breeding with the aid of molecular markers
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C.M. Mukankusi, R. A. Buruchara, A. Male, S. Sebuliba
Background: Common bean (Phaseolus vulgaris L.) plays an essential role in sustaining live-lihoods of smallholder farmers and their families in Africa. At farm level, beans are attacked by a combination of fungal, bacterial and viral diseases leading to poor yields (Fig. 1). The deployment of multiple disease resistant (MDR) varie-ties is probably the cheapest method of managing this problem. The use of MDR parents in breeding programs could speed up the development of MDR varieties. Marker Assisted Selection (MAS) o ers advantages within NARS breeding programs in targeting challenging breeding objectives requiring com-plex breeding schemes. However, the national programs lack the facilities and capacity to use these techniques. Taking advantage of the biotechnology facility at Kawanda in Uganda, and the available markers, CIAT-PABRA is utilizing molecular markers linked to resistance genes to three fungal diseases (Angular leaf Spot - ALS - Pythium root rots and Anthracnose) and one viral disease (Bean Common Mo-saic Virus (BCMV) and its necrotic strain Bean Common Mosaic Necrotic Virus (BCMNV) (Table 1) . The aim is to pyramid the resistance genes in common background in an e ort of developing MDR parents that can be utilized by NARS breeding programs.
Marker Assisted Selection (MAS) • MAS refers to the use of DNA markers that are tightly-linked to target
loci as a substitute for or to assist phenotypic screening • MAS is the most promising application of DNA markers for cultivar de-
velopment Materials and Methods:
• Single crosses between sources of resistance, aim of screening up to 1500 F2 plants per cross • MCM5001and MCM 1015 as sources of I and bc- 3 genes for
BCMV/ BCMNV • G2333—Co-4, Co-5 and Co-7 for resistance to anthracnose, • RWR719 and MLB-49-89A-Pythium root rot, • MEX54- phg for resistance to ALS.
• DNA extracted from leaves of 2 week old F2 plants • Two mm discs used as templates in PCR reactions using speci c mo-
lecular markers. • Plants positive for 2-3 gene combination selected and double crosses
conducted.
Results
To date 10 out of 340 plants have been found to possess four gene combina-tion; “Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/BCMNV (Fig 2 and 3).
Sixty two out of 103 F2 plants have been found to posses both the Pythium and Angular Leaf spot resistance genes (Fig 3).
Conclusions and Way Forward
• Crosses between Pythium/ ALS resistance plants and Anth/ BCMV/BCMNV plants have been conducted
• MDR parents developed will be made available to NARS for utilization in own breeding programs.
• We envisage a mechanism where the capacity of NARS in using MAS will be enhanced through this process with back up from continuous contact with the biotechnology facility in Kawanda.
• Some partners have been trained (MAS and Disease Phenotyping)
References Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann.
Rept. Bean Improv. Coop. 44:119-120. Buruchara et al. Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., Teran, H., and Beebe, S. 2004. Inheritance and charac-
terization of the angular leaf spot resistance gene in the common bean accession, G 10474 and identi cation of an AFLP marker linked to the resistance gene. Crop Science 44: 1817-1824.
Miklas, P.N., J.R. Smith, R. Riley, K.F. Grafton, S.P Singh, G. Jung, and D. P. Coyne. 2000. Marker-assisted breeding for pyramided resistance to common bacterial blight in common bean. Annu. Rep. Bean Improv. Coop. 43:39-40
Vallejo, V., and J.D. Kelly. 2001. Development of a SCAR marker linked to Co-5 gene in common bean. Ann. Rept. Bean Improv. Coop. 44:121-122.
Young, R.A., M. Melotto, R.O., Nodari and J.D. Kelly. 1998. Marker assisted dissection of the oligogenic anthracnose resistance in the common bean cultivar G2333. Theor. Appl. Genet. 96:87-94
For more information: [email protected]
Trait Markers Source
ALS OPE4709 MEX 54 (Mahuku et al., 2004)
PF9250 G10474 and G10909
Pythium root rot PYAA19 RWR719 (Buruchara et al)
PYB08 RWR 719
SAB-3 G2333 (Vallejo and Kelly, 2001)
SAS-13 G2333 (Young et al., 1998)
SBB-14 G2333 , AB 136 (Awale and Kelly, 2001)
SH-18 G2333 (Awale and Kelly, 2001)
BCMV ROC11 Various
BCMNV SW13 Various
CBB SAP820 Miklas et al., 2000, Deidre et al., 2007
Anthracnose
Figure 2: Part analysis of the plants from the cross G2333 X MCM1015 using the ROC 11 marker to detect presence of the recessive bc-3 gene. Sample 5 is positive control variety MCM 5001, Sample 1 is negative control RAB 487. Absence of a band indicates presence of the gene while presence of a band indicates absence of the gene. L is 100 bp molecular marker.
