A COSII genetic map of the pepper genome provides a detailed picture of synteny with tomato and new insights into recent chromosome evolution in the genus Capsicum

We report herein the development of a pepper genetic linkage map which comprises 299 orthologous markers between the pepper and tomato genomes (including 263 conserved ortholog set II or COSII markers). The expected position of additional 288 COSII markers was inferred in the pepper map via pepper–tomato synteny, bringing the total orthologous markers in the pepper genome to 587. While pepper maps have been previously reported, this is the first complete map in the sense that all markers could be placed in 12 linkage groups corresponding to the 12 chromosomes. The map presented herein is relevant to the genomes of cultivated C. annuum and wild C. annuum (as well as related Capsicum species) which differ by a reciprocal chromosome translocation. This map is also unique in that it is largely based on COSII markers, which permits the inference of a detailed syntenic relationship between the pepper and tomato genomes—shedding new light on chromosome evolution in the Solanaceae. Since divergence from their last common ancestor is approximately 20 million years ago, the two genomes have become differentiated by a minimum number of 19 inversions and 6 chromosome translocations, as well as numerous putative single gene transpositions. Nevertheless, the two genomes share 35 conserved syntenic segments (CSSs) within which gene/marker order is well preserved. The high resolution COSII synteny map described herein provides a platform for cross-reference of genetic and genomic information (including the tomato genome sequence) between pepper and tomato and therefore will facilitate both applied and basic research in pepper. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A detailed synteny map of the eggplant genome based on conserved ortholog set II (COSII) markers

We report herein the mapping of 115 PCR-based orthologous markers, including 110 conserved ortholog set or COSII markers, on the reference RFLP map of eggplant. The result permitted inference of a detailed syntenic relationship between the eggplant and tomato genomes. Further, the position of additional 522 COSII markers was inferred in the eggplant map via eggplant-tomato synteny, bringing the total number of markers in the eggplant genome to 869. Since divergence from their last common ancestor approximately 12 million years ago, the eggplant and tomato genomes have become differentiated by a minimum number of 24 inversions and 5 chromosomal translocations, as well as a number of single gene transpositions possibly triggered by transposable elements. Nevertheless, the two genomes share 37 conserved syntenic segments (CSSs) within which gene/marker order is well preserved. The high-resolution COSII synteny map described herein provides a platform for cross-reference of genetic and genomic information (including the tomato genome sequence) between eggplant and tomato and therefore will facilitate both applied and basic research in eggplant. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

Nicotiana chloroplast genome

Summary A physical map containing six restriction sites of the Nicotiana tabacum chloroplast genome, together with the BamHI maps of N. tabacum, N. otophora and N. knightiana, and the SmaI maps of N. acuminata, N. plumbaginifolia, N. langsdorffii, N. otophora, N. tabacum, N. tomentosiformis and N. knightiana was constructed. In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations. Deletions are also detected. Most of the observed changes are confined to one area of the large single copy region, which is designated as the hot spot. Based on the evidence obtained from Nicotiana chloroplast genomes, an origin of the inverted repeats in this genus is proposed. We suggest that the inverted repeats represent a vestige of what were once two identical, complete chloroplast genomes joined together in a head-to-head and tail-to-tail fashion, and that deletions generated the current chloroplast genome organization.     

Species-specific evolution of telomeric and rDNA repeats in the tobacco composite genome

In order to investigate possible interactions between parental genomes in the composite genome of Nicotiana tabacum we have analyzed the organization of telomeric (TTTAGGG)_n and ribosomal gene (rDNA) repeats in the progenitor genomes Nicotiana sylvestris and Nicotiana tomentosiformis or Nicotiana otophora. Telomeric arrays in the Nicotiana species tested are heterogeneous in length ranging from 20 to 200 kb in N. sylvestris, from 20 to 50 kb in N. tomentosiformis, from 15 to 100kb in N. otophora, and from 40 to 160kb in N. tabacum. The patterns of rDNA repeats (18S, 5.8S, 25S RNA) appeared to be highly homogeneous and speciesspecific; no parental rDNA units corresponding to N. sylvestris, N. tomentosiformis or N. otophora were found in the genome of N. tabacum by Southern hybridization. The results provide evidence for a species-specific evolution of telomeric and ribosomal repeats in the tobacco composite genome.     

