbred plant with Rf allele, (ii) inbred plant with rf allele, and

S. Kumar et al. / Scientia Horticulturae 111 (2007) 197–202 199

(iii) inbred plant still segregating for both Rf and rf alleles

(Table 1). Some of the morphological attributes of these

inbred lines are given (Table 1) and fruit types are shown

(Fig. 1). Among the 42 hot pepper lines, 37 (88.1%)

possessed restorer allele and, the remaining 5 (11.9%) lines

(JCA-9, EC-491094, PDC-49A, LCA-206 and G-5) segre-

Table 1

Morphological features and distribution of two RAPD markers in the identified re

ID no. Inbredsa Plant heightb (cm) Fruit lengthb (cm) Fruit width

1. EC-119457 58.2 � 3.31 6.38 � 0.14 1.04 � 0.09

2. EC-257216 71.4 � 2.90 6.08 � 0.10 1.08 � 0.02

3. EC-257716 77.2 � 3.68 7.96 � 0.91 0.90 � 0.04

4. EC-268216 65.2 � 2.06 4.86 � 0.31 0.94 � 0.02

5. EC-341074 70.5 � 3.01 4.50 � 0.41 0.93 � 0.10

6. EC-341075 62.2 � 2.33 5.32 � 0.45 0.86 � 0.06

7. EC-345629 69.4 � 2.11 9.46 � 0.02 1.18 � 0.02

8. EC-491094 62.0 � 2.81 5.54 � 0.12 1.24 � 0.02

9. Taiwan-2 60.6 � 1.60 2.12 � 1.40 0.82 � 0.02

10. G-4 68.0 � 2.39 6.28 � 0.13 0.88 � 0.02

11. G-5 68.0 � 3.00 3.60 � 0.10 1.50 � 0.20

12. Phule Sai 71.0 � 2.92 9.20 � 0.27 1.04 � 0.02

13. Punjab Lal 67.0 � 2.55 7.72 � 0.29 0.86 � 0.05

14. Pusa Jwala 58.0 � 2.21 8.58 � 0.41 1.1 � 0.03

15. DC-3 73.6 � 2.73 3.88 � 0.09 0.68 � 0.04

16. DC-5 74.4 � 1.69 4.66 � 0.21 0.92 � 0.04

17. K. Chanchal 53.4 � 2.54 3.10 � 0.10 0.82 � 0.02

18. Waialua 60.4 � 3.10 6.00 � 0.40 2.10 � 0.40

19. Assam-10 73.2 � 3.13 4.24 � 0.40 1.46 � 0.09

20. PBC-535 51.4 � 1.17 9.06 � 0.14 1.62 � 0.04

21. PBC-873 51.0 � 3.86 4.40 � 0.08 1.26 � 0.24

22. PBC-1512 48.6 � 3.31 11.04 � 0.5 2.78 � 0.25

23. Local Coll-1 49.6 � 2.13 6.92 � 0.17 1.08 � 0.04

24. PDG-1 47.6 � 2.04 5.32 � 0.09 0.80 � 0.05

25. Perennial 2A 85.8 � 2.96 4.80 � 0.13 1.06 � 0.02

26. F1-112-1 61.8 � 1.28 5.20 � 0.11 0.94 � 0.02

27. LCA-235 75.4 � 2.94 6.26 � 0.18 0.70 � 0.03

28. P-1649-1 66.8 � 2.89 4.84 � 1.12 0.74 � 0.02

29. P-1649-2 64.5 � 2.90 4.62 � 1.20 0.74 � 0.02

30. PDC-49A 58.0 � 1.90 4.80 � 0.09 1.10 � 0.05

31. PDC-53B 63.6 � 1.36 4.44 � 0.15 0.68 � 0.04

32. KA-2 35.0 � 1.41 5.20 � 0.14 0.70 � 0.03

33. KSPS-202* 48.0 � 2.70 5.64 � 0.16 4.98 � 0.05

34. KSPS-501* 33.3 � 2.40 7.56 � 0.31 4.62 � 0.17

35. Kaala* 54.4 � 1.69 5.82 � 0.14 4.06 � 0.07

36. C. Wonder* 35.0 � 2.50 6.90 � 0.90 5.14 � 0.71

37. ISPN # 2–3* 33.3 � 1.45 11.28 � 0.7 5.04 � 0.32

38. 9852-173 71.6 � 2.23 5.50 � 0.09 1.06 � 0.05

39. 97–7125-3 55.2 � 1.85 7.42 � 0.52 2.68 � 0.07

40. 97–7125-2 55.2 � 1.85 7.48 � 0.49 2.68 � 0.07

41. AKC-89/38 57.0 � 3.38 1.56 � 0.04 1.40 � 0.05

42. KDCS-810 33.6 � 2.64 3.88 � 0.05 0.86 � 0.05

43. JCA-9 47.0 � 9.07 6.60 � 0.16 1.22 � 0.12

44. Local Lal 40.1 � 1.10 5.23 � 0.20 1.50 � 0.60

45. Taiwan-1 58.5 � 2.29 7.16 � 0.12 1.22 � 0.02

46. CCA-4261 74.1 � 5.07 9.86 � 0.23 1.42 � 0.05

47. LCA-206 67.0 � 2.10 6.54 � 0.12 0.78 � 0.06

48. Pant C-1 54.4 � 1.69 3.56 � 0.19 0.94 � 0.04

a *Sweet pepper lines.b Average from 10 plants and fruits.c 10 green fruits.d Rf-fertility restorer gene and rf-fertility maintainer gene.e (+) Presence and (�) absence of bands.

