Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L.

Tobacco (Nicotiana tabacum L.) is an essential commercial crop and an ideal model plant for biological mechanism studies. As an allopolyploid species, tobacco harbors a massive and complex genome, which makes the application of molecular markers complicated and challenging. In our study, we performed whole-genome sequencing of an intraspecific recombinant inbred line (RIL) population, a F1 generation and their parents. With the Nicotiana tabacum (K326 cultivar) genome as reference, a total of 45,081 markers were characterized to construct the genetic map, which spanned a genetic distance of 3486.78 cM. Evaluation of a two-dimensional heat map proved the high quality of the genetic map. We utilized these markers to anchor scaffolds and analyzed the ancestral genome origin of linkage groups (LGs). Furthermore, such a high-density genetic map will be applied for quantitative trait locus (QTL) detection, gene localization, genome-wide association studies (GWAS), and marker-assisted breeding in tobacco.

Metabolome and transcriptome analyses of the molecular mechanisms of flower color mutation in tobacco.

BACKGROUND: Anthocyanins determinate the flower color of many plants. Tobacco is a model plant for studying the molecular regulation of flower coloration. We investigated the mechanism underlying flower coloration in tobacco by profiling flavonoid metabolites,expression of anthocyanin biosynthetic structural genes and their regulator genes in the pink-flowered tobacco cultivar Yunyan 87 and white-flowered Yunyan 87 mutant. RESULT: Significant down-accumulation of anthocyanins, including cyanidin 3-O-glucoside, cyanin, cyanidin 3-O-rutinoside, pelargonidin 3-O-beta-D-glucoside, cyanidin O-syringic acid, pelargonin, and pelargonidin 3-O-malonylhexoside (log2 fold change < - 10), endowed the flower color mutation in Yunyan 87 mutant. Transcriptome analysis showed that the coordinately down-regulated anthocyanin biosynthetic genes including chalcone isomerase, naringenin 3-dioxygenase, dihydroflavonol 4-reductase and UDP-glucose:flavonoid 3-O-glucosyltransferase played critical roles in suppressing the formation of the aforesaid anthocyanins. Several genes encoding MYB and bHLH transcription factors were also found down-regulated, and probably the reason for the suppression of structural genes. CONCLUSION: This is the first study of tobacco flower coloration combining metabolome and transcriptome analyses, and the results shed a light on the systematic regulation mechanisms of flower coloration in tobacco. The obtained information will aid in developing strategies to modify flower color through genetic transformation.

Genome-wide association study of leaf chemistry traits in tobacco.

Leaf chemistry traits are some of the key factors influencing tobacco quality, which can be significantly reduced by lower chemical components in cured leaf. To improve tobacco quality through breeding, genetic diversity analysis, population structure analysis, and genome-wide association studies were performed in a panel of 347 tobacco germplasms and the markers associated with five leaf chemistry traits, including total sugar (TS), reducing sugar (RS), total nitrogen (TN), nicotine (NIC), and total potassium (TP) contents were identified. Four groups were classified at a genetic distance of 0.316 by genetic diversity analysis based on coefficient parameter NEI72 using a program NTSYS-pc2.10e, whereas four well-differentiated subpopulations were postulated in the 347 tobacco accessions. A total of 47 target trait-associated SNPs was detected in at least three environments as well as the best linear unbiased predictions (BLUPs) across all environments, among which two, two, four, six, and one highly suggestive associated SNPs were repeatedly detected in all environments and BLUPs for TS, RS, TN, NIC, and TP, respectively. On the basis of the phenotypic effects of the alleles corresponding to suggestive associated SNPs, five tobacco accessions harboring favorable alleles with elite phenotypic performance in leaf chemistry traits were identified. The results could facilitate quality tobacco breeding for higher leaf chemistry trait contents through molecular marker-assisted approaches.

Effectors of Phytophthora pathogens are powerful weapons for manipulating host immunity.

MAIN CONCLUSION: This article provides an overview of the interactions between Phytophthora effectors and plant immune system components, which form a cross-linked complex network that regulates plant pathogen resistance. Pathogens secrete numerous effector proteins into plants to promote infections. Several Phytophthora species (e.g., P. infestans, P. ramorum, P. sojae, P. capsici, P. cinnamomi, and P. parasitica) are notorious pathogens that are extremely damaging to susceptible plants. Analyses of genomic data revealed that Phytophthora species produce a large group of effector proteins, which are critical for pathogenesis. And, the targets and functions of many identified Phytophthora effectors have been investigated. Phytophthora effectors can affect various aspects of plant immune systems, including plant cell proteases, phytohormones, RNAs, the MAPK pathway, catalase, the ubiquitin proteasome pathway, the endoplasmic reticulum, NB-LRR proteins, and the cell membrane. Clarifying the effector-plant interactions is important for unravelling the functions of Phytophthora effectors during pathogenesis. In this article, we review the effectors identified in recent decades and provide an overview of the effector-directed regulatory network in plants following infections by Phytophthora species.

