Annals of Sri Lanka Department of Agriculture. 2007.9:133-142.
TRANSFER OF YELLOW VEIN MOSAIC VIRUS RESISTANCE FROM A WILD RELATIVE (Abelmoschus angulosus L.) INTO CULTIVATED OKRA P.K. SAMARAJEEWA1, D.R. GIMHANI1, H.M. LIYANAGE1 and M.G.S.A. GUNARATHNA2 1 Plant Genetic Resources Centre, Gannoruwa, Peradeniya 2 Department of Biotechnology, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, Gonawila (NWP)
ABSTRACT Yellow Vein Mosaic Virus disease is a major problem in the cultivation of Okra (Abelmoschus esculentus). The economic loss due to the disease can vary from 50 to 94 percent depending on the growth stage of the crop. None of the recommended varieties are totally resistant to the disease. The importance of the wild relative, A. angulosus as a potential source of resistance has been documented earlier; but its incompatibility with okra together with high F 1 seed sterility presented a major difficulty to use the species in breeding. In the present work, backcrosses were done repeatedly on F1 plants derived from A. esculentus var MI 7 x A. angulosus cross. Later, by considering F1 plants as the pollen parent, three B 1F1 plants were raised. With the bud grafting experiments, they were confirmed to be virus resistant. They also showed some self-fertility. Further selfing enabled to establish a fertile selfed progeny (B 1F2). Segregation for virus resistance among the lines was assessed by both field screening and grafting techniques. Some RAPD markers that were identified were present in the wild type parent and the lines segregating for virus resistance. KEYWORDS: DNA marker, Virus resistance, Wild okra.
INTRODUCTION Okra (Abelmoschus esculentus L.) is one of the major vegetable crops grown in Sri Lanka as well as in many Asian and African countries. Owing to its wide adaptability, it is cultivated in many parts of the wet, intermediate, and dry zones of the country as a home garden crop or on a commercial scale (Samarajeewa and Rathnayaka, 2004). Fruits of okra are very rich in calcium amounting to around 90mg/100g fresh weight. (Markose and Peter, 1990) and therefore, provide a valuable supplementary nutrition in human diet. Among the production constraints affecting local okra cultivation, several disease problems have been identified. Yellow vein mosaic virus (YVMV) disease is the most serious problem in okra cultivation, especially in the wet zone (Samarajeewa and Rathnayaka, 2004). Heavy incidence of this disease causes loss in marketable yield up to 50-94%, depending on the stage of crop growth at which the infection occurs (Sastri and Singh, 1974). All locally grown recommended varieties of okra are
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susceptible to this disease except “Haritha” which has some field resistance (Giritharan and Arulandhy, 1995). However, due to low pod quality, farmer acceptance of the variety “Haritha” is poor. The causative organism of the YVMV disease is a DNA virus comprised of one or two single strands of DNA (Jose and Usha, 2000) and it is naturally transmitted by the white fly (Bemisia tabaci). The disease can also be transmitted by graft inoculation. Since Bemisia tabaci is resistant to insecticides, (Chague et al., 1997) breeding for resistance to YVMV appears to be a promising and ecological approach to control the disease. Wild relatives of crops have been recognized as an important source of useful resistance genes for breeding programmes. A number of characters present in wild relatives have been transferred to cultivated types through wide hybridization at the interspecific or intergeneric levels (Nomura and Makara, 1993). A wild type (Abelmoschus angulosus) has been previously identified as having complete resistance to the disease and some level of sexual compatibility with okra was also observed (Samarajeewa et al., 1999; Samarajeewa and Rathnayake, 2004). It has internal resistance to the virus. Therefore, it would be possible to use this as a resistance source to transfer the resistance traits in order to develop virus resistant varieties. Thus, this study was focused on transferring YVMV resistance from wild relative A. angulosus, into cultivated type of okra namely, MI-7. DNA markers have been widely accepted as potentially valuable tools in crop improvement via marker assisted selection (MAS) in many crops (McCouch and Doerge, 1995; Lee, 1995). However, no previous attempts have been made to identify DNA markers for virus resistance in okra. Segregating lines derived from wide hybridization can be conveniently used to develop a molecular marker for subsequent application in MAS. Polymerase Chain Reaction (PCR) based markers, such as random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) are the favourite selection in molecular breeding because of their simplicity and low cost (Kumar et al., 2005). Previously, RAPD has been used to characterize wild and cultivated types of okra germplasm (Samarajeewa and Rathnayake, 2004). In the present work, studies were also conducted to identify RAPD markers, which are potentially linked with YVMV resistance. MATERIALS AND METHODS Plant material Seeds of A. angulosus and cultivated okra variety MI-7 were obtained from the Gene Bank of Plant Genetic Resources Centre (PGRC) and grown in large plastic Wagnar pots and maintained in the green house of
