High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop
The M2 and S2 populations were used as the basis for genotypic screening. Leaves were collected from 754 juvenile M2 seedlings and 109 S2 individuals. DNA was extracted from fresh leaf tissue using a modified MagAttract 96 DNA Plant Protocol (Qiagen, Frankfurt, Germany) with one additional reverse osmosis purified water wash prior to being quantified using UV spectrophotometry at a wavelength of 260nm and 280nm (MWG Sirius Plate reader, MWG Biotech, Ebersberg, Germany). The DNA was normalised using Gibco Nuclease Free water to a concentration of 2ng/μl using the MWG Theonyx (MWG Biotech, Ebersberg, Germany). Prior to PCR amplification DNA was pooled from five individuals and 10ng of pooled template was used per PCR reaction. Stringent quality controls were applied during sample preparation. DNAwas quantified, normalised and pooled in equimolar proportions at each step in an attempt to maintain relative allele frequencies in the subsequent Illumina GAII sequence data.  Four candidate domestication related homologues were targeted for PCR amplification for this study (Supplementary Table 1). Homologues of granule bound starch synthase 1 (GBSS1), encoded by the Waxy gene [7], the Isa gene [34] and two gene homologues controlling seed shattering in rice, sh4/SHA1 and qSH1 [35]. PCR products were quantified by gel electrophoresis using Scion image (http://softwaretopic.informer.com/scion-image-free- software/, last accessed 02/08/11). Amplification products were combined in equimolar amounts to form homologue-specific pools of 109 and 754 M2 (mutant) individuals, respectively, in addition to a pool of 109 S2 (control) individuals. The homologue-specific pools for each gene were then quantified based on pico-green analysis and combined in equimolar amounts to form megapools representing the two mutant and control populations. These three megapools were run as individual lanes on the Illumina GAIIx platform (Illumina, San Diego, CA, USA) using a paired-end strategy with a fragment size of 400bp and a read length of 75bp.