Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum.
Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene-rich regions. Gene-enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene-enrichment strategy, we have compared assemblies using methyl-filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA-seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single-nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.
Laboratório de Biologia Molecular de Plantas, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373, CCS, Bl.L-29, Cidade Universitária, Rio de Janeiro, 21941-599, RJ, Brazil.
Clícia
C
Grativol
Michael
M
Regulski
Marcelo
M
Bertalan
W Richard
WR
McCombie
Felipe Rodrigues
FR
da Silva
Adhemar
A
Zerlotini Neto
Renato
R
Vicentini
Laurent
L
Farinelli
Adriana Silva
AS
Hemerly
Robert A
RA
Martienssen
Paulo Cavalcanti Gomes
PC
Ferreira
D022202:Chromosomes, Artificial, Bacterial; D032461:Chromosomes, Plant / Q000235:genetics*; D018556:Crops, Agricultural; D019175:DNA Methylation; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D018745:Genome, Plant / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D031786:Saccharum / Q000235:genetics*; D017421:Sequence Analysis; D045868:Sorghum / Q000235:genetics
D018744:DNA, Plant; D035683:MicroRNAs; D010940:Plant Proteins; D018749:RNA, Plant
Saccharum spp; de novo assembly; gene-rich regions; methylation filtration; sugarcane