RESUMEN

Premise: We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results. Methods: Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Results: Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Discussion: Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.

RESUMEN

As the global climate crisis continues, predictions concerning how wild populations will respond to changing climate conditions are informed by an understanding of how populations have responded and/or adapted to climate variables in the past. Changes in the local biotic and abiotic environment can drive differences in phenology, physiology, morphology and demography between populations leading to local adaptation, yet the molecular basis of adaptive evolution in wild non-model organisms is poorly understood. We leverage comparisons between two lineages of Calochortus venustus occurring along parallel transects that allow us to identify loci under selection and measure clinal variation in allele frequencies as evidence of population-specific responses to selection along climatic gradients. We identify targets of selection by distinguishing loci that are outliers to population structure and by using genotype-environment associations across transects to detect loci under selection from each of nine climatic variables. Despite gene flow between individuals of different floral phenotypes and between populations, we find evidence of ecological specialization at the molecular level, including genes associated with key plant functions linked to plant adaptation to California's Mediterranean climate. Single-nucleotide polymorphisms (SNPs) present in both transects show similar trends in allelic similarity across latitudes indicating parallel adaptation to northern climates. Comparisons between eastern and western populations across latitudes indicate divergent genetic evolution between transects, suggesting local adaptation to either coastal or inland habitats. Our study is among the first to show repeated allelic variation across climatic clines in a non-model organism.

Asunto(s)

Clima , Selección Genética , Frecuencia de los Genes/genética , Aclimatación , Adaptación Fisiológica/genética , Polimorfismo de Nucleótido Simple/genética , Variación Genética/genética

RESUMEN

PREMISE OF THE STUDY: Microsatellite primers were designed for Calochortus gunnisonii (Liliaceae), a montane lily species of the central and southern Rocky Mountains, using next-generation DNA sequencing. The markers will be used to investigate population structure, genetic diversity, and demographic history. METHODS AND RESULTS: Thirteen polymorphic microsatellite loci were isolated from C. gunnisonii using Illumina MiSeq next-generation DNA sequencing and bioinformatic screening. The mean number of alleles per locus ranged from 4.15 to 5.92 (avg. = 4.97). Observed and expected heterozygosity ranged from 0.077 to 0.871 and 0.213 to 0.782, respectively. The primers were also tested for cross-species amplification value with C. flexuosus, C. nuttallii, C. kennedyi var. kennedyi, and C. subalpinus. CONCLUSIONS: These primers will be useful for genetic and evolutionary studies across C. gunnisonii's range within the southern and central Rocky Mountains. Furthermore, these markers have proven valuable for cross-species amplifications within Calochortus.