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African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 4, Num. 2, 1996, pp. 145-150
African Crop Science Journal
Vol.5. No.2, pp. 145-150 1997

Genetic linkage of the aphid resistance gene, Rac, in cowpea

S. M. GITHIRI, P.M. KIMANI and R.S. PATHAK*

University of Nairobi, Department of Crop Science, P. O. Box 30197, Nairobi, Kenya
International Centre of Insect Physiology and Ecology (ICIPE), Mbita Point Field Station, P. O. Box 30, Mbita

*Present address: B31/35K-3A-1, Bhogabeer, Lanka, Varanasi, India

(Received 18 August, 1995; accepted 14 May, 1996)


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ABSTRACT

Linkage of the aphid resistance gene, Rac, with various polymorphic loci controlling morphological traits and aspartate amino-transferase isozyme (AAT) was studied in cowpea (Vigna unguiculata L. Walp) with the objective of identifying simply inherited and easily identifiable markers for aphid resistance and to distiguish between Rac1 and Rac2 reported by Pathak (1988). The F2 and F2 - derived F3 populations from crosses IT87S-1459 x Tvu 946 x ICV 5, and IT84S-2246 x Tvu 946 segragating for Rac1 and cross ICV 12 x Tvu 946 segragating for Rac2 were scored for various polymorphic morphological traits. Locus pd, controlling peduncle colour, was found to be linked to both Rac1 and Rac2 (P < 0.05). The recombination frequencies estimated by the maximum likehood method were 26 +/- 8.3% and 35 +/- 7.5% for Rac1-pd and Rac2-pd co-segregation, respectively, thus indicating that Rac1 and Rac2 were not different from one another. No linkage was found between aphid resistance genes and the genes controlling other polymorphic morphological traits or AAT isozyme.

Key Words: Aphid resistance, markers, genetic linkage, Vigna unguiculata

Resume

La relation entre le gene de resistance aux aphids, Rac, avec de nombreux loci polymorphiques qui controlent les traits morphologiques et l'isozyme asparate d'amino transferase (AAT) etait etudie chez le niebe dans l'optique d'identifier les marqueurs facilement heritables et facilement identifiables pour la resistance aux aphides et envue de faire la distinction entre Rac1 et Rac2 tels que precaunise par Pathak (1988). Les populations F2 et F2 - derivees de F3 des croisements IT87S-1459 x Tvu 946 segregues pour Rac2 ont ete cotees pour leurs differents traits morphologiques polymorphiques. Le locus pd, controllant la couleur de pedoncule etait associe au Rac1 et Rac2 (P<=0.05). Les frequences de recombination estimees par une methode de probabilite maximale etaient de 26 +/- 8.3% et de 35 +/- 7.5% pour la co-segregation des Rac1-pd et Rac-2-pd-respectivement; ce qui indique que la Rac2 n'etait pas differents l'une de l'autre. Aucune relation n'a ete revelee entre les genes de resistance aux aphides et les genes qui controllent les autres traits morphologiques polymorphiques ou l'isozyme AAT.

Mots Cles: Resistance aux aphides, marqueurs, lien genetique, Vigna unguiculata

INTRODUCTION

Cowpea aphid, Aphis cracccivora Koch, is an important pest of cowpea in Africa (Singh and Jackai, 1985). The pest causes direct damage on the crop by sucking plant sap, and indirect damage by transmitting aphid-borne cowpea mosaic viruses. The pest can be controlled by various methods including use of aphicides, cultural control and biological control (Singh and Jackai, 1985). However, growing of aphid resistant cultivars offers one of the simplest and most convenient methods of pest control (Dent, 1991). Sources of aphid resistance have been identified (IITA, 1983; ICIPE, 1986) and the resistance incorporated into a number of breeding lines.

In screening for aphid resistance, workers normally rely on actual aphid infestation of the test cultivars in the field or in the greenhouse. Aphid infestation, however, is highly erratic and influenced by environmental conditions, while planting of large segregating populations, as found in most breeding programmes, is not feasible in the greenhouse.