gated for both alleles (Rf and rf). Five sweet pepper lines had

maintainer allele (Table 1). The pollen stainability in male

fertile F1 progeny varied from 71.1% (CCA-4261 � EC-

345629) to 87.7% (CCA-4261 � EC-257716). Although, like

cms plants, pollen fertility in the male sterile (non-restorer)

F1 progenies ranged from 34.6% (CCA-4261 � California

storer and maintainer pepper (C. annuum) lines

b (cm) Fruits weightc (g) Identified gened Markerse

OPW 19800 OPP131400

42 Rf – –

25 Rf – –

42 Rf + –

41 Rf + –

40 Rf + –

20 Rf + –

25 Rf + +

30 Rf/rf + +

20 Rf + +

25 Rf + –

35 Rf/rf + +

25 Rf + –

32 Rf + +

47 Rf + +

41 Rf + +

27 Rf + +

10 Rf + –

210 Rf + +

25 Rf + +

75 Rf + �35 Rf + �

275 Rf � �42 Rf � �20 Rf � �20 Rf � �20 Rf � �15 Rf � �25 Rf � �25 Rf � �32 Rf/rf � �30 Rf � �34 Rf � �

655 rf � �550 rf � �410 rf � �450 rf � �355 rf � �50 Rf � �70 Rf � �70 Rf � �46 Rf � �15 Rf � �29 Rf/rf + �35 Rf � �60 Rf � �90 rf � �20 Rf/rf + �20 Rf + �

Fig. 1. Fruit size and shape variation in inbreds of pepper (C. annuum) characterized for restorer (Rf) and maintainer (rf) genes. Number corresponds to numbers and

names of inbreds in Table 1.

S. Kumar et al. / Scientia Horticulturae 111 (2007) 197–202200

Wonder) to 35.1% (CCA-4261 � KSPS-202), these progenies

produced only few pollen.

3.2. Promising hybrids

From the 47 cms-based crosses, two crosses, viz., CCA-

4261 � KA-2 and CCA-4261 � Pusa Jwala were selected for

evaluation along with a number of hybrids under multi-location

trials of All India Coordinated Improvement Projects on

Vegetable Crops. Based on two consecutive seasons evaluation

performances for yield and other traits (data not shown), CCA-

4261 � Pusa Jwala (CCH-2 synonym Kashi Surkh) has been

recommended and released for the commercial cultivation in

India (Rai et al., 2005).

3.3. Distribution of markers

The OPW19 primer produced a 800 bp fragment, as reported

previously (Zhang et al., 2000), during agarose gel electro-

Fig. 2. RAPD morphotypes of 23 pepper inbreds (C. annuum) obtained from primer

1–23 correspond to numbers and names of inbreds in Table 1.

phoresis (Fig. 2). This fragment was present in 17 hot pepper

restorer plants and five hot pepper plants segregating for both

Rf and rf alleles (Table 1). Similarly, OPP13 primer also

produced a band of 1400 bp, as reported previously (Zhang

et al., 2000), during agarose gel electrophoresis. However, this

band was present only in 10 hot pepper restorer plants (Table 1).

Both the markers were absent in all the five sweet pepper

maintainer lines and were male parent specific in the cross

CCA-4261 � Pusa Jwala.

3.4. Genetics of fertility restoration

The Pant C-1 was confirmed to have strong fertility

restoration based on the normal amount of selfed seed setting

in the F1 plants of CCA-4261 � Pant C-1. Segregation test

based on number of male fertile (restorer) and male sterile (non-

restorer) plants in F2 and testcross progeny derived from CCA-

4261 � Pant C-1, revealed monogenic, dominant gene control

for fertility restoration in Pant C-1 (Table 2).