Table 1. Molecular markers currently being utilized for Multiple disease resistance breeding in PABRA
Figure 3: Breeding scheme to develop Multiple Disease resistance parents to Angular leaf spot, Anthracnose, Pythium root rot and BCMV/BCMNV
Fig 1. Some of the most common diseases of beans in Africa
DEVELOPMENT OF MULTIPLE DISEASE RESISTANT BREEDING PARENTS WITH THE AID OF MOLECULAR MARKERS
C.M. Mukankusi, R. A. Buruchara, A. Male, S. Sebuliba
Background: Common bean (Phaseolus vulgaris L.) plays an essential role in sustaining live-lihoods of smallholder farmers and their families in Africa. At farm level, beans are attacked by a combination of fungal, bacterial and viral diseases leading to poor yields (Fig. 1). The deployment of multiple disease resistant (MDR) varie-ties is probably the cheapest method of managing this problem. The use of MDR parents in breeding programs could speed up the development of MDR varieties. Marker Assisted Selection (MAS) o ers advantages within NARS breeding programs in targeting challenging breeding objectives requiring com-plex breeding schemes. However, the national programs lack the facilities and capacity to use these techniques. Taking advantage of the biotechnology facility at Kawanda in Uganda, and the available markers, CIAT-PABRA is utilizing molecular markers linked to resistance genes to three fungal diseases (Angular leaf Spot - ALS - Pythium root rots and Anthracnose) and one viral disease (Bean Common Mo-saic Virus (BCMV) and its necrotic strain Bean Common Mosaic Necrotic Virus (BCMNV) (Table 1) . The aim is to pyramid the resistance genes in common background in an e ort of developing MDR parents that can be utilized by NARS breeding programs.
Marker Assisted Selection (MAS) • MAS refers to the use of DNA markers that are tightly-linked to target
loci as a substitute for or to assist phenotypic screening • MAS is the most promising application of DNA markers for cultivar de-
velopment Materials and Methods:
• Single crosses between sources of resistance, aim of screening up to 1500 F2 plants per cross • MCM5001and MCM 1015 as sources of I and bc- 3 genes for
BCMV/ BCMNV • G2333—Co-4, Co-5 and Co-7 for resistance to anthracnose, • RWR719 and MLB-49-89A-Pythium root rot, • MEX54- phg for resistance to ALS.
• DNA extracted from leaves of 2 week old F2 plants • Two mm discs used as templates in PCR reactions using speci c mo-
lecular markers. • Plants positive for 2-3 gene combination selected and double crosses
conducted.
Results
To date 10 out of 340 plants have been found to possess four gene combina-tion; “Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/BCMNV (Fig 2 and 3).
Sixty two out of 103 F2 plants have been found to posses both the Pythium and Angular Leaf spot resistance genes (Fig 3).
Conclusions and Way Forward
• Crosses between Pythium/ ALS resistance plants and Anth/ BCMV/BCMNV plants have been conducted
• MDR parents developed will be made available to NARS for utilization in own breeding programs.
• We envisage a mechanism where the capacity of NARS in using MAS will be enhanced through this process with back up from continuous contact with the biotechnology facility in Kawanda.
• Some partners have been trained (MAS and Disease Phenotyping)
References Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann.
Rept. Bean Improv. Coop. 44:119-120. Buruchara et al. Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., Teran, H., and Beebe, S. 2004. Inheritance and charac-
terization of the angular leaf spot resistance gene in the common bean accession, G 10474 and identi cation of an AFLP marker linked to the resistance gene. Crop Science 44: 1817-1824.
Miklas, P.N., J.R. Smith, R. Riley, K.F. Grafton, S.P Singh, G. Jung, and D. P. Coyne. 2000. Marker-assisted breeding for pyramided resistance to common bacterial blight in common bean. Annu. Rep. Bean Improv. Coop. 43:39-40
Vallejo, V., and J.D. Kelly. 2001. Development of a SCAR marker linked to Co-5 gene in common bean. Ann. Rept. Bean Improv. Coop. 44:121-122.
Young, R.A., M. Melotto, R.O., Nodari and J.D. Kelly. 1998. Marker assisted dissection of the oligogenic anthracnose resistance in the common bean cultivar G2333. Theor. Appl. Genet. 96:87-94
For more information: [email protected]
Trait Markers Source
ALS OPE4709 MEX 54 (Mahuku et al., 2004)
PF9250 G10474 and G10909
Pythium root rot PYAA19 RWR719 (Buruchara et al)
PYB08 RWR 719
SAB-3 G2333 (Vallejo and Kelly, 2001)
SAS-13 G2333 (Young et al., 1998)
SBB-14 G2333 , AB 136 (Awale and Kelly, 2001)
SH-18 G2333 (Awale and Kelly, 2001)
BCMV ROC11 Various
BCMNV SW13 Various
CBB SAP820 Miklas et al., 2000, Deidre et al., 2007
Anthracnose
Figure 2: Part analysis of the plants from the cross G2333 X MCM1015 using the ROC 11 marker to detect presence of the recessive bc-3 gene. Sample 5 is positive control variety MCM 5001, Sample 1 is negative control RAB 487. Absence of a band indicates presence of the gene while presence of a band indicates absence of the gene. L is 100 bp molecular marker.
Table 1. Molecular markers currently being utilized for Multiple disease resistance breeding in PABRA
Figure 3: Breeding scheme to develop Multiple Disease resistance parents to Angular leaf spot, Anthracnose, Pythium root rot and BCMV/BCMNV
Fig 1. Some of the most common diseases of beans in Africa
DEVELOPMENT OF MULTIPLE DISEASE RESISTANT BREEDING PARENTS WITH THE AID OF MOLECULAR MARKERS