Identification of simply inherited and easily distinguished markers closely linked to aphid resistance genes would be highly desirable in a breeding programme aimed at incorporating aphid resistance into high yielding cowpea cultivars. The marker(s) could be used for indirect selection of aphid resistant cultivars even in the absence of aphids.

Aphid resistance in cowpea is inherited as a monogenic dominant trait (Bata et al., 1987; Pathak, 1988). Pathak (1988) reported that the gene controlling aphid resistance in cultivars ICV 11 and ICV 12 was different from that in cultivars ICV 10 and Tvu 310. He assigned the gene symbols Rac1 for the gene in cultivars ICV 10 and Tvu 310 and Rac2 for the gene in cultivars ICV 11 and ICV 12.

Fery (1985) summarised results of genetic studies that have been conducted in cowpea since the 1900s. The majority of the traits studied based on various morphological characters, are those which are simply inherited and easily identifiable, such as coloration of various plant parts, plant type and others. Such traits are important in that they could be used as markers for aphid resistance genes in cowpea.

Several researchers have reported possible utilisation of isozyme markers in plant breeding (see Soltis and Soltis, 1989). Isoenzyme markers are important in marker-based selection in that, unlike morphological markers, they are expressed in a co-dominant manner and can thus distinguish between homozygotes and heterozygotes, need only a small tissue for analysis, and are not influenced by environmental conditions (Weeden and Wendel, 1989). Vaillancourt et al. (1992) and Githiri (1995) studied isozyme polymorphism in cowpea and observed that aspartate amino transferase (AAT) was polymorphic among cowpea cultivars.

The present study was undertaken to test for linkage between the aphid resistance genes, Rac1 and Rac2, and genes controlling various morphological traits and the aspartate amino transferase (AAT) isozyme in cowpea.

MATERIALS AND METHODS

Greenhouse and field experiments. This study was conducted at the Mbita Point Field Station (MPFS) of the International Centre for Insect Physiology and Ecology (ICIPE). Cowpea genotypes used in this study included three aphid resistant (ICV 12, IT87S-1459, and IT84S-2246) and two aphid susceptible (ICV 5 and Tvu 946) cultivars. ICV 12 and ICV 5 are advanced breeding lines obtained from ICIPE while Tvu 946, IT87S-1459 and IT87S-2246 are advanced breeding lines originally obtained from the International Institute for Tropical Agriculture (IITA). IT87S-1459 and IT84S-2246 carry resistance gene Rac1 while ICV 12 carries Rac2 (Pathak, 1988). All these cultivars have been maintained as pure lines at MPFS. Four crosses (IT87S-1459 x Tvu 946, IT87S-1459 x ICV 5, IT87S-2246 and ICV 12 x Tvu 946) were made in the green house in March-April 1992. These pairs of parents were selected because they differed with respect to various morphological attributes and/or isozyme AAT in addition to aphid resistance (Table 1).

Large F2 populations (over 100 plants) of each of the four crosses were grown in the field during the short rainy season of 1992. The seeds were sown at inter-and intra-row spacings of 60 cm and 30 cm, respectively. Individual plants were tagged and data recorded on each one of them for segregration in each of the contrasting traits found in the parents (Table 1).

Evaluation of reaction to aphid infestation for the F2 plants was done by progeny testing. To achieve this, 40-73 F2 plants of various crosses were selected and seed harvested separately for each plant. The seed of each F2 plant of cross ICV 12 x Tvu 946 was divided into two parts: one part was used for evaluation of progeny reaction to aphid infestation; the other part was used for isoenzyme (AAT) analysis.

Seedlings for progeny tests of reaction to aphid infestation were grown in the greenhouse. Seeds were sown in single rows, at inter- and intra-row spacings of 10 cm and 5 cm, respectively. Susceptible check (ICV 1) and resistant check (ICV 12) were grown in single rows after every ten rows of test material. At the two-leaf stage (three-four days after emergence) each seedling was infested with five, fourth-instar aphids. When all seedlings from the susceptible check row were killed, 14-16 days after infestation, the F3 progeny rows were scored as susceptible (>90% seedlings dead), resistant (<10% seedlings dead), or segragating (10-90% seedlings dead). The reaction of seedlings in the F3 population towards aphid infestation was thus used in deciding the genotype of the F2 plant.