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PGRC during 2004 yala season. A. angulosus was the resistant donor parent. MI-7 was treated as the susceptible parent. MI-7 plants were selfed to obtain seeds for experiment and the characters of the variety were confirmed. Hybridization between A. angulosus and MI-7 Following a standard hand pollination method, A. angulosus was crossed with MI-7 by using the wild relative as the pollen parent. The F 1 plants were raised in pots in the green house and backcrossed with MI-7. The resulting three backcrossed plants (B1F1) were maintained in the PGRC field during 2005/06 maha season. Recommended fertilizer application, spacing etc. were followed. Three backcrossed plants were then selfed separately to produce B1F2 plants and seeds were planted in the field along with MI-7 plants. Morphological characterization of progeny According to the keys given in a characterization catalogue on okra (PGRC, 1999) progeny resulting from first backcrossed plants (B1F1) and selfed progeny (B1F2) were characterized morphologically. Thirteen phenotypic characters were recorded at maturity and fruiting stages (Table 1). Screening for virus resistance Screening of parents and progeny lines for virus resistance was done by both graft inoculation and vector transmission. For graft inoculations and field infection, susceptible cultivar, MI-7 was maintained in the field as virus reservoir. Two to three months old plants were graft inoculated using about 2cm long scions obtained from YVMV infected plants (Samarajeewa and Rathnayaka, 2004). Four weeks after grafting, plants were observed for disease symptoms and development of yellowing in veins was recorded. Simultaneously, natural transmission of the virus to the hybrid plants by white flies was also allowed and development of YVMV symptoms was recorded. Molecular analysis DNA extraction Plant DNA was extracted from young fresh leaf samples (around 0.6g) of the parents (A. angulosus and MI-7), F1 plant, 3 B1F1 plants and 11 plants in selfed progeny (B1F2) according to the method described by Dellaporta et al., 1983.
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Assessment of quality and quantity of the DNA To check the quality of DNA, DNA samples were electrophoresed on 0.8% agarose gel in 0.5 x TBE at 5V/cm and the gel was visualized under UV light after staining with ethidium bromide (0.5µg/ml). Extracted DNA samples were quantified using Biomate 3 spectrophotometer. DNA samples were then diluted using TE buffer (pH 8.0) to a final concentration of 20ng/µl to be used in RAPD PCR reaction. RAPD analysis RAPD-PCR reactions were carried out using six 10 mer random primers namely OPA10, OPC10, OPC02, OPD20, OPK15 and OPM10 (Operon technologies Inc. USA) according to Williams et al. (1990). Analysis of PCR products RAPD-PCR products were separated by 1.4% agarose gel electrophoresis in 0.5 x TBE buffer at 5V/cm. After staining with ethidium bromide (0.5µg/ml), gel was visualized and analysed under UV light using a gel documentation system (BIO–RAD) equipped with quantity one software package. Reproducible bands unique to wild relative were selected by comparing the RAPD profiles of parents for each primer. Then, the presence of these unique bands in virus resistant individuals of the backcross progeny lines was recorded. The sizes of the RAPD products were estimated by comparison with 10kb DNA ladder (Sigma, Wide range DNA ladder). RESULTS AND DISCUSSION Hybridization between A. angulosus and MI-7 The wild relative, A. angulosus used in this study has been previously identified as having complete resistance to the disease (Samarajeewa, et al., 1999; Samarajeewa and Rathnayake, 2004). According to our previous studies A. angulosus could not be infected either by white fly or graft inoculation techniques indicating that it has both internal and external resistance to the virus infection. It also exhibits a very low level of sexual compatibility with the cultivated type MI-7. Though, incompatibility was observed even in F1 plants produced by the above cross, in the present work, it was possible to raise 3 backcrossed (B1F1) plants. According to the characterization data, these plants showed intermediate characters (Table 1). They also showed self-fertility to some extent. After selfing of B1F1 plants,
