Restriction site associated DNA (RAD) for de novo sequencing and marker discovery in sugarcane borer, Diatraea saccharalis Fab. (Lepidoptera: Crambidae)

We present the development of a genomic library using RADseq (restriction site associated DNA sequencing) protocol for marker discovery that can be applied on evolutionary studies of the sugarcane borer Diatraea saccharalis, an important South American insect pest. A RADtag protocol combined with Illumina paired-end sequencing allowed de novo discovery of 12 811 SNPs and a high-quality assembly of 122.8M paired-end reads from six individuals, representing 40 Gb of sequencing data. Approximately 1.7 Mb of the sugarcane borer genome distributed over 5289 minicontigs were obtained upon assembly of second reads from first reads RADtag loci where at least one SNP was discovered and genotyped. Minicontig lengths ranged from 200 to 611 bp and were used for functional annotation and microsatellite discovery. These markers will be used in future studies to understand gene flow and adaptation to host plants and control tactics.本研究开发了一套基于限制性酶切位点相关DNA测序（restriction site associated DNA sequencing, RADseq）技术的基因组文库构建方案，用于分子标记发掘，可应用于重要南美农业害虫甘蔗螟虫（Diatraea saccharalis）的演化生物学研究。结合RADtag技术与Illumina双端测序策略，本研究从6个供试个体中完成了122.8百万条双端测序读段的高质量组装，对应总测序数据量40Gb，并从头发掘得到12811个单核苷酸多态性（Single Nucleotide Polymorphism, SNPs）。针对至少发掘出一个单核苷酸多态性并完成基因分型的RADtag位点的首轮测序读段进行二次组装后，我们获得了分布于5289个微型重叠群（minicontigs）上、总长约1.7Mb的甘蔗螟虫基因组序列。该批微型重叠群的长度区间为200至611 bp，可用于功能注释与微卫星（microsatellite）标记发掘。上述分子标记将在未来研究中用于解析种群基因流、宿主植物适应性及害虫防控策略相关的生物学机制。