Development of a dependable microsatellite-based fingerprinting system for sugarcane.

摘要

For a long time, sugarcane breeders have been investing lots of efforts to develop a reliable molecular marker-based fingerprinting system that could aid in many ways the breeding programmes. The International Union for the Protection of New Varieties of Plants has also pursued such a system that, once it meets the standard of a distinctness, uniformity and stability test, would be used in addition to or to replace morphological characterization used to guarantee the breeders' property rights until now. A large EST database was screened and ~37 000 SSR motifs were identified in 33 000 sequences. Candidate clusters (112 loci) were selected and validated (primer design, PCR amplification and PAGE/DNA sequencer analyses) in a group of sugarcane accessions sharing diverse levels of genetic relationship. These loci were classified according to their PIC values and visual quality of molecular profiles. The top loci were elected to form a "Sugarcane Microsatellite-based Fingerprinting System". Three loci discriminated a collection of 1205 entries of a germplasm bank with at least 2 differences (discriminatory alleles). This system is being routinely used in the laboratory in several process, such as quality control of the tissue culture facility to avoid varietal mislabeling; identification of duplicated accessions in the germplasm bank; determination of genetic similarity indices to aid the selection of crosses to be performed; determination of selfing levels in seed samples; identification of male progenitor of clones originated from polycrosses; and identification of hybrids. In the near future,this method can be used as an additional tool for the protection of cultivars. The system has passed several evaluations for stability and repeatability. Protocols for high throughput genotyping in DNA sequencer have been developed. It is believe that this system will discriminate all cultivars and related sugarcane genotypes existing in the world, qualifying it to become an important tool for all those involved in breeding and property rights of sugarcane cultivars.