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only 11 plants (B1F2) resulted from one of them and B1F2 plants were successfully grown in the field for further characterization and evaluation. Morphological characterization of progeny Table 1 presents the 13 recorded morphological characters of the B1F1, B1F2 progenies and their parents. Characterization of progeny lines is helpful to identify the plants with useful characters, and to determine the level of seed formation. According to the morphological data, most of the plants were similar to the recurrent parent (MI-7), except for few characters. Some characters were intermediate to the parents. For all the progeny lines three morphological characters, namely growth habit, leaf colour and number of ridges per fruit were similar to the parents. Branching habit, leaf shape and fruit pubescence of progeny plants showed close affinity to their wild parent. However, when characters such as stem colour, stem pubescence, size of the fruit and shape of the fruit are considered, they varied from intermediate to characters of MI-7. Screening for virus resistance According to evaluation for YVMV resistance, by both graft inoculation and field infection, all three individuals resulting from the first backcross (B1F1) showed YVMV resistance. The 11 plants of the B1F2 progeny produced by selfing of backcross plants (B1F1) also showed field resistance. However, due to rainy weather conditions the graft inoculation experiments were not successful on all the plants in B1F2 progeny and hence require further evaluation. Observations made even with a few progeny plants indicate that the virus resistance in A. angulosus may be inherited in a dominant manner and apparently controlled by a major gene. However, to confirm the nature of inheritance, i.e. whether the trait was controlled by a major gene or by QTL, a large population, segregating for the resistance trait will have to be analyzed (Paterson and Lander, 1988). Sufficient number of backcrosses using MI-7 (the recurrent parent) could produce a large population. Molecular analysis Segregating lines derived from wide hybridization could be conveniently used to develop a molecular marker for subsequent application in MAS. A similar set of molecular data can be used to establish genetic linkage maps and assigning resistance loci onto chromosomes (Mohan, 1997).
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Table 1. Morphological characteristics of parents (A. angulosus-wild type and A. esculentus -variety MI-7) and their progenies.
Characters
A.angulosus (Wild type)
A..esculentus (Cultivated variety MI-7)
F1 Hybrids
Backcross 1 (B1F1)
B1 F2 generation
1 2
Growth habit Branching
Erect Orthotropic
3 4
Stem pubescence Stem color
Erect Moderately branched Conspicuous Green
5
Leaf shape
Deeply lobed (Catalogue no. 8)
Leaf color Fruit color at table use
Green Green
Erect Moderately branched Slight Green with red patches Intermediate to both parents Green green
Erect Orthotropic to moderately branched Slight to Absent Green to Green with red patches Intermediate to both parents
6 7
Erect Moderately branched Slight Green with red patches Intermediate to both parents Green Green
8 9
5 3
9 2
16 9.2
10
Fruit length at maturity (cm) Fruit width at the widest point (cm) Fruit shape
11 12
No. of ridges per fruit Fruit pubescence
5-7 Slightly rough
Long pods (Catalogue no. 1) 5-7 Downy
Intermediate to both parents 5-7 Prickly
13
No. of seeds/pod
55-60
100-200
0-13
Intermediate to both parents 5-7 Slightly rough 70-80
Small pods (Catalogue no. 14)
Slight Green with red patches Shallowly lobed (Catalogue no. 4) Green Yellowish green 23 8
Green Green to Yellowish green 9.1 to 23 8 to 11 Intermediate to both parents 5-7 Downy to prickly 10-50
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In the present work, susceptible parent (MI–7) and resistant parent for YVMV (A. angulosus) were tested along with the individuals of the backcrossed (B1F1) and selfed (B1F2) progenies using RAPD. Preliminary work was carried out to identify potential RAPD marker/s for the YVMV resistance in okra. All the random primers used in this study were able to generate amplification products except for the primer OPK 15, which produced few bands for some samples. Out of the six primers tested, OPC02, OPA10, OPC10, OPM10 (Fig. 1) and OPD20 amplified reproducible bands unique to wild relative (resistant parent) (Table 2). These unique bands were present in all the segregating individuals, which showed field resistance to YVMV indicating the potential of RAPD technique to identify markers for the YVMV resistance. Table 2. RAPD bands generated by selected primers. Primer Total Total No. of No. of unique bands No. of Polymorphic in A. angulosus and bands in bands between resistant individuals parents parents in progenies OPM10 OPD20 OPC02 OPC10 OPA10
11 20 19 7 17
9 11 13 6 7
1 2 3 3 2
Approximate size in bp
1200 390, 1250 800, 900, 1000 1000, 1400, 2500 1350, 1550
However, RAPD markers that resulted in the present study could be or could not be linked with resistance and therefore they have to be confirmed with the subsequent backcross populations of the A. angulosus x MI-7 cross. Moreover, present molecular marker based study has to be conducted further using more polymorphic primers and a sufficient number of individuals segregating for the trait in order to identify the RAPD marker/s, which may be tightly linked to the YVMV resistance. One of the prerequisites for the identification of a DNA marker linked to the disease resistance is to have a segregating population for the trait. F2 populations derived from F1 hybrids and backcross populations derived from the F1 hybrid to one of the parents are the simplest types of populations that can be used for this purpose (Collard et al., 2005). In this study, F2 population could not be raised due to high sterility of F 1 plants. Therefore, a backcross population and the selfed progeny of the backcross population were used.
