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DEVELOPMENT OF MULTIPLE DISEASE RESISTANT
                                   BREEDING PARENTS WITH THE AID DISEASE RESISTANT
                                        DEVELOPMENT OF MULTIPLE OF MOLECULAR MARKERS
                                    BREEDING PARENTS WITHA. Buruchara, A. MOLECULAR MARKERS
                                             C.M. Mukankusi, R. THE AID OF Male, S. Sebuliba
                                                               C.M. Mukankusi, R. A. Buruchara, A. Male, S. Sebuliba currently being utilized for Multiple
                                                                                             Table 1. Molecular markers
Background:                                                                                      disease resistance breeding in PABRA
                                                                                                Table 1. Molecular markers currently being utilized for Multiple
  Background:                                                                                   disease resistance Markers
                                                                                                 Trait             breeding in PABRA
                                                                                                                                 Source
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                Trait                        Markers 709             Source 54 (Mahuku et al., 2004)
are attacked by aPhaseolus vulgaris L.) plays an essentialviral diseases leading to
  Common bean ( combination of fungal, bacterial and role in sustaining live-                     ALS                           OPE4                    MEX

poor yields (Fig. 1). The farmers and their families in Africa. At farm(MDR)beans
  lihoods of smallholder deployment of multiple disease resistant level, varie-
                                                                                                 ALS                            PF9250
                                                                                                                              OPE4709                   G10474 and G10909
                                                                                                                                                      MEX 54 (Mahuku et al., 2004)
ties is probably the cheapest method of managing and problem. The use of to
  are attacked by a combination of fungal, bacterial this viral diseases leading
                                                                                                   Pythium root rot             PYAA19                  RWR719 (Buruchara et al)
  poor yields (Fig. 1). The deploymentcould speed up the development ofvarie-
MDR parents in breeding programs        of multiple disease resistant (MDR) MDR                                               PF9250                  G10474 and G10909
  ties is probably the cheapest method of managing this problem. The use of                                                     PYB08                 RWR719 719
                                                                                                                                                        RWR (Buruchara et al)
varieties. Marker Assisted Selection (MAS) o ers advantages within NARS                          Pythium root rot             PYAA19
  MDR parents in breeding programs could speed up the development of MDR
breeding programs in targeting challenging breeding objectives requiring com-                      Anthracnose                  SAB-3
                                                                                                                              PYB08                   RWR 719 (Vallejo and Kelly, 2001)
                                                                                                                                                        G2333
  varieties. Marker Assisted Selection (MAS) o ers advantages within NARS
plex breeding schemes.
  breeding programs in targeting challenging breeding objectives requiring com-                  Anthracnose                    SAS-13
                                                                                                                              SAB-3                   G2333 (Vallejo and Kelly,1998)
                                                                                                                                                        G2333 (Young et al., 2001)
  plex breeding schemes.                                                                                                        SBB-14                G2333 (Young et al., 1998) and Kelly, 2001)
                                                                                                                                                        G2333 , AB 136 (Awale
However, the national programs lack the facilities and capacity to use these                                                  SAS-13
techniques. Taking advantage of lackbiotechnology facility at Kawanda in
  However, the national programs the the facilities and capacity to use these                                                 SBB-14
                                                                                                                                SH-18                 G2333 , AB 136 (Awale and Kelly, 2001)
                                                                                                                                                        G2333 (Awale and Kelly, 2001)
Uganda, andTaking advantage of the biotechnologyutilizing molecular markers
  techniques. the available markers, CIAT-PABRA is facility at Kawanda in                          BCMV                       SH-18
                                                                                                                                ROC11                 G2333 (Awale and Kelly, 2001)
                                                                                                                                                        Various
  Uganda, and the available markers, CIAT-PABRA is (Angular leaf Spot - ALS -
linked to resistance genes to three fungal diseases utilizing molecular markers
Pythium root rots and Anthracnose)fungal diseases (Angular leaf Spot - ALSMo-
  linked to resistance genes to three and one viral disease (Bean Common -                       BCMV
                                                                                                  BCMNV                       ROC11
                                                                                                                                SW13                  Various
                                                                                                                                                         Various
saic Virus root rots and its necrotic strain one viral disease (Bean CommonVirus
  Pythium (BCMV) and Anthracnose) and Bean Common Mosaic Necrotic Mo-                            BCMNV
                                                                                                  CBB                         SW13 820
                                                                                                                               SAP                    Various et al., 2000, Deidre et al., 2007
                                                                                                                                                         Miklas
(BCMNV) (Table 1) .and its necroticpyramid the resistance genes Necrotic Virus
  saic Virus (BCMV) The aim is to strain Bean Common Mosaic in common                            CBB                          SAP820                  Miklas et al., 2000, Deidre et al., 2007
background in an e . ort of developing MDR parents that can bein common
  (BCMNV) (Table 1) The aim is to pyramid the resistance genes utilized by
NARS breeding programs. developing MDR parents that can be utilized by
  background in an e ort of                                                                     Conclusions and Way Forw ard
  NARS breeding programs.                                                                      Conclusions and Way Forward
                                                                                                   •        Crosses between Pythium/ ALS resistance plants and Anth/ BCMV/
                                                                                               •           Crosses between have been conducted plants and Anth/ BCMV/
                                                                                                            BCMNV plants Pythium/ ALS resistance
                                                                                                           BCMNV plants have been conducted
                                                                                                   •        MDR parents developed will be made available to NARS for utilization in
                                                                                               •           MDR parents developed will be made available to NARS for utilization in
                                                                                                            own breeding programs.
