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Testing validity of fertility restorer (Rf ) gene associated RAPD markers in newly identified restorer and maintainer lines of pepper (Capsicum annuum L.)

Sanjeet Kumar, Vineeta Singh, Major Singh, Sanjeev Kumar, G. Kalloo and Mathura Rai

Indian Institute of Vegetable Research, P.B. # 5002, Varanasi-221 005, India, www.iivr.org Email: sanjeetk1@sify.com

Abstract

Validity of two RAPD markers (OPP131397 and OPW19800) associated with fertility restorer (Rf ) gene was tested in a panel of 47 newly identified restorer and maintainer inbred plants of pepper (Capsicum annuum L.). Among the 37 restorer lines identified during this study, OPP131397 and OPW19800 fragments were although repeatable and consistent, they were present only in 17 and 10 restorer lines, respectively. Thus the presence of these two markers often did not coincide with the presence of Rf gene, which suggest that the distribution and origin of both the markers are narrow. Hence search for more widely distributed Rf gene associated markers would be required to exercise marker assisted selection (MAS) for restorer and maintainer plants of pepper. The case specific applications of both the repeatable and consistent RAPD markers in cms heterosis breeding of pepper have been described.

Media summary

The efficient and feasible utilization of molecular markers (RAPDs) in hybrid breeding and hybrid seed purity testing of hot pepper has been demonstrated.

Key words

Pepper, fertility restoration, RAPD markers, cms line

Introduction

Pepper (Capsicum annuum L.) is an important commercial crop of India, cultivated for vegetable, spice and industrial (oleoresin extraction) purposes. In the recent years, hybrid cultivars have become popular and many farmers are producing hybrid seeds of hot pepper based on nuclear male sterility (Dash et al. 2001), but hybrid seed production based on cytoplasmic-nuclear male sterility (cms) would be more cost effective. Due to the limited availability of maintainer (rf ) gene in hot pepper lines (Shifriss 1997), process of nuclear diversification of cms line is delayed, leading to restriction in the choice of the parents to obtain heterortic hybrids. Molecular marker aided selection (MAS) using tightly linked (co-segregating) marker(s) with restorer (Rf) gene is expected to facilitate: (i) rapid screening of inbred lines for Rf/rf gene without developing and evaluating testcross progenies, (ii) accelerated transfer of rf gene in hot pepper female parent (maintainer breeding) and (iii) accelerated transfer of Rf gene in sweet pepper male parent (restorer breeding) without progeny testing of selected plant(s) after each backcross generation. In the light of availability of tightly linked RAPD markers with Rf gene in pepper (Zhang et al. 2000) and to examine their feasible utilization in cms based heterosis breeding, validity of already reported two Rf gene associated RAPD markers in a panel of newly identified restorer and maintainer lines were tested and results are described in this communication.

Materials and methods

Plant materials

A total of 42 hot and 5 sweet pepper inbred lines with varying fruit size/shape and morphological features (Table1) were crossed on a hot pepper cms line (CCA-4261). Kaala (hot pepper) and Waialua (sweet pepper) inbred lines derived from a single cross (Takeda et al. 1996) were deliberately included in the study to examine consequences of segregation of Rf and rf genes along with segregation of fruit size and pungency.

Determination of fertility restoration

The fertility restoration ability of each inbred line was determined by analysing male fertility of 5 to 10 plants of F1 derived from cms (CCA-4261) and respective inbred lines through (i) visual observation of 5-10 fully developed flowers of each plant at three times during the crop stand, for the presence (male fertile) or absence (male sterile) of pollen, (ii) selfing (bagging with muslin cloth bags) of one branch of each plant and examining ability of plant to produce selfed seeds (male fertile) or non-ability of plants to produce selfed seeds (male sterile) and (iii) staining of pollen in 2% carmine prepared in 45% acetic acid and counting of stained pollen (male fertile) and non-stained pollen (male sterile).

DNA extraction and selection of primers

The total DNA from the young leaves of 5 plants of CCA-4261 (cms line) and individual plant of 47 inbred lines were isolated using DNeasy Plant Mini Kit (Qiagen) according to the previously developed protocol (Kumar et al. 2002). For the validation of RAPD markers, two primers, viz., OPP13 and OPW19 were selected based on their association with a major Rf gene (Baoxi et al. 2000).

PCR reaction mixture and electrophoresis

The master mix consisted of 1.0 μl dNTPs, 2.5 mM MgCl2, 0.4 unit Taq polymerase with the supplied polymerase buffer, 0.5 μM decamer primer and 210 ng genomic DNA. The amplification profile consisted of 42 cycles of 20 sec. at 94C for denaturation, 40 sec. at 36C for primer annealing and 1 min. 20 sec. at 72C for primer extension and DNA synthesis. At the beginning of cycling profile the reaction was held for 3 min. at 94C and final cycle was extended for 5 min. at 72C (Kumar et al. 2002). The amplification products were dissolved in 1.2% agarose gel (Kumar et al. 2002) as well as in 4% natural polyacrylamide gel with ethidium bromide staining (Zhang et al. 2000). The gels were analysed using a Gel Documentation Alpha Imager and inbuilt software (Alpha Innotech, USA).