Do potatoes and tomatoes have a single evolutionary history, and what proportion of the genome supports this history?

Background  

Phylogenies reconstructed with only one or a few independently inherited loci may be unresolved or incongruent due to taxon and gene sampling, horizontal gene transfer, or differential selection and lineage sorting at individual loci. In an effort to remedy this situation, we examined the utility of conserved orthologous set (COSII) nuclear loci to elucidate the phylogenetic relationships among 29 diploid Solanum species in the sister clades that include tomato and potato, and in Datura inoxia as a far outgroup. We screened 40 COSII markers with intron content over 60% that are mapped in different chromosomes; selected a subset of 19 by the presence of single band amplification of size mostly between 600 and 1200 bp; sequenced these 19 COSII markers, and performed phylogenetic analyses with individual and concatenated datasets. The present study attempts to provide a fully resolved phylogeny among the main clades in potato and tomato that can help to identify the appropriate markers for future studies using additional species.     

Comparison of the mitochondrial genome of Nicotiana tabacum with its progenitor species

Summary Mitochondrial DNAs from Nicotiana tabacum, an amphiploid, and its putative progenitor species, N. sylvestris and N. tomentosiformis were compared in structure and organization. By using DNA transfer techniques and cloned fragments of known genes from maize and N. sylvestris as labeled probes, the positions of homologous sequences in restriction digests of the Nicotiana species were analyzed. Results indicate that the mitochondrial DNA of N. tabacum was inherited from N. sylvestris. Conservation in organization and sequence homology between mtDNAs of N. tabacum and the maternal progenitor, N. sylvestris, provide evidence that the mitochondrial genome in these species is evolutionarily stable. Approximately one-third of the probed restriction fragments of N. tomentosiformis mtDNA showed conservation of position with the other two species. Pattern variations indicate that extensive rearrangement of mtDNA has occurred in the evolution of these Nicotiana species.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.     

Genome evolution in allotetraploid Nicotiana

The nuclear cytoplasmic interaction (NCI) hypothesis of genome evolution and speciation in plants states that newly formed allopolyploids pass through a bottleneck of sterility and the fertile plants that emerge are fixed for species‐specific chromosome translocations. These translocations restore fertility and reduce negative effects of the maternal cytoplasm on an alien paternal genome. Using fluorescent in situ hybridization and genomic in situ hybridization and by reviewing published data, we test the NCI hypothesis using three natural Nicotiana allotetraploids (all 2n = 4x = 48, N. arentsii, N. rustica and several genotypes, including a feral plant and cultivars, of N. tabacum (tobacco)). We compare these data with three synthetic tobacco plants (Th37) that are F3 descendent progeny of an allotetraploid formed from ♀N. sylvestris (2n = 24) ×♂N. tomentosiformis (2n = 24). No intergenomic translocations were observed in N. arentsii and N. rustica. An analysis of subtelomeric tandem repeats in these allotetraploids and their putative parents shows minimal genetic changes; those that do occur may reflect evolution in the diploids or the polyploids subsequent to allopolyploidy. All natural N. tabacum genotypes have intergenomic translocations. This may reflect a large ‘genomic‐shock’ generated by allopolyploidy involving widely diverged parental species. Two of three synthetic tobacco plants had a translocation similar to that found in all cultivars of tobacco. This translocation may be significant in tobacco fertility and may have been fixed early in tobacco's evolution. But it is lacking in the feral tobacco, which might indicate a polyphyletic origin or early divergence from all cultivars examined. Overall, only in tobacco is there any evidence that NCI may have influenced genome evolution, and here further data are required to verify chromosome identity. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 599–606.     