                                                                                                           own breeding programs.
                                                                                                   •        We envisage a mechanism where the capacity of NARS in using MAS will
                                                                                               •           We envisage a mechanism where the capacity of NARS in using MAS contact
                                                                                                            be enhanced through this process with back up from continuous will
                                                                                                           be enhanced through this facility in Kawanda. from continuous contact
                                                                                                            with the biotechnology process with back up
                                                                                                           with the biotechnology facility in Kawanda.
                                                                                                   •        Some partners have been trained (MAS and Disease Phenotyping)
                                                                                               •           Some partners have been trained (MAS and Disease Phenotyping)




Fig 1. Some of the most common diseases of beans in Africa
  Fig 1. Some of the most common diseases of beans in Africa

Marker Assisted Selection (MAS)
 Marker Assisted Selection (MAS)                                                                         Figure 2: Part analysis of the plants from the cross G2333 X MCM1015 using the ROC 11
                                                                                                       Figure 2: Part analysis of the plants recessive bc-3G2333 Sample 5 is positive control 11
                                                                                                         marker to detect presence of the from the cross gene. X MCM1015 using the ROC variety
                                                                                                       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
                                                                                                       MCM 5001, Sample 1 is negativeband indicates absence of the gene. indicatesbp molecular
                                                                                                         the gene while presence of a control RAB 487. Absence of a band L is 100 presence of
••       MAS refers to the use of DNA markers that are tightly-linked to target
          MAS refers to the use of DNA markers that are tightly-linked to target                       the gene while presence of a band indicates absence of the gene. L is 100 bp molecular
                                                                                                         marker.
                                                                                                       marker.
         loci as aasubstitute for or to assist phenotypicscreening
          loci as substitute for or to assist phenotypic screening
••       MAS is the most promising application of DNA markers for cultivar de-
          MAS is the most promising application of DNA markers for cultivar de-
         velopment
          velopment

Materials and Methods:
 Materials and Methods:

••       Single crosses between sources of resistance, aim of screening up to
          Single crosses between sources resistance, aim of screening up to
         1500 FF2plants per cross
          1500 2 plants per cross
         ••     MCM5001and MCM 1015 as sources of IIand bc- 3 genes for
                 MCM5001and MCM                sources of and bc- 3 genes for
                 BCMV/ BCMNV
                BCMV/ BCMNV
         ••     G2333—Co-4,,Co-5 and Co-7 for resistance to anthracnose,
                 G2333—   Co-4 Co-5 and           resistance to anthracnose,
         ••     RWR719 and MLB-49-89A-Pythiumroot rot,
                 RWR719 and MLB-49-89A-Pythium root rot,
         ••      MEX54- phg for resistance
                MEX54- phg for resistance to ALS.
••       DNA extracted from leaves of 2 week old F2 plants
          DNA extracted from leaves of              2 plants
••       Two mm discs used as templates in PCR reactions using speci cc mo-
          Two mm discs used as templates in PCR reactions using speci mo-
          lecular markers.
         lecular markers.                                                                     Figure 3: 3: Breeding schemedevelop Multiple Disease resistance parents to Angular leaf spot, spot, Anthracnose,
                                                                                                Figure Breeding scheme to to develop Multiple Disease resistance parents to Angular leaf Anthracnose,
                                                                                              Pythium root rot rot and BCMV/BCMNV
                                                                                                Pythium root and BCMV/BCMNV
••       Plants positive for 2-3 gene combination selected and double crosses
          Plants positive for 2-3 gene combination selected and double crosses
          conducted.
         conducted.
