Breeding Pole Type French Bean (Phaseolus vulgaris) for Rust (Uromyces phaseoli Reben Wint) Resistance using Molecular Markers  

Bapuji Divya1 , T.S. Aghora2 , N. Mohan2 , D.C. Lakshman Reddy2 , C. Aswath2
1. University of Horticultural Sciences, PG Center, Bengaluru-560065, India
2. Indian Institute of Horticultural Research, Bengaluru- 560 089, India
Author    Correspondence author
International Journal of Horticulture, 2014, Vol. 4, No. 10   doi: 10.5376/ijh.2014.04.0010
Received: 14 Apr., 2014    Accepted: 21 Apr., 2014    Published: 07 May, 2014
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Divya et al., 2014, Breeding Pole Type French Bean (Phaseolus vulgaris) for Rust (Uromyces phaseoli Reben Wint) Resistance using Molecular Markers, International Journal of Horticulture, 2014, Vol.4, No.10 50-52 (doi: 10.5376/ijh.2014.04.0010)

Abstract

French bean (Phaseolus vulgaris L.) 2n=14 is an important legume vegetable grown for its tender green pods which are rich in proteins, minerals and dietary fibre. Though it is a short duration crop, it is prone to several biotic and abiotic stresses. Among the biotic stresses, rust caused by Uromyces phaseoli (Reben Wint) has become epidemic in bean growing areas and the yield loss to the tune of 80% is reported. Rust fungi have wide variability in pathogenicity and also demonstrate frequent sexual recombination and more than 150 physiological races of the pathogen are reported. Hence, the induction of genetic resistance is always preferred because it is least expensive and has no adverse environmental effects. Inheritance of rust resistance was studied in F2 generation by using molecular markers. Resistance was confirmed in parent genotype IC525236 by using SK 14. In IC525236 resistance to rust is controlled by single dominant gene. 

Keywords
French bean; Rust resistance; Single dominant gene; SK 14

French bean, Phaseolus vulgaris L. (2n=22) is a member of the family Fabaceae. It is an important legume vegetable grown for its tender green pods either for fresh consumption or for processingIn world, french bean is grown over an area of 1.48 million ha with annual production of 17.65 million MT and the productivity of 11.95 t/ha. In India, its cultivation is in 0.22 million ha with production of 0.68 million MT and productivity of 2.8 t/ha (1). Like any other crops, french bean is also susceptible to various biotic and abiotic stresses. Among the biotic stresses, rust (Uromyces phaseoli Reben Wint.) has become epidemic in bean growing areas. The yield loss due to rust ranges from 18% to 78% (2 and 3). Recently, the trend in french bean cultivation is changing from bush type to pole type beans because of their long yielding period and high yield. Popular pole bean varieties like NZ, NZ Super King, US-2, HAPB-4 etc, though high yielding, they are all susceptible to rust disease causing heavy loss to farmers. Thus, the present study was under taken to incorporate rust resistance into quality french bean cultivars using molecular markers (SCAR) linked to rust resistance.
Result and Discussion
Confirmation of rust resistance in parents
Four parental genotypes viz., IC52236, IIHRPB-1, IIHRPB-2 and IIHRPB-7 were used for confirmation of rust resistance in french bean using validated SCAR markers viz., SK 14. Initially, genomic As the banding pattern was intact in all the genotypes, primer SK 14 was used to study the polymorphism for rust resistance. SK14 showed resistant banding pattern at 620 bp (Table 1; Figure 1).


Table 1 Estimates for rust resistance in several crosses of F1 population


Figure 1 PCR amplification of DNA from Resistance and Susceptible parents using SCAR marker SK 14


Screening of F1 population for rust resistance
F1’s obtained from each cross combination were raised in field and at first trifoliate leaf stage, DNA was extracted. F1’s were screened using molecular marker, SK 14. F1’s involved IC525236 as resistant parents showed the resistant banding pattern 620 bp (Table 1; Figure 1).
Screening of F2 population for rust resistance
SCAR marker, SK 14 assisted foreground selection for rust resistant gene was done in the F2 (Table 2; Figure 2). Initially, the parental DNA with SCAR markers linked to rust resistance was amplified. The PCR conditions for SCAR primers were optimized for DNA amplification to obtain a good amplification with clear bands at optimum annealing temperature. SK 14620 amplified at 620 bp in resistant parent IC-525236 and no amplification was observed in IIHRPB-7 (susceptible). In F2 population from the cross IIHRPB-7 X IC-525236, out of 100 plants screened, 71 plants showed the resistant banding pattern at 620 bp indicating the presence of target gene. These resistant lines were selected for further advancing. This resistant marker was also used for screening F1 population, wherein all the lines showed the presence of the above marker associated with rust resistance. Similarly, Vikas et al. (2012) incorporated blast resistance genes in rice viz., Piz-5 and Pi54, from the donor lines C101A51 and Tetep into the genetic background of PRR78 through marker assisted foreground selection. Foreground selection for these genes was done using tightly linked molecular markers, AP5930 and RM206. But even though IIHRPB-1 and IIHRPB-2 were highly susceptible to rust showed amplification as like in resistant parent IC-525236 at 620 bp. This showed that resistance to individual rust races in bean line IC-525236 (Ur-3) is conditioned by single dominant gene linked in coupling that appear to be inherited as a complex linkage block. The apparent linkage of an additional unnamed gene from the suggested that the Ur-3 region may contain an even greater complex of linked genes than Ur-3 alone. Similar finding were reported by Pastor and Corrales (2003).