Results and discussion

Distribution of Rf/rf genes in inbred lines

Forty-seven cms based F1s derived from the 42 hot pepper and 5 sweet pepper inbred plants were evaluated and based on their fertility restoration reaction, 47 inbred plants were classified in three categories: (i) inbred plant with restorer (Rf) allele, (ii) inbred plant with maintainer (rf) allele and (iii) inbred plant still segregating for both restorer and maintainer (Rf/rf) alleles (Table 1). Among the 42 hot pepper inbred plants, 37 (88.10%) had restorer allele. In the remaining 5 (11.90%) (JCA-9, EC-491094, PDC-49A, LCA-206 and G-5) plants both the alleles (Rf and rf) were segregating because among the F1 plants derived from these lines, male sterile plants were also observed. All the 5 sweet pepper lines had maintainer alleles (Table 1). Except California Wounder, all the inbred lines were characterised for the first time with respect to their ability to restore fertility of sterile cytoplasm. These results reveal and support the previous reports (Shifriss 1997), wherein more frequent distribution of Rf allele in hot pepper lines has been reported. Since Waialua (hot pepper) produced pungent and small fruits and Kaala (sweet pepper) produced non-pungent semi-bell type fruits, the presence of Rf gene in Waialua and rf gene in Kaala provide partial support to the hypothesis suggesting linkage of the genes controlling sweet pepper fruit traits (larger fruit size, non-pungency) with the rf gene and linkage of genes controlling hot pepper traits (pungency and smaller fruits) with Rf gene (Shifriss 1997, Zhang et al. 2000) and vice versa. We are currently analysing such linkage relationship utilizing RILs derived from a restorer hot pepper (small fruited) and maintainer sweet pepper (large fruited) line (Kumar et al. 2000).

Distribution of Rf gene associated markers in inbred lines

The OPW19 produced a unique, sharp and similar band (804 bp) reported previously (Zhang et al. 2000), when amplification products were separated on agarose gel. However, out of 37 restorer plants identified during this study, this band (OPW19800) was present only in 17 hot pepper restorer plants and 5 hot pepper plants segregating for both restorer and maintainer traits. As expected OPP13 also produced a unique and similar band (1397 bp) reported previously (Zhang et al. 2000), when separated on agrose gel. This band (OPP131400) was also present only in limited (10) restorer plants. Both the RAPD markers (OPW19800 and OPP131400) were absent in all the 5 sweet pepper maintainer plants (Table 1, Fig.1) as also observed by Zhang et al. (2000). Thus the presence of the both the markers often not coincided with the presence of Rf gene in the investigated restorer plants, which indicate that the origin and distribution of both the markers is narrow. Therefore, in spite of consistent and highly reproducible results with both the RAPD markers, they could not be utilized in MAS for fertility restoration or for direct screening of inbred lines of pepper for the presence/ absence of Rf/rf genes. Nevertheless, these reproducible RAPD markers may have case specific utilization in our cms based hybrid breeding program. For instance, in one of the identified promising hybrids (CCA-4261 x Pusa Jwala), which has been selected for further evaluation and promotion, both the markers are male specific, hence could be exploited in hybrid seed purity testing. The absence of both these markers in all the five sweet pepper maintainer lines may be useful during transfer of Rf gene in these sweet pepper lines (restorer breeding). Albeit practical use of RAPD markers in plant breeding has been questioned due to its poor reproducibility across the lab (Mohan et al. 1997), they have efficiently and successfully been utilized for hybrid seed purity testing of pepper (Ilbi 2003), cabbage (Crockett et al. 2000) and in MAS for resistance against root knot of tomato (Williamson 1998) and common bacterial blight of bean (Yu et al. 2000).

Table 1. Morphological features and distribution of two RAPD markers in the identified restorer and maintainer pepper (C. annuum) lines

ID #

Plant height (cm)
Mean SE

Fruit length (cm)
Mean SE

Fruit width (cm)
Mean SE

10 fruits (green) weight (g)

Identifi-ed gene

Plant height (cm)
Mean SE

4Markers

             

