Poster for CIAT 2009 Knowledge Sharing Week

1. Adaptation and use of SCAR markers for BCNMV resistance
(bc-3 and dominant I genes ) in an Andean bean breeding program.
M.W. Blair, H. F. Buendia, M. Castaño, G.E. Santana, F. Morales
CIAT – International Center for Tropical Agriculture, A.A. 6713, Cali, Colombia
Abstract Figures and Tables Results and Discussion
Marker assisted selection for BCMV resistance Crosses: Triple crosses and backcrosses were
was begun for Andean bush and climbing beans. made between BCMV resistant, bc3-containing, bush-
Two markers were evaluated: ROC11 for the bc-3 type small red or red mottled beans and mid-altitude
climbing (MAC) bean advanced lines bred recently at
gene and SW13 for the dominant I gene with on
CIAT as well as the landraces G2333 and G685. The
almost 2000 breeding lines. The bc-3 marker MAC climbing beans included red and cream mottled
was found to work well in Andean genotypes selections, namely SEL1445, 1446, 1447, 1448, 1452,
especially for screening of climbing beans, where 1453 and 1454. The BCMNV sources included
it resulted in substantial savings in planting area BRB29, BRB 32 and BRB191.
and agronomic effort. Breeding method: Gamete selection was applied to
Figure 1. Symptoms of BCMV / BCMNV in susceptible beans the F1 plants from these triple crosses and the
Introduction 1 kb size populations were then advanced by mass and
SW13 marker ROC11 marker Std. pedigree selection to the F4 and F5 generation when
Although virus resistance can be screened single plant selections were made and evaluated for
phenotypically (Figure 1), the frequency of escape, the the resistance gene marker.
complex interaction of multiple genes and the recessive Parental survey: A total of 74 parents involved in the
nature of most of these makes marker assisted crosses described above were evaluated with both
selection (MAS) the best option for rapidly breeding - - + - - + - + + + + - + + - - + - - - ++-++-+-+- - - -+- - - - - -+-+
Figure 2. SCAR markers and resistance gene genotype (+/-) for viral inoculations and the SCAR markers to determine
resistant varieties. The need to protect the crop from the dominant I gene (SW13) and the recessive bc-3 gene (ROC11). if they contained the recessive bc-3 or dominant I
virus diseases is great, especially seed borne and resistance genes and the expected marker alleles
easily transmitted viral diseases such as bean common Table 1. Parents evaluated for BCMV/BCMNV resistance in the (Table 1; Figure 2). Polymorphism was easily
mosaic virus (BCMV) and its necrotic strains (BCMNV). greenhouse and for the SCAR marker for the bc-3 resistance gene.
observable in the Andean genepool for the bc-3 gene,
A number of BCMV / BCMNV resistance genes have Parents SCAR bc-3
ROC11 Gene
Virus response (Greenhouse) Parents SCAR bc-3
ROC11 Gene
Virus response (Greenhouse)
since the majority of susceptible Andean genotypes
been tagged including the dominant I gene, as well as N=
necrosis
M = mosaic O = bc-3
symptoms resistance
N= M = mosaic
necrosis symptoms
O = bc-3
resistance produce a band for the ROC11 SCAR, while the
the recessive bc-3, bc-2 and bc-12 genes. The genes AFR298 A + - - 15 G12582 A + - 11 - majority of bc-3 resistant Andean genotypes do not.
BRB029 A + - - 15 G12727 A + - 12 -
can be distinguished by inoculation with different viral BRB032 A + 1 - 14 G19833 P - - 15 - Likewise, the band for the SW13 SCAR for the
BRB151 A + - - 14 G2333 A + - 14 -
isolates. Although most BCMV and BCNMV resistance BRB181 P - - - 15 G23604 A + - 14 - dominant I gene is present in resistant parents but not
BRB183 P - - - 14 G23614 A + - 11 -
genes have been tagged with SCAR markers very little BRB189 A + - - 15 KABOON A + - 10 - in susceptibles. Therefore in Andean breeding
BRB197 A + - - 14 MAM49 A + 17 - -
work has been done to validate and utilize the markers BRB203 A + - - 15 MDRK A + - 14 - populations segregation for presence or absence of
BRB204 A + - - 15 MICHELITE A + - 14 -
in applied breeding programs. Therefore the objective BRB211
BRB217
A
A
+
+
-
-
-
-
15
15
PVA773
S31465
P
A
-
+
-
-
15
15
-
- the SCARs was expected to predict well the presence
of this research was to validate SCAR markers for the CAL096
CAL143
A
P
+
-
-
-
12
12
-
-
SEL1445 P
SEL1446 P
-
-
-
-
15
15
-
- or absence of the resistance alleles.
