Poster for CIAT 2009 Knowledge Sharing Week

1. Marker Assisted Selection of apomixis in Brachiaria breeding through SCAR-N14 and
SCAR-
FTA® DNA technology
FTA®
Vargas J., Bernal D., Giraldo M., Quintero C., Chacon J., Miles J.W. and Tohme J.
J.
Conservation and Use of Tropical Genetic Resources, CIAT, AA 6713 , Cali, Colombia
6713
INTRODUCTION
Brachiaria species are the most widely planted commercial forage grasses in On 1996 Tohme et al. identified a molecular marker that is closely linked to the
tropical America, with approx. 40-50 million hectares of Brachiaria pastures apomictic phenotype in B. decumbens. This molecular marker was used to design
sown in Brazil alone (Miles et al. 1996). A very commonly used species is B. the Sequence Characterized Amplified Region (SCAR) N14. Further screenings,
decumbens, an apomitic tetraploid, which given its apomictic reproductive type such as Vargas et.al., (2007), have shown that the SCAR N14 co-segregates more
cannot be crossed with other cultivars, eliminating the possibility of using than 90% with the apomictic phenotype as long as the apomictic phenotype
conventional plant breeding. To overcome this situation, B. ruziziensis, a diploid donor is B. decumbens CIAT accession 606 (Miles, personal comm.). In the
sexual species was tetraploidized with colchicine (Swenne et. al., 1981), which present poster we describe the procedure to genotype 7031 individuals with
rendered it cross compatible with the commercial B. decumbens. Thus it is now SCAR N14 in less than 2 months, which is allowing the forage breeding program
possible to combine desirable agronomic traits from different Brachiaria species to take to field trials plants, which are mostly true breeding individuals.
in true breeding apomitic cultivars.
MATERIALS AND METHODS
B. decumbens (CIAT 606) x Brachiaria (175 individuals) FTA® purification reagent washes and PCR preparation
FTA®
Apomictic tetraploids Sexual tetrapolids on 384-well plates using TECAN Genesis workstation
(6856 Individuals) F1 plants
384-well plate
384-well plate
Plant material collection with Whatman FTA® for Plant DNA with FTA®
with FTA®
plant discs
plant discs
PCR reaction on 384-well plate containing FTA® plant discs
PCR reaction on 384-well plate containing FTA® plant discs
FTA® plant card cover
labeled with sample
ID’s on the backside
UV visualization and data recording
SCAR-N14 present band SCAR-N14 absent band
HS
band
Agarose gel electrophoresis
Agarose gel electrophoresis
system from Apelex ® compatible
system from Apelex ® compatible
with multi-channel pipettes
with multi-channel pipettes
Disk removal and translocation to 384-well plates
Electrophoresis of 384 samples in 1.5% agarose gel, stained with EtBr and
visualized with UV light. The PCR reaction performed was a multiplex of
SCAR-N14 and “Herbicide Safener” specific primers (HS), which are
known to produce a band present in every individual (red arrows).
Consequently the multiplex reaction informs whether or not a PCR reaction
is taking place adequately. The yellow arrows show the presence/absence of
the band obtained for SCAR-N14. The condition of this band was recorded
in EXCEL files.
RESULTS AND DISCUSSION CONCLUSIONS AND PERSPECTIVES
7031 plants were evaluated for the presence or absence of SCAR N14 in Around 15% of the selected plants will be submitted to the field progeny test,
7 weeks of full time work of 4 research assistants. which will show us what fraction of the selected plants are true breeding
The Whatman FTA® for Plant DNA technology, replaced the traditional genotypes (i.e. have apomictic reproduction).
DNA extraction protocol. The SCAR N14 was used to implement a PCR-based marker-assisted
It permitted the use of 384-well plates and of the TECAN genesis work selection (MAS) as a new tool for screening large populations of Brachiaria
station to carry out the process of FTA purification and PCR to identify individuals with an apomictic mode of reproduction at the
preparation in a semi-automatized way. plantlet stage. This procedure reduces the time and cost for the forage-
It reduced approximately by 4 times the time and a significant part of the breeding program at CIAT.
plastic ware spent for DNA extractions and storage.
REFERENCES
The agarose gel electrophoresis system from Apelex® compatible with
Miles, J.W., Maass, B.L. and Valle, C.B. (1996). Brachiaria: Biology, Agronomy, and Improvement. CIAT/Embrapa,
multi-channel pipettes allowed the evaluation of 384 individuals per gel CIAT Publication No. 259, Cali, Colombia
Swenne, A., Louant, B.P. and Dujardin, M.T. (1981). Induction par la colchicine de formes autotetraploides chez Brachiaria
by using 8 combs of 52 teeth. ruziziensis Germain et Evrard (Graminee). Agronomie Tropicale 36(2):134-141
Tohme, J., Palacios, N., Lenis, S., and Roca, W. (1996). Aplications of Biotechnology to Brachiaria. In Miles JW, Maass
The reproductive mode of 175 plants had been previously determined as BL, Valle CB (1996). Brachiaria: Biology, Agronomy, and Improvement. CIAT/Embrapa, CIAT Publication No. 259, Cali,
Colombia
sexual by a field progeny test. None of them presented the band Vargas, J., Gallego, G. and Tohme, J. (2007). Using of Sequence characterized amplified Regions SCAR N14 or marker-
assisted selection in Brachiaria-breeding to select for apomixis at International Center of Tropical Agriculture. Posters VI
corresponding to the SCAR-N14 which satisfactorily supports the work. Encuentro Latinoamericano y del Caribe de Biotecnologia Agropecuaria. Viña del Mar, Chile 22-26 de Octubre de 2007.