Chromosome evolution and the genetic basis of agronomically important traits in greater yam

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an ‘orphan crop.’ Here we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using our new genomic tools we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Methods
Fresh leaf samples were collected from the field at both IITA and NRCRI. DNAs were extracted using CTAB protocols and sent to DArT (Canberra, Australia) or Integrated Genotyping Service and Support (IGSS) at BecA-ILRI for high-density genotyping using the DArTseq platform. 

Samples from IITA were collected on ice. 100 mg of leaf sample was placed in a 2.0 mL Eppendorf tube, and ground with liquid nitrogen. To remove secondary metabolites, it was washed 2–3 times by adding 1000 µl HEPES buffer [0.1 M HEPES, PVP, L-ascorbic acid, 2-mercaptoethanol, and sterile distilled water], mixing thoroughly, centrifuging at 13,000 rpm for 2 min, and decanting the supernatant. Next, 800 µl of freshly prepared CTAB buffer [1M Tris HCl pH 8, 0.5 EDTA pH 8, 5M NaCl pH 8, 1% mercaptoethanol, 3% CTAB] was added and mixed well to ensure homogenization, and samples were incubated for 30 min at 65 °C in a water bath. 600 µl of chloroform-isoamyl alcohol (24:1) was added and the samples were mixed gently and centrifuged at 13,000 rpm for 5 min. The aqueous phase was transferred into freshly labeled 1.2 mL tubes. DNA was precipitated with the addition of ⅔ vol ice-cold isopropanol, mixed by inverting, incubated at −20 °C for 1 h, and centrifuged at 13,000 rpm for 5 min. The supernatant was decanted carefully, and the DNA pellet was washed twice with 500 µl of cold 70% ethanol. The ethanol was drained completely and the pellet dried at 37 °C for 30 min. DNA was resuspended in 50 µl of low TE (1 mM Tris, 0.1 mM EDTA) and 3 µl RNase, incubated at 37 °C for 1 h, and stored at 4 °C.

Samples from NRCRI were lyophilized and ground to powder in a Qiagen TissueLyser LT for 1 min at a rate of 1500 strokes/min and transferred to 2 mL microtubes. The ground tissue was homogenized in 800 µl of CTAB buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 1% polyvinyl pyrrolidone, 2% 2-mercaptoethanol, 3% CTAB), then incubated for 30 min at 65 °C. 600 μl of an equal volume of chloroform and isoamyl alcohol (24:1 vol/vol) was added to the tube and centrifuged for 10 min at 13000 rpm. The nucleic acid in the aqueous phase was precipitated out with cold isopropanol, and the pellets washed by centrifuging at 13000 rpm with 70% ethanol. The pellets were further suspended in 50 µl of sterile water and treated with 3 µl of RNAse A (20 mg/mL) for 1 h at 37 °C. Finally, the samples were stored at −20 °C until use. The DNA samples were quantified using a NanoDrop 1000 (Thermo Scientific) and their integrity assessed by agarose gel electrophoresis.