Genome-wide analysis of the HAK potassium transporter gene family reveals asymmetrical evolution in tobacco (Nicotiana tabacum).

Being an essential mineral nutrient, potassium (K+) plays numerous important roles in plant growth and development and determines the yield and quality of crop products. The cellular level of K+ is controlled to a large extent by the K+ transporter, which belongs to the KT/HAK/KUP (HAK) family. However, little is known about these genes in tobacco. In this study, we surveyed the tobacco genome and identified 41 putative NtHAK genes (NtHAKS1-NtHAKS21 and NtHAKT1-NtHAKT20). Investigation of the cis-elements in upstream regions of these NtHAK genes suggests that members of this family respond to environmental cues and phytohormones. Expression data mining reveals that NtHAK genes showed clear sub-genome dominance. In all, these results will provide molecular insights into K+ transporter research in tobacco.

Large-scale development of SSR markers in tobacco and construction of a linkage map in flue-cured tobacco.

Tobacco (Nicotiana tabacum L.), particularly flue-cured tobacco, is one of the most economically important nonfood crops and is also an important model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping, and breeding. Simple sequence repeats (SSR) are one of the most widely-used molecular markers, having significant advantages including that they are generally co-dominant, easy to use, abundant in eukaryotic organisms, and produce highly reproducible results. In this study, based on the genome sequence data of flue-cured tobacco (K326), we developed a total of 13,645 mostly novel SSR markers, which were working in a set of eighteen tobacco varieties of four different types. A mapping population of 213 backcross (BC1) individuals, which were derived from an intra-type cross between two flue-cured tobacco varieties, Y3 and K326, was selected for mapping. Based on the newly developed SSR markers as well as published SSR markers, we constructed a genetic map consisting of 626 SSR loci distributed across 24 linkage groups and covering a total length of 1120.45 cM with an average distance of 1.79 cM between adjacent markers, which is the highest density map of flue-cured tobacco till date.

A tonoplast-localized TPK-type K+ transporter (TPKa) regulates potassium accumulation in tobacco.

Potassium ion (K+) is one of the most essential nutrients for the growth and development of tobacco (Nicotiana tabacum L.), however, the molecular regulation of K+ concentration in tobacco remains unclear. In this study, a two-pore K (TPK) channel gene NtTPKa was cloned from tobacco, and NtTPKa protein contains the unique K+ selection motif GYGD and its transmembrane region primarily locates in the tonoplast membrane. The expression of NtTPKa gene was significantly increased under low-potassium stress conditions. The concentrations of K+ in tobacco were significantly increased in the NtTPKa RNA interference lines and CRISPR/Cas9 knockout mutants. In addition, the transport of K+ by NtTPKa was validated using patch clamp technique, and the results showed that NtTPKa channel protein exclusively transported K+ in a concentration-dependent manner. Together, our results strongly suggested that NtTPKa is a key gene in maintaining K+ homeostasis in tobacco, and it could provide a new genetic resource for increasing the concentration of K+ in tobacco.

Genome-wide characterization, evolution, and expression profiling of FBA gene family in response to light treatments and abiotic stress in Nicotiana tabacum.

Fructose 1,6-bisphosphate aldolase (FBA) as a key enzyme play crucial roles in glycolysis, gluconeogenesis and Calvin cycle processes in plants. However, limited information is known regarding FBA genes in Nicotiana tabacum. In this study, 16 FBAs were identified and characterized in Nicotiana tabacum. Phylogenetic analysis revealed that these genes can be categorized as type I (NtFBA1-10 located in chloroplast) and type II (NtFBA11-16 located in cytoplasm) subfamilies. According to the conserved motifs and gene structure analysis, NtFBA protein sequences had the highly homologous to FBAs in other species. Most members of the NtFBA gene family responded positively to NaHCO3 stress, especially the expression of NtFBA13/14 increased by 642%. In addition, the expression results of NtFBAs under five abiotic stress (light, NaCl, NaHCO3, drought, and cold) conditions were showed that NtFBA13/14 were highly up-regulated. qRT-PCR results showed that most of the NtFBAs expressed higher in leaves. NtFBA7/8 and NtFBA13/14 have important significance in photosynthesis and abiotic stress, respectively. This study provides a basis foundation for further elucidating the function of NtFBAs and the N. tabacum mechanism of resistance under abiotic stress.

Important Roles of Key Genes and Transcription Factors in Flower Color Differences of Nicotianaalata.

