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Poster79: Marker assisted selection of apomixis in Brachiaria breeding through SCAR-N14 and FTA  DNA technology
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Poster79: Marker assisted selection of apomixis in Brachiaria breeding through SCAR-N14 and FTA DNA technology

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Poster for CIAT 2009 Knowledge Sharing Week …

Poster for CIAT 2009 Knowledge Sharing Week

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  • 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.

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