Extensive macrosynteny between Medicago truncatula and Lens culinaris ssp. culinaris

The first predominantly gene-based genetic linkage map of lentil (Lens culinaris ssp. culinaris) was constructed using an F₅ population developed from a cross between the cultivars Digger (ILL5722) and Northfield (ILL5588) using 79 intron-targeted amplified polymorphic (ITAP) and 18 genomic simple sequence repeat (SSR) markers. Linkage analysis revealed seven linkage groups (LGs) comprised of 5–25 markers that varied in length from 80.2 to 274.6 cM. The genome map spanned a total length of 928.4 cM. Clear evidence of a simple and direct macrosyntenic relationship between lentil and Medicago truncatula was observed. Sixty-six out of the 71 gene-based markers, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the Lens c. ssp. culinaris and M. truncatula genomes. However, there was evidence of moderate chromosomal rearrangements which may account for the difference in chromosome numbers between these two legume species. Eighteen common SSR markers were used to connect the current map with the most comprehensive and recent map that exists for lentil, providing the syntenic context of four important domestication traits. The composite map presented, anchored with orthologous markers mapped in M. truncatula, provides a strong foundation for the future use of genomic and genetic information in lentil genetic analysis and breeding. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Construction of a high-density SNP genetic map in flue-cured tobacco based on SLAF-seq

Tobacco (Nicotiana tabacum L., 2n = 48) is an important agronomic crop and model plant. Flue-cured tobacco is the most important type and accounts for approximately 80 % of tobacco production worldwide. The low genetic diversity of flue-cured tobacco impedes the construction of a high-density genetic linkage map using simple sequence repeat (SSR) markers and warrants the exploitation of single nucleotide polymorphic (SNP) markers from genomic regions. In this article, initially using specific locus-amplified fragment sequencing, we discovered 10,891 SNPs that were subsequently used as molecular markers for genetic map construction. Combined with SSR markers, a final high-density genetic map was generated containing 4215 SNPs and 194 SSRs distributed on 24 linkage groups (LGs). The genetic map was 2662.43 cM in length, with an average distance of 0.60 cM between adjacent markers. Furthermore, by mapping the SNP markers to the ancestral genomes of Nicotiana tomentosiformis and Nicotiana sylvestris, a large number of genome rearrangements were identified as occurring after the polyploidization event. Finally, using this novel integrated map and mapping population, two major quantitative trait loci (QTLs) were identified for flue-curing and mapped to the LG6 of tobacco. This is the first report of SNP markers and a SNP-based linkage map being developed in tobacco. The high-density genetic map and QTLs related to tobacco curing will support gene/QTL fine mapping, genome sequence assembly and molecular breeding in tobacco.     

The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.)

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2–5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5–14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of Nicotiana tabacum PIN genes identifies NtPIN4 as a key regulator of axillary bud growth

The plant‐specific PIN‐FORMED (PIN) auxin efflux proteins have been well characterized in many plant species, where they are crucial in the regulation of auxin transport in various aspects of plant development. However, little is known about the exact roles of the PIN genes during plant development in Nicotiana species. This study investigated the PIN genes in tobacco (Nicotiana tabacum) and in two ancestral species (Nicotiana sylvestris and Nicotiana tomentosiformis). Genome‐wide analysis of the N. tabacum genome identified 20 genes of the PIN family. An in‐depth phylogenetic analysis of the PIN genes of N. tabacum, N. sylvestris and N. tomentosiformis was conducted. NtPIN4 expression was strongly induced by the application of exogenous indole‐3‐acetic acid (IAA), but was downregulated by the application of ABA, a strigolactone analogue, and cytokinin, as well as by decapitation treatments, suggesting that the NtPIN4 expression level is likely positively regulated by auxin. Expression analysis indicated that NtPIN4 was highly expressed in tobacco stems and shoots, which was further validated through analysis of the activity of the NtPIN4 promoter. We used CRISPR‐Cas9 technology to generate mutants for NtPIN4 and observed that both T₀ and T₁ plants had a significantly increased axillary bud growth phenotype, as compared with the wild‐type plants. Therefore, NtPIN4 offers an opportunity for studying auxin‐dependent branching processes.     