Nicotiana alata is an ornamental horticultural plant with a variety of flower colors and a long flowering period. The genes in four different colored N. alata (white, purple, red, and lemon green) were analyzed to explain the differences in flower color using transcriptomes. A total of 32 differential expression genes in the chlorophyll biosynthesis pathway and 41 in the anthocyanin biosynthesis pathway were identified. The enrichment analysis showed that the chlorophyll biosynthesis pathway and anthocyanin biosynthesis pathway play critical roles in the color differences of N. alata. The HEMA of the chlorophyll biosynthesis pathway was up-regulated in lemon green flowers. Compared with white flowers, in the red and purple flowers, F3H, F3'5'H and DFR were significantly up-regulated, while FLS was significantly down-regulated. Seventeen differential expression genes homologous to transcription factor coding genes were obtained, and the homologues of HY5, MYB12, AN1 and AN4 were also involved in flower color differences. The discovery of these candidate genes related to flower color differences is significant for further research on the flower colors formation mechanism and color improvements of N. alata.

Cloning, structural features, and expression analysis of resistance gene analogs in tobacco.

Using degenerate primers based on the conserved nucleotide binding site (NBS) and protein kinase domain (PKD), 100 resistance gene analogs (RGAs) were isolated from tobacco variety Nicotiana repanda. BLASTx search against the GenBank database revealed that 27 belong to the NBS class and 73 belong to the protein kinase (PK) class. Cluster analysis and multiple sequence alignment of the deduced protein sequences indicate that RGAs of the NBS class can be divided into two groups: toll/interleukin receptor (TIR) and non-TIR types. Both types possess 6 conserved motifs (P-loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, GLPL). Based on their sequence similarity, the tobacco RGAs of the PK class were assigned to 8 subclasses. We examined their expression after infection with either Tobacco mosaic virus (TMV) or the tobacco black shank pathogen (Phytophthora parasitica var. nicotianae). The expression levels of 4 RGAs of the PK class were significantly elevated by TMV and 1 RGA of the PK class and 3 RGAs of the NBS class were up-regulated by P. parasitica var. nicotianae. The expression of two RGAs of the PK class was induced by P. parasitica var. nicotianae. Infection by either TMV or P. parasitica var. nicotianae enhanced the expression of NtRGA2, a RGA of the PK class. The present study shows that RGAs are abundant in the tobacco genome and the identification of tobacco RGAs induced by pathogens should provide valuable information for cloning related resistance genes in tobacco.

Short Tandem Repeats in plants: genomic distribution and function prediction

BACKGROUND: Short Tandem repeats (STRs) existed as popular elements in both eukaryotic and prokaryotic genomes. RESULTS: In this study, we analyzed the characteristics, distributions, and motif features of STRs within whole-genomes of 140 plant species. The results showed that STR density was negatively correlated with the genome size. Hexanucleotide repeat was the most abundant type of STRs. The distribution of algae shows a preference different from that of other plants. By analyzing GC contents of STRs and genome, it was concluded that STR motif was influenced by GC contents. Analysis of the long STRs in genome (length 1000 bp) found that dicots have the more long STRs. For STR types, di- and tri-nucleotide accounted for the highest proportion. Analyzing and designing long STRs in CDS (length 500 bp) was to verify the role of long STRs in Gossypium hirsutum TM-1 and Solanum tuberosum. By comparing the long STRs found in Fragaria x ananassa with other species, some evolutionary characteristics of the long STRs were obtained. CONCLUSIONS: We got the characteristics, distribution, and motif features of STRs in the whole genome of 140 plants and obtained some evolutionary characteristics of long STRs. The study provides useful insights into STR preference, characteristics, and distribution in plants.

[Genes of metal-binding proteins and their application in bioremediation of heavy metals].

Heavy metal pollution has become a global environmental hazard. The use of microorganisms and plants for the decontamination of heavy metals is recognized as a low lost and high efficiency method for cleaning up metal contamination. It shows that various metal-binding proteins such as metallothioneins (MTs) or phytochelatines (PCs) play an important role in defense systems and detoxification to heavy metals in organisms. Many genes of MTs and PCs have been cloned and utilized successfully in genetically modified bacteria and plants for increasing remediation capacity. These transgenic organisms have been displayed a great potential in bioremediation and phytoremediation of heavy metals.

The two-component signal system in rice (Oryza sativa L.): a genome-wide study of cytokinin signal perception and transduction.

In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice (Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) (OsHK1-4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) (OsHP1-5), 15 type-A RRs (response regulators) (OsRR1-15), 7 type B RR genes (OsRR16-22), and 5 predicted pseudo-response regulators (OsPRR1-5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.