Search

•Present the dynamic transcriptome in tobacco genotypes resistant or susceptible to black shank disease and understand the defense response of tobacco to P. nicotianaeinfection.•A new statistical approach in analyzing time-series RNA-Seq data, which identifies variable transcripts with a standard deviation of log2(TPM + 1) > 1 among samples, and identified 7261 variable transcripts were enriched in terms to be related to defense responses against pathogen infection.•We have provided a systematic analysis scheme that can be followed by other researchers who are dealing with such type of RNA-Seq data.

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement. Currently available genome-editing tools have a limited number of targets, restricting their application in genetic research. In this study, we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors, eight donor vectors, four destination vectors, and one primer-design software package. By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sgRNA expression cassettes, the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci. A rice knockout vector containing 49 sgRNA expression cassettes was assembled and a high co-editing efficiency was observed. This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

Tobacco (Nicotiana tabacumL.) is an important model system, which has been widely used in plant physiological studies and it is particularly useful as a bioreactor. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping and breeding. Restriction-site associated DNA sequencing (RAD-seq) is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis through linkage mapping. We constructed a RAD library using genomic DNA from a BC1backcross population. Sequencing of 196 individuals was performed on an Illumina HiSeq 2500. Two linkage maps were constructed, one with a reference genome and another, termed as de novo identification of single nucleotide polymorphism (SNP) by RAD-seq, without a reference genome. Overall, 4138 and 2162 SNP markers with a total length of 1944.74 and 2000.9 cM were mapped to 24 linkage groups in the genetic maps based on reference genome and without reference, respectively. Using two different SNP discovery methods based on next generation RAD sequencing technology, we have respectively mapped 2162 and 4318 SNPs in our backcross population. This study gives an excellent example for high density linkage map construction, irrespective of genome sequence availability, and provides saturated information for downstream genetic investigations such as quantitative trait locus analyses or genomic selection (e.g. bioreactor suitable cultivars).

Black shank, caused by Phytophthora nicotianae, is typically the most important disease affecting tobacco (Nicotiana tabacumL.) production in the United States. Pedigree information suggests that most black shank resistance was derived from the cigar tobacco cultivar Florida 301. This resistance is thought to be polygenic in nature. The objectives of the current experiment were to (i) evaluate lines from a recombinant inbred line population derived from a cross between Florida 301 and the black shank‐susceptible cultivar Hicks for partial resistance using replicated field and greenhouse testing, (ii) genotype the population and use quantitative trait loci (QTL) analyses to identify Florida 301 genomic regions associated with resistance, and (iii) compare results with those obtained from a previous QTL analysis of a population derived from a cross involving ‘Beinhart 1000’. A total of 11 QTL affecting area under the disease progress curve were identified in both the field and greenhouse experiments. The QTL with the largest effect explained 16.9 and 18.6% of the phenotypic variation in the field and greenhouse experiments, respectively. This QTL was also found to have the largest effect on resistance in a Beinhart 1000 × Hicks doubled haploid mapping population. A major QTL found to affect resistance on linkage group 15 in the latter population, however, was not found to be important in the current population. Quantitative trait loci identification using greenhouse data was comparable to, if not superior to, use of field data.