OPW19 showing presence/absence of Rf gene associated 800 bp band. The lanes

Table 2

Segregation of F2 (3:1) and testcross (1:1) hot pepper (C. annuum) for fertility

restoration in cross CCA-4261 � Pant C-1 during 2001–2002 and 2002–2003

Season Ratio

tested

Segregation (no. of plants) Chi-square

Male fertile Male sterile Total

2001–2002 3:1 82 24 106 0.31

1:1 44 38 82 0.44

2002–2003 3:1 60 16 76 0.63

1:1 24 30 54 0.41

Pooled 3:1 142 40 182 0.88

1:1 68 68 136 0.00

S. Kumar et al. / Scientia Horticulturae 111 (2007) 197–202 201

4. Discussion

Fertility restoration analysis of testcross progeny suggests

that the Rf allele is widely distributed in the hot pepper lines

examined (Table 1). Except California Wonder, which was

already known to be maintainer (Greenleaf, 1986), all other

inbreds have been characterized for the first time with respect to

constitution at fertility restoration locus. The majority of hot

pepper (comparatively small and pungent fruits) and sweet

pepper (large and non-pungent fruits) lines previously

examined been found to be restorer and maintainer lines,

respectively (Greenleaf, 1986; Shifriss, 1997). This is likely

due to a linkage between the genes controlling hot pepper traits

such as small fruits and pungency and Rf locus (Shifriss, 1997).

The inbred lines found to be segregating for restorer and

maintainer traits in other hot pepper germplasm, could be used

for the selection of maintainer plants to facilitate development

of new pair of A and B lines more rapidly (without maintainer

breeding). This germplasm development would allow for a

broader choice of parental lines during cms-based heterosis

breeding in hot pepper.

The distribution of Rf gene associated with both the

markers, viz., OPW19800 and OPP131400 (Zhang et al., 2000)

is not frequent because band presence often does not coincide

with the presence of Rf gene in the identified restorer plants.

Both the markers were represented by band presence in

Waialua and band absence in Kaala, which are restorer line

and maintainer line, respectively. Interestingly, these hot and

sweet pepper varieties are derivatives of a cross between hot

pepper (Chabai Mirah: cayenne-type fruits) and sweet pepper

(Keystone Resistant Giant: bell-type fruits) (Takeda et al.,

1996). Although the restoration ability of the parental lines of

Kaala and Waialua was not tested herein, Chabai Mirah likely

contributed the Rf allele to Waialua and Keystone Resistant

Giant contributed the rf allele to Kaala. Since Waialua

produces pungent, small fruits and Kaala produces non-

pungent, semi-bell type fruits (number 18 and 35 in Fig. 1),

the presence of Rf allele in Waialua and rf allele in Kaala

provides partial support to the hypothesed linkage between

the genes controlling sweet pepper fruit traits (larger fruit

size, non-pungency) with the rf allele and genes controlling

hot pepper fruit traits with Rf allele (Shifriss, 1997; Zhang

et al., 2000).

The action of a single, dominant gene controlling fertility

restoration in Pant C-1 was repetitively confirmed over the two

growing seasons. The control of fertility restoration by a major

dominant gene (Peterson, 1958), by associated complementary

genes (Novak et al., 1971) has previously been described in

pepper. The discrepancy concerning genetic control of fertility

restoration (single versus complementary gene action) in

pepper may be due to the differences in the paternal genotypes

at Rf locus used for experimentation. Since the stability of most

of the cms lines is governed by modifier/minor genes (Shifriss,

1997), the single gene control fertility restoration in Pant C-1

can be used for efficient transfer of the Rf gene into sweet

pepper lines.

The presence/absence of RAPD band morphotypes

(OPW19800 and OPP131400) was consistent and repeatable in

the inbred lines examined. However, the narrow distribution of

both these markers in restorer inbreds precludes their use in

MAS for the screening of inbred lines for Rf gene. A search for

more widely distributed marker–trait association among

restorer lines may facilitate the indirect screening for restorer

and maintainer genes. In elite inbreds, these markers, however,

likely be case specific in their utilization in cms-based hybrid

breeding. For instance, in a commercial hybrid described herein

(CCA-4261 � Pusa Jwala), both the markers are male specific

and hence could be useful for hybrid seed purity testing.

Likewise a band absence morphotype for both putative marker

loci in all the five sweet pepper maintainer lines suggests that

they may assist in Rf gene transfer in these lines during restorer

breeding.

Acknowledgement

The financial support in the form of research grant to the

corresponding author, obtained from the Indian Council of

Agricultural Research (ICAR), New Delhi is thankfully

acknowledged. Thanks are also due to Professor G. Kalloo,

DDG (Hort. & Crop Sci.), ICAR, New Delhi.

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