OPW
19804

OPP
131397

1

EC-119457

EC-119457

6.380.14

1.040.09

42

Rf

-

-

2

EC-257216

EC-257216

6.080.10

1.080.02

25

Rf

-

-

3

EC-257716

EC-257716

7.960.91

0.900.04

42

Rf

+

-

4

EC-268216

EC-268216

4.860.31

0.940.02

41

Rf

+

-

5

EC-341074

EC-341074

4.500.41

0.930.10

40

Rf

+

-

6

EC-341075

EC-341075

5.320.45

0.860.06

20

Rf

+

-

7

EC-345629

EC-345629

9.460.02

1.180.02

25

Rf

+

+

8

EC-491094

EC-491094

5.540.12

1.240.02

30

Rf/rf

+

+

9

Taiwan-2

Taiwan-2

2.121.40

0.820.02

20

Rf

+

+

10

G-4

G-4

6.280.13

0.880.02

25

Rf

+

-

11

G-5

G-5

3.600.10

1.500.20

35

Rf/rf

+

+

12

Phule Sai

Phule Sai

9.200.27

1.040.02

25

Rf

+

-

13

Punjab Lal

Punjab Lal

7.720.29

0.860.05

32

Rf

+

+

14

Pusa Jwala

Pusa Jwala

8.580.41

1.10.03

47

Rf

+

+

15

DC-3

DC-3

3.880.09

0.680.04

41

Rf

+

+

16

DC-5

DC-5

4.660.21

0.920.04

27

Rf

+

+

17

K. Chanchal

K. Chanchal

3.100.10

0.820.02

10

Rf

+

-

18

Waialua

Waialua

6.000.40

2.100.40

210

Rf

+

+

19

Assam-10

Assam-10

4.240.40

1.460.09

25

Rf

+

+

20

PBC-535

PBC-535

9.060.14

1.620.04

75

Rf

+

-

21

PBC-873

PBC-873

4.400.08

1.260.24

35

Rf

+

-

22

PBC-1512

PBC-1512

11.040.5

2.780.25

275

Rf

-

-

23

Local Coll-1

Local Coll-1

6.920.17

1.080.04

42

Rf

-

-

24

PDG-1

PDG-1

5.320.09

0.800.05

20

Rf

-

-

25

Perennial 2A

Perennial 2A

4.800.13

1.060.02

20

Rf

-

-

26

F1-112-1

F1-112-1

5.200.11

0.940.02

20

Rf

-

-

27

LCA-235

LCA-235

6.260.18

0.700.03

15

Rf

-

-

28

P-1649-1

P-1649-1

4.841.12

0.740.02

25

Rf

-

-

29

P-1649-2

P-1649-2

4.621.20

0.740.02

25

Rf

-

-

30

PDC-49A

PDC-49A

4.800.09

1.100.05

32

Rf/rf

-

-

31

PDC-53B

PDC-53B

4.440.15

0.680.04

30

Rf

-

-

32

KA-2

KA-2

5.200.14

0.700.03

34

Rf

-

-

33

KSPS-202*

KSPS-202*

5.640.16

4.980.05

655

rf

-

-

34

KSPS-501*

KSPS-501*

7.560.31

4.620.17

550

rf

-

-

35

Kaala*

Kaala*

5.820.14

4.060.07

410

rf

-

-

36

C. Wonder*

C. Wonder*

6.900.90

5.140.71

450

rf

-

-

37

ISPN # 2-3*

ISPN # 2-3*

11.280.7

5.040.32

355

Rf/rf

-

-

38

9852-173

9852-173

5.500.09

1.060.05

50

Rf

-

-

39

97-7125-3

97-7125-3

7.420.52

2.680.07

70

Rf

-

-

40

97-7125-2

97-7125-2

7.480.49

2.680.07

70

Rf

-

-

41

AKC-89/38

AKC-89/38

1.560.04

1.400.05

46

Rf

-

-

42

KDCS-810

KDCS-810

3.88 0.05

0.860.05

15

Rf

-

-

43

JCA-9

JCA-9

6.600.16

1.220.12

29

Rf/rf

+

-

44

Local Lal

Local Lal

5.230.20

1.500.60

35

Rf

-

-

45

Taiwan-1

Taiwan-1

7.160.12

1.220.02

60

Rf

-

-

46

CCA-4261

CCA-4261

9.860.23

1.420.05

90

rf

-

-

47

LCA-206

LCA-206

6.540.12

0.780.06

20

Rf/rf

+

-

48

Pant C-1

Pant C-1

3.560.19

0.940.04

20

Rf

+

-

1: *sweet pepper lines; 2: average of 10 green fruits; 3: Rf-fertility restorer gene and rf-fertility maintainer gene; 4: + presence and – absence of bands.

Figure 1. RAPD pattern generated through primer OPW19, showing presence and absence of Rf gene specific 800 bp fragment. The lane M is molecular weight marker (Lambda DNA EcoRI+HindIII Double Digested). The lanes 1 to 24 crosspond to numbers and names of inbred mentioned in table 1.Conclusions

The results on validation of Rf gene associated two RAPD markers in pepper revealed narrow distribution of both the markers in a panel of newly identified restorer and maintainer lines. The results showed potentiality of case specific utilization of RAPD markers in hybrid seed purity testing of one promising hybrid selected for further evaluation and promotion.

References

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Ilbi H (2003). RAPD markers assisted varietal identification and genetic purity test in pepper, Capsicum annuum. Scientia Horticulturae 97, 211-218.

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Williamson VM (1998). Root-knot resistance genes in tomato and their potential for future use. Ann. Rev. Phytopathology 36, 277-293.

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Zhang, BX, Huang S, Yang G, Guo J (2000). Two RAPD markers linked to a major fertility restorer gene in pepper. Euphytica 113, 155-161.

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