                                                                                             References
                                                                                              References
                                                                                             Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann.
                                                                                               Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann.
 Results                                                                                               Rept. Bean Improv. Coop. 44:119-120.
Results                                                                                                    Rept. Bean Improv. Coop. 44:119-120.
                                                                                             Buruchara et al.
                                                                                               Buruchara et al.
                                                                                             Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., T   eran, H., and Beebe, S. 2004. Inheritance and charac-
  To date 10 out of 340 plants have been found to possess four gene combina-                   Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., T in the and Beebe, S. 2004. Inheritance and charac-
                                                                                                       terization of the angular leaf spot resistance gene H., common bean accession, G 10474 and
                                                                                                                                                        eran,
To date 10 out of 340 plants have been found to possess four gene combina-                             identi cation of anangular leaf spot resistance gene in the common bean accession, G 10474 and
                                                                                                           terization of the AFLP marker linkedto the resistance gene. Crop Science 44: 1817-1824.
  tion; “ Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/                 Miklas, P.N., J.R. Smith, R.of an AFLP Grafton,linkedto the resistance D. P. Coyne.Science 44: 1817-1824.
                                                                                                           identi cation Riley, K.F. marker S.P Singh, G. Jung, and gene. Crop 2000. Marker-assisted
tion; “ Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/                     Miklas,breeding for pyramided resistance to common bacterial blight in D. P. Coyne. 2000. Marker-assisted
                                                                                                        P.N., J.R. Smith, R. Riley, K.F. Grafton, S.P Singh, G. Jung, and common bean. Annu. Rep. Bean
  BCMNV (Fig 2 and 3).
BCMNV (Fig 2 and 3).                                                                                   Improv. Coop. 43:39-40 resistance to common bacterial blight in common bean. Annu. Rep. Bean
                                                                                                           breeding for pyramided
                                                                                                           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.
 Sixty two out of 103 F2 plants have been found to posses both the Pythium and                 Vallejo,Rept. Bean Improv.2001. Development of a SCAR marker linked to Co-5 gene in common bean. Ann.
                                                                                                         V., and J.D. Kelly. Coop. 44:121-122.
Sixty two out of 103 F2 plants have been found to posses both the Pythium and                Young, R.A.,Rept. Bean Improv. Coop. 44:121-122.
 Angular Leaf spot resistance genes (Fig 3).                                                                M. Melotto, R.O., Nodari and J.D. Kelly. 1998. Marker assisted dissection of the oligogenic
Angular Leaf spot resistance genes (Fig 3).                                                    Young, R.A., M. Melotto, R.O., Nodaricommon bean cultivar G2333. assisted dissection96:87-94
                                                                                                       anthracnose resistance in the and J.D. Kelly. 1998. Marker Theor. Appl. Genet. of the oligogenic
                                                                                                           anthracnose resistance in the common bean cultivar G2333. Theor. Appl. Genet. 96:87-94
                                                                                                                                             For more information: c.mukankusi@cgiar.org
