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Black shank, caused by Phytophthora nicotianae, is typically the most important disease affecting tobacco (Nicotiana tabacum L.) 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.

ABSTRACTBackgroundNicotine biosynthesis is mainly regulated by jasmonate (JA) signaling cascade in Nicotiana tabacum. As an allotetraploid species, the regulation of nicotine biosynthesis has been genetically verified via two unlinked NIC loci (named as NIC1 and NIC2) which are possibly originated from its two ancestral diploids. Previously, a N. tomentosiformis originated ethylene response factor (ERF) gene cluster was identified as the NIC2-locus which has been demonstrated positively regulates nicotine accumulation in N. tabacum.ResultsHere, we describe the genetic mapping of NIC1-locus, the major nicotine regulatory locus, by using a NIC1-locus segregating population through bulked segregant analysis. We identified two linkage marker TM23004 and TM22038 were delimited the NIC1-locus within a ~34.3-Mb genomic region at pseudochromosome 07 of tobacco genome. Genomic scan within this region revealed a NIC2-like locus ERF gene cluster exist in. To verify this ERF gene cluster is the genetically called “NIC1-locus”, different functional experiments based on most of the ERFs in regulating nicotine biosynthesis and their influences on alkaloid accumulations have been carried out. Collinearity analysis showed that NIC1-locus ERF genes are originated from N. sylvestris and exclusively expressed in root tissues. In addition, transcriptomic results indicate that NIC1-locus ERF genes are coexpressed with the NIC2-locus ERF genes and other nicotine biosynthetic genes and regulators after JA induction. Furthermore, the suppressed expression of four ERFs of the NIC1-locus genes corresponding with decreased NtPMT and NtQPT expression in NtMYC2-RNAi lines indicates the selected NIC1-locus ERFs function in downstream of NtMYC2 in the JA signaling cascades. In the meanwhile, the alkaloid levels are also determined by the amplitude of the four ERF gene expressions in both wild type and LA mutant. Additionally, in vitro binding assays, transient activation assays, and ectopic expression in transgenic plants demonstrate that these ERF genes are able to bind the GCC-box elements residing in the step-limiting gene promoters (such as NtPMT2, NtQPT2) and functional redundant but quantitatively transactivate nicotine biosynthetic gene expression. For nic1-locus mutation, two different sizes of deletions (nic1-S and nic1-B) were identified which occurred at the surrounding regions of the NIC1-locus gene cluster, which might disrupt, to some extent, chromosomal microenvironment and change gene expression around the deletion regions (including NIC1-locus ERFs), resulting in the decreased expression levels of NIC1-locus ERFs (such as NtERF199) and reduced alkaloid accumulation in the nic1-locus mutant.ConclusionsOur findings not only provide insight in to the mechanism of the NIC1-locus ERFs in the regulatory network of nicotine biosynthesis, but also unraveled the theoretical basis of the nic1-locus mutation in low nicotine mutant. These functional verified NIC1-locus ERF genes can be further used as potential target(s) for ethyl methanesulfonate-based mutagenesis to manipulate nicotine level in tobacco variety in tobacco breeding program.

Brown spot (BS) caused by Alternaria alternata is one of the most destructive foliar diseases affecting tobacco (Nicotiana tabacum L.) production and quality in China. Breeding of BS-resistant cultivars is difficult because the resistance has proved to be quantitatively inherited. To facilitate marker-assisted selection, we carried out a study of mapping quantitative trait loci (QTLs) for BS resistance. We developed an F 2 population consisting of 213 individuals from a cross between a BS-susceptible cultivar ÔChangbohuangÕ (CBH) and a BSresistant cultivar ÔJinyehuangÕ (JYH) and constructed a genetic map consisting of 196 simple sequence repeat (SSR) markers based on this population. Using disease index (DI) as the indicator of BS resistance, we detected three QTLs located between SSR markers TM20534 and TM10737, TM10589 and TM10216, and TM10443 and PT60669, respectively. The resistant alleles of the three QTLs were all from the resistant parent JYH. The three QTLs together could explain $86% of the DI difference between the two parents in total, with $61% explained by their additive effects. Therefore, the three QTLs will be useful for BS-resistance breeding.