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L
P 1 P 2 F1 1 2
3 4 5 6 7 8 9 10 11 12 13
bp
2000 1550 1000
~1200 bp
750 500 400 300 200 100 50
B1F1
B1F2
Figure 1. RAPD profile generated by primer OPM10. Lane L – 10 kb DNA ladder, Lane P1 - MI-7 (YVMV susceptible parent), Lane P2 - A. angulosus (YVMV resistant parent), Lane F1- F1 hybrid of the A. angulosus x MI-7 cross, Lane 1 and 2 - resulted 2 individuals of B1F1 progeny, Lane 3 - 13 – resulted 11 individuals in B1F2 progeny, Arrow - unique band present in resistant parent and resistant individuals in B1F1 and B1F2 progeny.
During this study, extraction of DNA from okra leaves was a difficult task, since okra is a highly mucilaginous plant. Mucilage in okra is an acidic polysaccharide composed of galacturonic and glucuronic acids associated with minerals (Jose and Usha, 2000). Polysaccharides have viscous, glue like texture and make the DNA unmanageable in pipetting and unsuitable for PCR since they inhibit Taq DNA polymerase activity. They can also co- precipitate with DNA after alcohol addition during DNA isolation to form highly viscous solutions (Do and Adams, 1991). However, in this study, it was able to extract okra DNA free from polysaccharides, using the protocol described by Dellaporta et al. (1983). Here, protein and polysaccharides were precipitated simultaneously by potassium acetate in the presence of SDS. In present study, confirmation of the virus infection was carried out by field infection and graft inoculation. However, use of molecular diagnostic techniques would greatly facilitate quick and accurate detection of the infection enabling to distinguish resistant individuals from susceptible lines. There is a possibility to establish a molecular diagnostic method based on PCR using a virus specific primer pair (Jose and Usha, 2000).
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CONCLUSIONS YVMV resistance could be transferred into cultivated type of okra, MI-7 from Abelmoschus angulosus. However, further backcrossing should be carried out using MI-7 as a recurrent parent to produce a virus resistant line close to MI-7. The random primers, OPC02, OPA10, OPC10, OPD20 and OPM10 amplified unique bands that could be potentially used in identification of resistant segregants. ACKNOWLEDGEMENTS The authors wish to acknowledge the SL-USDA programme for making the laboratory facilities acquired through the programme, which were available for DNA work. The support given by the staff and non-staff officers of the PGRC is also highly appreciated. REFERENCES Chague, V., J.C. Mercier, M. Guenard, A. de Courcel and F. Vedel. 1997. Identification of RAPD markers linked to a locus involved in quantitative resistance to TYLCV in tomato by bulked segregant analysis. Theor. Appl. Genet. 95: 671-677. Collard, B.C.Y., M.Z.Z. Jahufer, J.B. Brouwer and E.C.K. Pang. 2005. An introduction to markers, quantitative trait loci mapping and marker assisted selection for crop improvement. The basic concepts. Euphytica 142:169-196. Dellaporta, S.L., J. Wood and J. Hicks. 1983. A plant DNA minipreparation version 11. Plant Mol. Biol. Rep. 1 (4): 19-21. Do, N. and R.P. Adams. 1991. A simple technique of removing plant polysaccharides contaminants from DNA. Biotechniques 10: 162-166. Giritharan, D. and V. Arulnandhy. 1995. Studies on hybrid vigour in the crosses of okra (Abelmoschus esculentus) (L.) (Monech) genotypes of diverse origin. Tropical Agriculturist 150: 65-69. Jose, J. and R. Usha. 2000. Extraction of Geminiviral DNA from a highly Mucilaginous Plant (Abelmoschus esculentus). Plant Molecular biology reporter 18: 349- 355. Kumar, B., S.M. Gomez, N.M. Boopathi, S.S. Kumar, D. Kumaresan, K.R. Biji, B.K.Babu, N.S.R. Prasad, P. Shanmugasundaram and R.C. Babu. 2005. Identification of microsatellite markers associated with drought tolerance in rice (Oryza sativa L.) using bulked line analysis. Tropical Agricultural Research 17: 39 - 47. Lee, M. 1995. DNA markers and plant breeding programmes. Advance Agronomy 55: 265344.
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