Development of a seed DNA-based genotyping system for marker-assisted selection in maize

Abstract

Leaf collection from the field, labeling and tracking back to the source plants after genotyping are rate limiting steps in leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications. A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F 2 populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue. Extraction from 30 mg of endosperm yields 3-10 lg DNA, which is sufficient for analysis of 200-400 agarose-gel PCR-based markers, with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals from an F 2 population, genotyping errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%, respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field conditions the germination rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of markerassisted selection and phenotypic selection within and between generations.

High-throughput SNP genotyping with the GoldenGate assay in maize

“…Thus, the whole process of DNA extraction, genotyping and data analysis must be completed within 4-6 weeks for each cycle. The recent development of a single seed-based DNA extraction system (Gao et al 2008), should greatly assist largescale genome-wide screening systems to routinely complete 3-cycles of selection per year. The CIMMYT global maize program is currently introducing genome-wide selection to assist drought tolerance breeding.…”

Endosperm genotyping as a strategy to differentiate the allele source in maize heterozygous progeny

“…This also reduces the total amount of isolated DNA, since, according to Ramos et al (2006), part of these molecules cannot be separated from other organic compounds and are thus discarded. However, in a protocol developed by Gao et al (2008) for maize seed DNA extraction, 30 mg of endosperm fragment yielded enough high-quality DNA for around 200 PCR reactions. Therefore, DNA extraction from seeds may not be a critical issue for the wide utilization of this genotyping strategy.…”

Australian Cotton Germplasm Resources

“…To overcome this problem a number of companies have developed non-destructive seed based screening methodologies to screen markers. 'Seed chippers' [96] use complicated robotics and liquid handling equipment to remove small portions of embryo, extracting DNA and performing genotyping. This technology was first demonstrated in maize and then soybean but is beginning to be adapted to work in cotton.…”