Table 2 χ2 estimates for rust resistance in several crosses of F2 population


Figure 2 PCR amplification of DNA from F2 individuals of IIHRPB-7×IC-525236 using SCAR marker SK 14


In cross involving IC-525236, a
ll the plants in F1’s exhibited polymorphism at 620 bp which indicated that the resistance to rust is dominant in nature in IC-525236. In F2 progeny of cross IIHRPB-7× IC-525236, out of 100 plants, 71 plants showed polymorphic DNA at 620 bp for SK 14, remaining 29 plants did not exhibit polymorphism. χ2 values also indicated that the ratio for observed to expected was not significant. These results indicated that the segregation was in the pattern of Mendelian ratio of 3:1 which confirmed that the inheritance to rust resistance is monogenic dominant (Aghora et al., 2007; 2008). Therefore, it would be easy to transfer the resistant gene to any desirable background. Thus, resistance can be easily incorporated with any other commercial cultivars through back cross breeding using resistant parent as donor. These findings are in accordance with Yuebin (1993); Sayler et al.(1995) and Aghora et al. (2007; 2008).
Material and Methods
Experiments on breeding pole type french beans (Phaseolus vulgaris L.) for resistance to rust using molecular markers were carried out at Division of Vegetables crops and Division of Biotechnology, Indian Institute of Horticultural Research, Bengaluru, 2012 to 2013. Experimental material consisted of resistant parent, IC525236 and three susceptible pole type good pod quality lines namely, IIHRPB-1, IIHRPB-2 and IIHRPB-7. Crossing was carried out by emasculation and hand pollination between resistant and susceptible parents to develop F1 and F2 populations. DNA of these genotypes were extracted using CTAB method.
The present study aims with three SCAR markers namely, SK 14, SA 14 and SBC 6 used as foreground selection to integrate the rust resistant genes into genetic background of susceptible or elite cultivar (Table 3). Amplification of DNA from parents, F1’s and F2’s was carried out.Each PCR programme was carried out at different annealing temperatures as shown in the Table 3.


Table 3 SCAR markers linked to rust (Uromyces phaseoli) resistance used in present study


Authors' contributions
BD: Participated in carrying out the entire work as M. Sc research program; TSA: Participated in the designing of the study and acted as chairperson to carry out the work under his guide lines; NM: guided in designing the research work and to study the inheritance pattern in French bean crosses; DCLR and CA: helped in identifying the molecular markers for rust resistance.
References
Aghora T.S, Mohan N., Somkuwar R.G., and Girija Ganeshan, 2007, Breeding french bean (Phaseolus vulgaris L.) for resistance to rust (Uromyces phaseoli Reben Wint), Journal of Horticultural Sciences, 2: 104-107
Aghora T.S., Madalageri M.B., and Mohan N.,2008, Genetics of Resistance to Rust (Uromyces Phaseoli Reben Wint) in French bean (Phaseolus vulgaris), Vegetable Science,35: 79-80
Anonymous, 2011, FAOSTAT., http://www.fao.org/site/567/default.aspx.
Pastor-Corrales M.A., 2003, Sources, genes for resistance, and pedigrees of 52 rust and mosaic resistant dry bean germplasm lines released by the USDA bean project in collaboration with the Michigan, Nebraska and North Dakota Agricultural Experimental Stations, Annu. Rep. Bean Improv. Coop., 46: 252-257
Sayler R.J., Ewing J.D., and McClean P.E., 1995, Monogenic and epistatic resistance to bean rust infection in common bean, Physiol. Mol. Plant. Pathology, 47: 173-184
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Vikas K.S., Atul S., Singh S.P., Ranjith K. E., Vikas C., Sarkel S., Devinder S., Gopala S., Krishnan M.N., Vinod K.K., Singh U.D., Rathore R., Prashanthi S.K., Agrawal P.K., Bhatt J.C., Mohapatra T., Prabhu K.V., and Singh A.K., 2012, Incorporation of blast resistance into “PRR78”, an elite Basmati rice restorer line through marker assisted backcross breeding, Field Crops Research., 128: 8-16
http://dx.doi.org/10.1016/j.fcr.2011.12.003


Yuebin, 1993, Preliminary studies on kidney bean rust resistant breeding, Acta Horticulture, 402: 115-119

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