                                                                                                                                               For more information: c.mukankusi@cgiar.org

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Breeding with the aid of Molecular Markers

  • 1. DEVELOPMENT OF MULTIPLE DISEASE RESISTANT BREEDING PARENTS WITH THE AID DISEASE RESISTANT DEVELOPMENT OF MULTIPLE OF MOLECULAR MARKERS BREEDING PARENTS WITHA. Buruchara, A. MOLECULAR MARKERS C.M. Mukankusi, R. THE AID OF Male, S. Sebuliba C.M. Mukankusi, R. A. Buruchara, A. Male, S. Sebuliba currently being utilized for Multiple Table 1. Molecular markers Background: disease resistance breeding in PABRA Table 1. Molecular markers currently being utilized for Multiple Background: disease resistance Markers Trait breeding in PABRA Source 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 Trait Markers 709 Source 54 (Mahuku et al., 2004) are attacked by aPhaseolus vulgaris L.) plays an essentialviral diseases leading to Common bean ( combination of fungal, bacterial and role in sustaining live- ALS OPE4 MEX poor yields (Fig. 1). The farmers and their families in Africa. At farm(MDR)beans lihoods of smallholder deployment of multiple disease resistant level, varie- ALS PF9250 OPE4709 G10474 and G10909 MEX 54 (Mahuku et al., 2004) ties is probably the cheapest method of managing and problem. The use of to are attacked by a combination of fungal, bacterial this viral diseases leading Pythium root rot PYAA19 RWR719 (Buruchara et al) poor yields (Fig. 1). The deploymentcould speed up the development ofvarie- MDR parents in breeding programs of multiple disease resistant (MDR) MDR PF9250 G10474 and G10909 ties is probably the cheapest method of managing this problem. The use of PYB08 RWR719 719 RWR (Buruchara et al) varieties. Marker Assisted Selection (MAS) o ers advantages within NARS Pythium root rot PYAA19 MDR parents in breeding programs could speed up the development of MDR breeding programs in targeting challenging breeding objectives requiring com- Anthracnose SAB-3 PYB08 RWR 719 (Vallejo and Kelly, 2001) G2333 varieties. Marker Assisted Selection (MAS) o ers advantages within NARS plex breeding schemes. breeding programs in targeting challenging breeding objectives requiring com- Anthracnose SAS-13 SAB-3 G2333 (Vallejo and Kelly,1998) G2333 (Young et al., 2001) plex breeding schemes. SBB-14 G2333 (Young et al., 1998) and Kelly, 2001) G2333 , AB 136 (Awale However, the national programs lack the facilities and capacity to use these SAS-13 techniques. Taking advantage of lackbiotechnology facility at Kawanda in However, the national programs the the facilities and capacity to use these SBB-14 SH-18 G2333 , AB 136 (Awale and Kelly, 2001) G2333 (Awale and Kelly, 2001) Uganda, andTaking advantage of the biotechnologyutilizing molecular markers techniques. the available markers, CIAT-PABRA is facility at Kawanda in BCMV SH-18 ROC11 G2333 (Awale and Kelly, 2001) Various Uganda, and the available markers, CIAT-PABRA is (Angular leaf Spot - ALS - linked to resistance genes to three fungal diseases utilizing molecular markers Pythium root rots and Anthracnose)fungal diseases (Angular leaf Spot - ALSMo- linked to resistance genes to three and one viral disease (Bean Common - BCMV BCMNV ROC11 SW13 Various Various saic Virus root rots and its necrotic strain one viral disease (Bean CommonVirus Pythium (BCMV) and Anthracnose) and Bean Common Mosaic Necrotic Mo- BCMNV CBB SW13 820 SAP Various et al., 2000, Deidre et al., 2007 Miklas (BCMNV) (Table 1) .and its necroticpyramid the resistance genes Necrotic Virus saic Virus (BCMV) The aim is to strain Bean Common Mosaic in common CBB SAP820 Miklas et al., 2000, Deidre et al., 2007 background in an e . ort of developing MDR parents that can bein common (BCMNV) (Table 1) The aim is to pyramid the resistance genes utilized by NARS breeding programs. developing MDR parents that can be utilized by background in an e ort of Conclusions and Way Forw ard NARS breeding programs. Conclusions and Way Forward • Crosses between Pythium/ ALS resistance plants and Anth/ BCMV/ • Crosses between have been conducted plants and Anth/ BCMV/ BCMNV plants Pythium/ ALS resistance BCMNV plants have been conducted • MDR parents developed will be made available to NARS for utilization in • MDR parents developed will be made available to NARS for utilization in own breeding programs. own breeding programs. • We envisage a mechanism where the capacity of NARS in using MAS will • We envisage a mechanism where the capacity of NARS in using MAS contact be enhanced through this process with back up from continuous will be enhanced through this facility in Kawanda. from continuous contact with the biotechnology process with back up with the biotechnology facility in Kawanda. • Some partners have been trained (MAS and Disease Phenotyping) • Some partners have been trained (MAS and Disease Phenotyping) Fig 1. Some of the most common diseases of beans in Africa Fig 1. Some of the most common diseases of beans