Anchoring 323 Mbp of the Pinus taeda Genome Assembly v1.01 to a Single-Tree Sub-Centimorgan Genetic Map of 2,469 DArT-Seq Markers and Microsatellites

A total of 323 Mbp of sequence scaffolds of the Pinus taeda reference genome assembly v1.01 (PineRefSeq) were anchored to a high density genetic map built with microsatellites and sequence based (DArT-seq) markers genotyped in 288 haploid megagametophytes of Pinus taeda clone 7-56. DArT-seq carried out by genome complexity reduction using double digest with PstI-HhaI, yielded 4,367 high quality markers (69 bp tags). Map construction (Joinmap 3.0) resulted in 2,469 ordered markers covering 1,226.5 cM, marker density of 0.62cM and an additional set of 1,516 markers grouped at LOD>20 but yet to be ordered. A key feature of the DArT-seq markers is that they preferentially target low copy gene-rich regions as a result of the complexity reduction method. A total of 3,858 DArT-seq tags were aligned to a set of 14,412,986 scaffolds of P. taeda assembly, under rigorous alignment criteria using BLAT (99% identity; 69 bp alignment) to provide the most robust linkage to physical anchoring information. A scaffold was anchored if all DArT-seq markers on it mapped to the same linkage group and the median absolute deviation of the map coordinates of the markers was < 5 cM. A total of 1,120 DArT-seq markers captured a total of 1,399 scaffolds, 582 of them >1Mbp and nine >4Mbp, totaling 323,268,536 bp. Anchored scaffolds included 816 high quality gene models annotated in PineRefSeq with 790 of them successfully captured by unambiguous tag alignments. Although only a small fraction of the P. taeda genome (1.3%) could be aligned to the high-density map, illustrating the challenge of anchoring such a megagenome, the fact that  979 DArT-seq markers targeted a single scaffold with no ambiguity, should make this information increasingly valuable as assembled sequence scaffolds increase in size, therefore contributing to upcoming assembly efforts particularly in gene-rich regions.