Abi 3730xl capillary

Complete genomic sequencing was conducted using a Roche GS FLX system (454 Life Sciences Corp, Branford, CT, USA; Margulies et al., 2005 (link)). A total of 267,232 reads amounting 128 Mb of raw data (average read length of 478 bp), were obtained, resulting in 29-fold genome coverage. Assembly was performed using the GS de novo Assembler software from SFF file with default parameters, which will remove reads shorter than 50 bp. A total of 113 contigs ranging from 500 to 203,016 bp were produced. The relationships among the contigs were determined by multiplex polymerase chain reaction (PCR) (Tettelin et al., 1999 (link)). Then, gaps were filled in by sequencing the PCR products using ABI 3730XL capillary sequencers (Applied Biosystems, Waltham, MA, USA). Phred, Phrap, and Consed software packages (http://www.phrap.org/phredphrapconsed.html) were used for final assembly and editing, and the low-quality regions (the bases with phred quality score <20) of the genome were re-sequenced. The final sequencing accuracy was 99.99%.

DNA was extracted from single wheat seedlings using the CTAB extraction method (Doyle and Doyle, 1990 ). All 256 lines were screened with published markers for 11 stem rust resistance genes using the published PCR protocols (Supplementary Table 2). Fluorescently labeled primers allowed sequence-based fragment detection on an ABI3730xl capillary instrument (Applied Biosystems, Foster City, CA, USA) applied at the Central Analytical Facility of Stellenbosch University, South Africa. GeneScan™ 500 LIZ® or GeneScan™ 1200 LIZ® (Applied Biosystems) was used as an internal size standard. Data were analyzed using GeneMapper v4.0 (Thermofischer, formally Applied Biosystems). A stem rust resistance gene was called as present if the marker-allele at all markers associated with the Sr gene were the same as the control line. Controls for known stem rust resistance genes and gene complexes included: Cranbrook (Sr2), Sr22B (Sr22), Palmiet (Sr2, Sr24), Avocet S (Sr26), Federation^*4/Kavkaz (Sr31), RL5405 (Sr33), Mq12/5^*G2191_Rsr35 (Sr35), SrTt1Sr36 (Sr36), RL6082 (Sr39/Lr35), Kariega (Lr34/Yr18/Sr57) and Pavon 76 (Lr46/Yr29/Sr58).