Parental origin of the allotetraploid tobacco Nicotiana benthamiana

Nicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.     

Nuclear DNA,heterochromatin and phylogeny of Nicotiana amphidiploids

There are significant differences in nuclear DNA amount between both diploid and amphidiploid species of Nicotiana. Owing to the higher DNA density in the interphase nuclei of the amphidiploids DNA amounts tend to be underestimated by microdensitometry. After applying necessary corrections to amphidiploid readings it was found that: (1) The nuclear DNA amount in the tetraploid N. rustica is not significantly different from the sum of nuclear DNA amounts in reputed diploid parents, N. undulata and N. paniculata. (2) It is well established that N. sylvestris is one of the diploid progenitors of N. tabacum. The sum of the nuclear DNA amounts in N. sylvestris and N. tomentosiformis is not significantly different from that of the amphidiploid N. tabacum. In contrast the sum of the DNA amounts in N. sylvestris and N. otophora is significantly higher than that in N. tabacum. Observations and measurements of the amount and distribution of heterochromatin in interphase nuclei of the diploid and tetraploid species give further support to the conclusion that N. tomentosiformis rather than N. otophora is the second diploid progenitor of N. tabacum.     

Analysis of an introgressed Nicotiana tomentosa genomic region affecting leaf number and correlated traits in Nicotiana tabacum

Germplasm from closely related diploid relatives of tobacco (Nicotiana tabacum L.) could be of value for continued genetic modification of this species and for mapping quantitative trait loci (QTLs). We examined near isogenic tobacco lines and hybrids differing for an introgressed genomic region from N. tomentosa Ruiz and Pavon designated as Many Leaves that exhibits a large influence on leaf number and correlated traits. Within a ‘Red Russian’ genetic background, the region acted in an additive to partially dominant fashion to delay flowering time, and increase leaf number, plant height, and green leaf yield. Evidence of epistasis was observed as the region affected these traits to varying degrees in diverse near isogenic hybrids. Fifteen amplified fragment length polymorphism (AFLP) markers of N. tomentosa origin were mapped within a single linkage group of 34.5 cM using a population of 207 BC₁F₁ individuals segregating for Many Leaves. Composite interval mapping produced 2–LOD confidence intervals for likely QTL positions influencing leaf number (3.1 cM region), plant height (2.9 cM region), and days to flowering (3.3 cM region). These intervals were overlapping. Results demonstrate that genomic regions with large genetic effects can be transferred to tobacco from closely related diploid relatives, and that sufficient recombination within these regions may permit mapping of genes controlling quantitative traits. Materials and results described here may be useful in future research to gain insight on the genetic control of the transition from vegetative to reproductive development in Nicotiana.     

High resolution synteny maps allowing direct comparisons between the coffee and tomato genomes

Tomato (Solanum lycopersicum) and coffee (Coffea canephora) belong to the sister families Solanaceae and Rubiaceae, respectively. We report herein the mapping of a common set of 257 Conserved Ortholog Set II genes in the genomes of both species. The mapped markers are well distributed across both genomes allowing the first syntenic comparison between species from these two families. The majority (75%) of the synteny blocks are short (<4 cM); however, some extend up to 50 cM. In an effort to further characterize the synteny between these two genomes, we took advantage of the available sequence for the tomato genome to show that tomato chromosome 7 is syntenic to half of the two coffee linkage groups E and F with the putative break point in tomato localized to the boundary of the heterochromatin and euchromatin on the long arm. In addition to the new insight on genome conservation and evolution between the plant families Solanaceae and Rubiaceae, the comparative maps presented herein provide a translational tool by which coffee researchers may take benefit of DNA sequence and genetic information from tomato and vice versa. It is thus expected that these comparative genome information will help to facilitate and expedite genetic and genomic research in coffee.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.