Molecular tools for marker-assisted breeding of buffelgrass

The increasing availability of molecular tools is facilitating marker-assisted selection (MAS) in plant improvement programs. The objectives of this research were to: 1) populate the framework buffelgrass genome map with additional molecular markers, 2) develop polymerase chain reaction (PCR)-based markers from selected, informative restriction fragment length polymorphism (RFLP) markers on the buffelgrass genome map, and 3) increase marker resolution near the locus conferring apomixis (PApo1). Buffelgrass [Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L.] (2n=4x=36), a highly polymorphic, apomictic, perennial forage grass, is well-suited for genetic linkage analyses. One hundred and seventy one probes from an apomictic, spikelet-specific, complementary deoxyribonucleic acid (cDNA) library and 70 expressed sequence tag simple sequence repeats (EST-SSRs) from apomictic pistil cDNAs were evaluated and added to the framework buffelgrass genome map. The improved linkage map contains 851 markers from 11 grass species and covers approximately 80-85% of the buffelgrass genome. Two RFLPs from the buffelgrass genome map were converted to PCR-based markers for both the identification of hybrids and quantification of sexual versus apomictic reproduction. A gel-free, high-throughput technique was developed to analyze these markers directly in 96-well plates. Five additional markers were placed onto the buffelgrass linkage group with the PApo1 apomixis locus through comparative mapping of candidate orthologs from the sorghum genome map and bulked-segregant analysis of amplified-fragment-lengthpolymorphisms (BSA-AFLP). Increasing the mapping population size did not increase map resolution in the PApo1 region. Association mapping revealed that the recombination suppression near PApo1 is moderate and would complicate comparative map-based cloning efforts of the orthologous region in sorghum.