Isozyme variation. Variation at the AAT locus was studied using horizontal starch gel electrophoresis of the parents and F2-derived F3 progenies of the cross ICV 12 x Tvu 946. This cross was selected because, when staining for the isozyme AAT in the parent cultivars, it was observed that cultivar Tvu 946 had a none allele while cultivar ICV 12 stained for one band (Githiri, 1995). Techniques for isozyme analyses were adapted from Glaszmann et al. (1988) and Weeden and Wendel (1988).

Cowpea seeds soaked on wet filter paper for 24-30 hr at room temperature were used for enzyme extraction. The extract was absorbed onto sewing thread wicks and inserted in the appropriate slits of starch gel (8% gels made with hydrolysed starch from Sigma Chemical Co.). After loading the samples, the gel was mounted onto the electrode trays containing lithium-borate electrode buffer (Weeden and Wendel, 1989) and connected to a LKB continous-current power supply. Electrophoresis was conducted at constant current (250 mA, 38 W) for about 3 hr at 4o C. Aspartate amino transferase (AAT) (EC 2.6.1.1) activity was revealed by immersing the gel slices in a stain assay composed of 20 mg L-aspartic acid, 10 mg -ketoglutaric acid, 15 mg fast blue BB salt, 0.2 mg pyridoxal-5'-phosphate and 15 ml lithium-borate electrode buffer, pH 8.1, for about one hour at room temperature. The zymograms were scored as present or absent.

Statistical analyses. Chi-square analysis in a contingency table was used to test the goodness-of-fit of observed ratios with expected ratios following Mendelian segregation. For each morphological trait and aphid resitance, 3:1 or 1:2:1 distribution was tested; for co-segregation between aphid resistance gene, Rac, and the genes controlling each of the morphological traits, an expected 9:3:3:1 distribution was tested, except in the case of loci Pal, Pus, and Pup where an expected 3:6:3:1:2:1 ratio was tested. When the Chi-square values for co-segragation were significant, linkage between such gene pairs was suspected. Percent recombination between linked genes were calculated by the method of maximum likelihood (Allard, 1956). When Chi-square values for co-segregation were non-significant, such gene pairs were considered to be independent of each other.

RESULTS AND DISCUSSION

Segregation of qualitative traits. Results of inheritance of some selected morphological traits, AAT isozyme and aphid reistance from four crosses indicated that all these traits were simply inherited (monogenic) and they segregated either in a 3:1 or 1:2:1 ratio (data not shown). Results of pooled populations from different crosses, where applicable, also gave good fits for monogenic inheritance.

Linkage between Rac1 and 13 loci. Results of the tests for linkage between the aphid resistance gene Rac1 (Pathak, 1988) and 13 morphological traits in three different crosses are presented in Table 2.

Cross IT84S-2246 x Tvu 946. Significant Chi-square values (P < 0.05) were obtained for the joint segregation of Rac1 and each of the loci Pbr, Pd, and Pt. An estimate of recombination frequency for Rac1-Pd co-segregation was 26+/-8.3% thus indicating that the two loci were linked. Recombination frequency estimates for Rac1-Pbr and Rac1-Pt co-segregation were over 50% thus indicating that these loci were not in the same linkage group. Non-significant Chi-square values (P>0.05) were obtained for the joint segregation of Rac1 and each of loci Hg and Bk, indicating that they were independent of each other.

Cross IT87S-1459 x Tvu 946. A significant Chi-square value (P < 0.05) was obtained for Rac1 - Gn co-segregation. An estimate of recombination frequencies gave a value of over 50%, thus indicating that these loci were independent of each other. Non-significant Chi-square values (P>0.05) were obtained for the joint segregation of Rac1 and all other loci tested.

Cross IT87S-1459 x ICV 5. Significant Chi-square values (P<0.05) were obtained for Rac1-Pup, and Rac1-Pus co-segregation. Estimates of recombination fractions for the joint segregation of Rac1 with each of the loci gave values of over 50% recombinants thus indicating that Rac1 was not linked to any one of the these loci.

