Extraction of high molecular weight DNA suitable for next-generation sequencing from the fiber crop abaca

Deoxyribonucleic acid (DNA) extraction in abaca (Musa textilis Nee), the source of the strongest natural fiber (Manila hemp), is difficult due to its fibrous nature, high cellulose content, and polyphenol compounds. Thus an optimized DNA extraction method is required for extracting high quality abaca DNA for next-generation sequencing applications. This study, hence, aimed to test and compare five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional cetyl trimethylammonium bromide (CTAB) method (Protocol 1), the CTAB with polyvinylpyrrolidone (PVP) method (Protocol 2), the CTAB with 0.3 % beta-mercaptoethanol method (Protocol 3), sodium dodecyl sulfate method (Protocol 4), and CTAB with Triton X-100 and PVP method (Protocol 5). The use of a high throughput homogenizer (TissueLyserII) was also tested in tandem with select extraction protocols for applications in high-throughput DNA extraction. DNA from two abaca varieties were extracted via Protocol 3 and were sent for sequencing based on the Illumina Novaseq platform. After passing the quality control parameters for library preparation, Protocol 3 was found to be the simplest and most consistent method for extracting average yield DNA with high quality for next-generation sequencing applications, while Protocol 4 was determined to have the shortest processing time. Together with TissueLyserII-facilitated homogenization, Protocol 4 is the most appropriate combination for high-throughput extraction of abaca samples which will be useful for genotyping-by-sequencing (GBS) strategies as a molecular tool for plant breeding.

Automated summary

This study aimed to compare five different methods for extracting high molecular weight DNA from abaca leaves, with Protocol 3 (CTAB with 0.3% beta-mercaptoethanol) found to be the simplest and most consistent method for producing high quality DNA for sequencing applications. Protocol 4 (sodium dodecyl sulfate) was determined to have the shortest processing time, and when combined with TissueLyserII-facilitated homogenization, it is the most suitable combination for high-throughput extraction of abaca samples for genotyping-by-sequencing strategies.