in Africa Marker Assisted Selection (MAS) Marker Assisted Selection (MAS) Figure 2: Part analysis of the plants from the cross G2333 X MCM1015 using the ROC 11 Figure 2: Part analysis of the plants recessive bc-3G2333 Sample 5 is positive control 11 marker to detect presence of the from the cross gene. X MCM1015 using the ROC variety 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 MCM 5001, Sample 1 is negativeband indicates absence of the gene. indicatesbp molecular the gene while presence of a control RAB 487. Absence of a band L is 100 presence of •• MAS refers to the use of DNA markers that are tightly-linked to target MAS refers to the use of DNA markers that are tightly-linked to target the gene while presence of a band indicates absence of the gene. L is 100 bp molecular marker. marker. loci as aasubstitute for or to assist phenotypicscreening loci as substitute for or to assist phenotypic screening •• MAS is the most promising application of DNA markers for cultivar de- MAS is the most promising application of DNA markers for cultivar de- velopment velopment Materials and Methods: Materials and Methods: •• Single crosses between sources of resistance, aim of screening up to Single crosses between sources resistance, aim of screening up to 1500 FF2plants per cross 1500 2 plants per cross •• MCM5001and MCM 1015 as sources of IIand bc- 3 genes for MCM5001and MCM sources of and bc- 3 genes for BCMV/ BCMNV BCMV/ BCMNV •• G2333—Co-4,,Co-5 and Co-7 for resistance to anthracnose, G2333— Co-4 Co-5 and resistance to anthracnose, •• RWR719 and MLB-49-89A-Pythiumroot rot, RWR719 and MLB-49-89A-Pythium root rot, •• MEX54- phg for resistance MEX54- phg for resistance to ALS. •• DNA extracted from leaves of 2 week old F2 plants DNA extracted from leaves of 2 plants •• Two mm discs used as templates in PCR reactions using speci cc mo- Two mm discs used as templates in PCR reactions using speci mo- lecular markers. lecular markers. Figure 3: 3: Breeding schemedevelop Multiple Disease resistance parents to Angular leaf spot, spot, Anthracnose, Figure Breeding scheme to to develop Multiple Disease resistance parents to Angular leaf Anthracnose, Pythium root rot rot and BCMV/BCMNV Pythium root and BCMV/BCMNV •• Plants positive for 2-3 gene combination selected and double crosses Plants positive for 2-3 gene combination selected and double crosses conducted. conducted. References References Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann. Awale, H.E., and J.D. Kelly. 2001. Development of SCAR markers linked to Co-42 gene in common bean. Ann. Results Rept. Bean Improv. Coop. 44:119-120. Results Rept. Bean Improv. Coop. 44:119-120. Buruchara et al. Buruchara et al. Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., T eran, H., and Beebe, S. 2004. Inheritance and charac- To date 10 out of 340 plants have been found to possess four gene combina- Mahuku, G., Montoya, C., Henríquez, M.A., Jara, C., T in the and Beebe, S. 2004. Inheritance and charac- terization of the angular leaf spot resistance gene H., common bean accession, G 10474 and eran, To date 10 out of 340 plants have been found to possess four gene combina- identi cation of anangular leaf spot resistance gene in the common bean accession, G 10474 and terization of the AFLP marker linkedto the resistance gene. Crop Science 44: 1817-1824. tion; “ Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/ Miklas, P.N., J.R. Smith, R.of an AFLP Grafton,linkedto the resistance D. P. Coyne.Science 44: 1817-1824. identi cation Riley, K.F. marker S.P Singh, G. Jung, and gene. Crop 2000. Marker-assisted tion; “ Co-4”, Co-5, I and bc3” resistance genes for anthracnose and BCMV/ Miklas,breeding for pyramided resistance to common bacterial blight in D. P. Coyne. 2000. Marker-assisted P.N., J.R. Smith, R. Riley, K.F. Grafton, S.P Singh, G. Jung, and common bean. Annu. Rep. Bean BCMNV (Fig 2 and 3). BCMNV (Fig 2 and 3). Improv. Coop. 43:39-40 resistance to common bacterial blight in common bean. Annu. Rep. Bean breeding for pyramided 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. Sixty two out of 103 F2 plants have been found to posses both the Pythium and Vallejo,Rept. Bean Improv.2001. Development of a SCAR marker linked to Co-5 gene in common bean. Ann. V., and J.D. Kelly. Coop. 44:121-122. Sixty two out of 103 F2 plants have been found to posses both the Pythium and Young, R.A.,Rept. Bean Improv. Coop. 44:121-122. Angular Leaf spot resistance genes (Fig 3). M. Melotto, R.O., Nodari and J.D. Kelly. 1998. Marker assisted dissection of the oligogenic Angular Leaf spot resistance genes (Fig 3). Young, R.A., M. Melotto, R.O., Nodaricommon bean cultivar G2333. assisted dissection96:87-94 anthracnose resistance in the and J.D. Kelly. 1998. Marker Theor. Appl. Genet. of the oligogenic anthracnose resistance in the common bean cultivar G2333. Theor. Appl. Genet. 96:87-94 For more information: c.mukankusi@cgiar.org For more information: c.mukankusi@cgiar.org