most important BCNMV resistance genes, I and bc-3, CALIMA
COS16
P
P
-
-
-
14
12
-
-
-
SEL1447 A
SEL1448 P
+
-
7
12
6
-
-
-
and use these markers to introgress the genes into new DOR476 P - 15 - - SEL1449 P - - 14 - Marker-assisted selection: The SCAR marker for bc-
DOR482 P - 10 - - SEL1450 A + - 14 -
bush bean and climbing bean germplasm being EMP122 P - - 14 - SEL1451 P - - 15 - 3 was applied on a mass scale at two locations as part
EMP250 P - 14 - - SEL1452 A + - 15 -
produced at CIAT. EMP320 A + - 15 - SEL1453 A + - 11 - of the bush and climbing bean breeding program
EMP364 P - 14 - - SEL1454 A + - 15 -
EMP496 P - 14 - - SEQ1033 A + - 15 - (Table 2). Of the total of 1923 F4 lines evaluated,
G685 A + - 14 - SEQ1040 A + 15 - -
Materials and Methods 1481 were climbing beans (246 from simple crosses
and 1235 from triple crosses), and 442 were bush
Table 2. Advanced lines evaluated for the ROC11 - SCAR marker.
Viral inoculations: were carried out in the greenhouse on 20 beans. In advanced generations, it was easy to score
Location Type of cross Climbers Total Bush Total Total Total lines
seeds of the F1:2 or F4 families using a necrotic strain of BCMNV + - Climbers + - Bush + - evaluated the resistant and susceptible genotypes based on
to determine resistance or susceptibility. After approximately 10 Location 1 (D) Simple Crosses 142 104 246 ---- ---- ---- 142 104 246
Triple Crosses 386 275 661 90 2 92 476 277 753 absence or presence of the PCR product / band as
days, the plants were scored for necrotic hypersensitivity (I gene Total Lines / location 907 92 999
resistance = N), susceptiblity (mosaic symptoms = M) or immune shown in Figure 3. The marker and the gene are
resistance (presence of the bc3 gene = 0).
Location 2 (P) Simple Crosses
Triple Crosses
---- ----
100 474 574
---- ----
159 191 350
0 0
259 665
0
924
tightly linked, although some cross-over events have
Total Lines / location 574 350 924 been observed.
DNA extraction: was carried out on young bean plants in the
Grand total 1923
field before flowering. Vegetative tissue was collected for Conclusion: bc-3 resistance is very important in
alkaline DNA extraction from four leaflets of four individual plants Africa where necrotic strains are prevalent and marker
per line using a hole-puncher. The leaf disks were placed
Gel 1 assisted selection will be an effective way to
directly into 96 well plates that were kept on ice while in the field 1st loading
and until subsequent alkaline extraction (60 ml of 0.25M NaOH, incoporate the gene into new African varieties.
-
2nd loading
heating to 100°C for 2 min., neutralizing with 60 ml 0.25M HCl,
and 30 ml of 0.5M pH 8.0 Tris- HCl buffer, followed by heating to Gel 2 References
1st loading
100°C for 2 min.). The crude DNA extract was transferred to a 2nd loading 1. Johnson W, Guzman P, Mandala D, Mkandawere A, Temple S,
clean 96 well plate and diluted 1:1 with sterile water. 3rd loading Gilbertson R, Gepts P. (1997) Molecular tagging of the bc-3 gene for
PCR amplification: 5 ul of the diluted DNA was used for PCR introgression in Andean common bean. Crop Sci. 37: 248-254
Gel 3
amplification of two sequence characterized amplified region 2. Melotto M, Afanador L, Kelly JD (1996) Development of a SCAR
1st loading
(SCAR) markers: ROC11 used to detect the bc-3 resistance 2nd loading marker linked to I gene in common bean. Genome 39:1216-1219.
gene (Johnson et al. (1997) and SW13 used to detect the I gene 3rd loading
(Melotto et al., 1996). PCR products were visualized on 1.5% Acknowledgements
agarose gels run in 0.5X TBE buffer. Products were run for 45 Figure 3. The SCAR marker ROC11 used to evaluate the bc-3 re- Funding for this work came from BID/IICA/Fontagro
minutes at 150 volts. Gels (Figure 2) were photographed and the sistance gene in a total of 378 segregating progeny. Agarose gels
presence/absence of a band was evaluated for each genotype. and the Rockefeller foundation.
(1.5 %) were consecutively loaded.