Development of genomic resources for Nothofagus species using next‐generation sequencing data

Abstract

Using next-generation sequencing, we developed the first whole-genome resources for two hybridizing Nothofagus species of the Patagonian forests that crucially lack genomic data, despite their ecological and industrial value. A de novo assembly strategy combining base quality control and optimization of the putative chloroplast gene map yielded ~32,000 contigs from 43% of the reads produced. With 12.5% of assembled reads, we covered ~96% of the chloroplast genome and ~70% of the mitochondrial gene content, providing functional and structural annotations for 112 and 52 genes, respectively. Functional annotation was possible on 15% of the contigs, with ~1750 potentially novel nuclear genes identified for Nothofagus species. We estimated that the new resources (13.41 Mb in total) included ~4000 gene regions representing ~6.5% of the expected genic partition of the genome, the remaining contigs potentially being nongenic DNA. A high-quality single nucleotide polymorphisms resource was developed by comparing various filtering methods, and preliminary results indicate a strong conservation of cpDNA genomes in contrast to numerous exclusive nuclear polymorphisms in both species. Finally, we characterized 2274 potential simple sequence repeat (SSR) loci, designed primers for 769 of them and validated nine of 29 loci in 42 individuals per species. Nothofagus obliqua had more alleles (4.89) on average than N. nervosa (2.89), 8 SSRs were efficient to discriminate species, and three were successfully transferred in three other Nothofagus species. These resources will greatly help for future inferences of demographic, adaptive and hybridizing events in Nothofagus species, and for conserving and managing natural populations.

Searching for molecular insight on hybridization in Nothofagus spp. forests at Lagunas de Epulauquen, Argentina

“…A higher sample size was not required as genetic diversity of molecular makers from both species has been previously determined supporting the power of selected markers to discriminate both species (Manos 1997, El Mujtar et al 2014, Sola et al 2016. Samples representing putative parental species have homozygote genotypes for Adh isozyme alleles, N. obliqua Adh 1 -Adh 1 and N. alpina Adh 2 -Adh 2 and therefore they were also used to test species specificity of isozyme alleles.…”

“…Twelve SSRs (table 2) were selected from those developed by Torales et al (2012) andEl Mujtar et al (2014), considering the presence of species-specific alleles for N. obliqua and N. alpina and the number of detected alleles. In addition, 16 samples from Lácar watershed (Argentina) previously genotyped at ten out of these 12 SSRs were included in the analysis: eight natural hybrids between N. obliqua and N. alpina (NoxNa), four N. obliqua and four N. alpina individuals were selected based on their genotypes, with hybrids showing alleles from both species at more than one loci (Sola et al 2016).…”

“…This means that it is possible that the isozyme Adh-2 allele detected in N. obliqua populations differs in its nucleotide constitution from that of N. alpina populations, and therefore isozyme heterozygote genotypes (Adh-1/Adh-2) should reveal intra-specific variability instead of signals of hybridization. A nucleotide sequence analysis of nuclear regions showed low to moderate intraspecific diversity combined with high divergence between N. obliqua and N. alpina, revealing a strong potential of nucleotide polymorphisms for the identification of species and hybrids (El Mujtar et al 2014). Among low-copy nuclear genes, Adh has proven to be useful for inter and intraspecific analyses and to provide evidence of the hybrid origin for some species (Järvinen et al 2004, Pan et al 2007.…”

De novo assembly and characterization of leaf and floral transcriptomes of the hybridizing bromeliad species (Pitcairnia spp.) adapted to Neotropical Inselbergs

“…Unigenes larger than 8000 bases were analysed at NCBI because they exceeded the length limit imposed by blast2go pro (Mujtar et al . ).…”

“…A BLASTX search was performed with an E-value of 1e-06 against the NCBI nonredundant protein database (NR), followed by attribution of Gene Ontology (GO) terms, Enzyme Commission (EC), InterProScan domains and mapping of annotations to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Database. Unigenes larger than 8000 bases were analysed at NCBI because they exceeded the length limit imposed by BLAST2GO PRO (Mujtar et al 2014).…”