Linkage between Rac2 and eight loci. Results on tests for linkage between Rac2 and eight loci controlling morphological traits and AAT isozyme are presented in Table 3.

Cross ICV 12 x Tvu 946. A significant Chi-square value (P<0.05) was obtained for the Rac2 -pd co-segregetation in this cross. An estimate of the recombination fractions between the two loci was 35 +/- 7.5%. The results suggested that locus Rac2 is in the same linkage group with locus Pd. Non-significant Chi-square values (P<0.05) were obtained for the joint segregation of Rac2 with each of the loci Ndt, Gh, Pt, Gn, Bk, Pal, AAT and Er. These results suggested that locus Rac2 was independent of all these loci.

Pathak (1988) reported that two independent loci Rac1 and Rac2 are involved in the expression of resistance to aphids in cowpea. Results from this study based on two crosses indicate that both Rac1 and Rac2 are linked to a common locus, pd, controlling peduncle colour. Rac2 in cultivar IT87S-1459 was not linked to Pd. This information was contradicting since both cultivars IT87S-1459 and IT84S-2246 are from the same breeding programme and carry the same gene for resistance to aphids. However, recent studies on allelic relationships among sources of aphid resistance indicated that cultivars ICV 12, IT87S-1459 and IT84S-2246 carry the same gene for resistance to aphids (Githiri et al., 1996). The difference between cross IT87S-1459 x Tvu 946 and other crosses in this study might have arisen from other factors, among which is the small size of the F2 populations used, modifiers present in the cultivars, or simply sampling errors. In view of this, therefore, these results have to be taken as preliminary until confirmatory experiments using larger F2 populations have been conducted.

ACKNOWLEDGEMENTS

This work was conducted at the Mbita Point Field Station of ICIPE with funds availed to the first author from the German Academic Exchange Programme (DAAD). The techinical assistance of Mr. James Onduru is appreciated.

REFERENCES

Allard, R.W. 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24:235-278. Bata, H.D., Singh, B.B., Singh, S.R. and Ladeinde, T.A.O. 1987. Inheritance of resistance to aphids in cowpea. Crop Science 27:892-894.

Dent, D. 1991. Insect Pest Management. CAB Wallingford, UK.

Githiri, S.M. 1995. Aphid Resistance in Cowpea and its Relationships With Morphologial and Biochemical Characters. Ph.D. Thesis, University of Nairobi, Kenya.

Githiri, S. M., Ampong-Nyarko, K., Osir, E. O. and Kimani, P. M. 1996.

Genetics of resistance to Aphis craccivora in cowpea. Euphytica 89:371-376.

Glaszmann, J.C., de los Reyes, B.G. and Khush, G.S. 1988. Electrophoretic variation of isozymes in plumules of rice (Oryza sativa L.) - A Key to the identification of 76 alleles at 24 loci. IRRI Research Paper Series No 134.

International Centre for Insect Physiology and Ecology (ICIPE) 1986. Annual Report 1985. ICIPE, Nairobi, Kenya. International Institute of Tropical Agriculture (IITA). 1984. Annual Report 1983. IITA, Ibadan, Nigeria.

Pathak, R.S. 1988. Genetics of resistance to aphid in cowpea. Crop Science 28:474-476.

Pathak, R.S. and Olela, J.C. 1986. Registration of 14 cowpea cultivars. Crop Science 26:

Singh, S.R. and Jackai, L.E.N. 1985 Insect pests of cowpea in Africa: Their life cycle, economic importance, and potential for control. In: Cowpea Research, Production, and Utilization. Singh, S.R. and Rachie, K.O. (Ed.), pp. 217-231. John Wiley & Sons, New York, USA.

Soltis, D.E. and Soltis P.S. 1989. Isozymes in Plant Biology. Dioscorides Press, Portland, Oregon, USA.

Wendel, J.F. and Weeden, N.F. 1989. Visualisation and interpretation of plant isozymes. In: Isozymes in Plant Biology. Soltis, D.E. and Soltis, P.S. (Ed.), pp. 5-45. Dioscorides Press, Portland, Oregon, USA.

Copyright 1996 The African Crop Science Society


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