Genomic sequencing combined with marker-assisted breeding effectively eliminates potential linkage drag of a target gene: a case study in tobacco

Linkage drag frequently impedes the utilization of beneficial genes from wild species in crop improvement. The N gene from Nicotiana glutinosa confers strong resistance to tobacco mosaic virus (TMV) but introduces linkage drag when introgressed into cultivated tobacco (Nicotiana tabacum). To address this issue, we sequenced the TMV-resistant flue-cured tobacco line 0970A and carried out comparative genomic analysis. Additionally, we used molecular markers to screen a BC₄F₁ population derived from the cross between 0970A and an elite flue-cured tobacco variety CB-1 (recurrent parent). As a result of sequencing 0970A, the N gene was located at the end of chromosome Nt11. The comparative genomic analysis showed that 0970A inherited approximately 3.74 Mb of N. glutinosa DNA (N-fragment) from its donor, Coker 176. From screening the BC₄F₁ population with molecular markers, a recombinant was identified. This recombinant had a significantly reduced N-fragment (~270 kb), which minimized the linkage drag while still maintaining resistance to TMV. This research indicates that the combination of genome sequencing and marker-assisted breeding can be successfully applied to reduce linkage drag. The findings offer valuable resources for breeding tobacco with resistance to TMV.

1 Introduction

Introducing favorable genes from wild species into cultivated varieties via sexual hybridization is a prevalent practice in plant breeding. Nevertheless, when transferring the target beneficial genes, some linked unfavorable genes may also be introduced, a phenomenon referred to as linkage drag. Hence, eliminating linkage drag (that is, breaking the linkage between favorable and unfavorable genes) stands as a crucial task in leveraging wild germplasm resources for plant breeding (Huang et al., 2022). However, given that it remains unclear which of the linked genes are unfavorable, all the linked genes from wild species are regarded as potentially harmful. Therefore, an effective strategy to alleviate linkage drag is to minimize the genomic segments from wild species that are co-introduced with the target gene. To achieve this, it is necessary to accurately identify the alien fragment introgressed along with the target gene from the wild-species parent (Huang et al., 2022).

Tobacco mosaic virus (TMV) is a single-strand sense RNA virus that causes a common disease in cultivated tobacco (Nicotiana tabacum) known as tobacco mosaic disease, resulting in significant economic losses in tobacco production (Creager, 2022). Developing TMV-resistant tobacco varieties is the most cost-effective and efficient method to manage tobacco mosaic disease. The dominant N gene from Nicotiana glutinosa (Holmes, 1938) is the most commonly used TMV-resistant gene in tobacco breeding due to its strong resistance, simple inheritance, and easy identification, although there are some other genes discovered for TMV resistance in N. rustica sp (Holmes, 1937; Nifong, 2008) and N. repanda (Burk, 1967; Gwynn, 1986; Bates, 1990).

The N gene confers resistance to TMV in plants by initiating a hypersensitivity response, preventing the spread of the virus (Marathe et al., 2002). In 1914, Allard first observed necrotic lesions on the leaves of N. glutinosa infected with TMV. Later, through classical genetic experiments, Holmes (1938) demonstrated that this necrotic response was controlled by a dominant gene, which he named N. Whitham et al. (1994) successfully cloned the N gene by utilizing the insertion mutation caused by the maize Ac transposon. The N protein consists of three domains. The first domain is the N-terminal TIR domain, which shares similarities with the cytoplasmic domain of Toll and interleukin-1 receptor (IL-1R) in drosophila and humans. The second domain is the nucleotide-binding (NB) domain located in the middle. The third domain is the C-terminal leucine-rich repeat (LRR) domain. Therefore, the N gene falls under the R gene classification of the TIR-NB-LRR class (Baker et al., 1997; Marathe et al., 2002).

Different researchers have introduced the N gene into N. tabacum through interspecific hybridization and multi-generation backcross, as documented in several studies (Holmes, 1938; Gerstel, 1943; Mallah, 1943; Gerstel, 1945a, 1946). This genetic modification led to the development and widespread cultivation of TMV-resistant burley tobacco varieties (Chaplin and Mann, 1978; Legg et al., 1979; Lewis et al., 2005). However, the TMV-resistant flue-cured tobacco varieties obtained through the same gene have exhibited reduced yield and quality, failing to gain popularity among farmers. Research indicates that the negative impact of introducing the N gene is attributed to linkage drag rather than the gene’s inherent pleiotropic nature (Chaplin et al., 1961, 1966; Chaplin and Mann, 1978; Legg et al., 1979; Lewis et al., 2007).

With the cloning of the N gene, researchers have explored introducing it into tobacco using transgenic technology to circumvent linkage drag issues (Lewis et al., 2007). Nonetheless, the limited commercial use of transgenic tobacco necessitates reducing or eliminating N gene linkage drag through backcrossing and molecular marker-assisted selection. To effectively address the linkage drag associated with the N gene, understanding the location and size of the foreign fragment carrying the N gene (referred to as N-fragment here for convenience) from N. glutinosa in the N. tabacum genome is essential.

The N-fragment introduced into N. tabacum by Holmes (1938) was found to be integrated on chromosome H (Gerstel, 1945b), but the N-fragments introduced into N. tabacum by other researchers might be integrated on different chromosomes (Lewis et al., 2005). The N. tabacum genome has been sequenced (Sierro et al., 2014; Edwards et al., 2017; Sierro et al., 2024; Wang et al., 2024). However, it is unclear which chromosome corresponds to the chromosome H. In addition, the specific integration position of the N-fragment on the chromosome also remains uncertain.

Several N. tabacum varieties or lines that are resistant to TMV have been developed from the original N-gene introgressed lines. Lewis et al. (2005) utilized AFLP markers to examine 15 TMV-resistant N. tabacum materials and found significant variation in the size of N-fragments among them. Among these materials, Coker 176 and Burley 21 appear to have the smallest N-fragment. Therefore, these two varieties are the optimal candidates as an N gene donor parent for breeding TMV-resistant tobacco. However, the study by Lewis et al. (2005) was unable to determine the precise size of the N-fragment in each material.

Line 0970A is a flue-cured tobacco inbred line resistant to TMV. It was developed by the Institute of Tobacco Science, Fujian Tobacco Monopoly Administration, China, with Coker 176 serving as the source of the N gene. This line functions as a donor parent for TMV resistance in flue-cured tobacco breeding. Cuibi-1 (abbreviated as CB-1) is a major elite cultivated variety of flue-cured tobacco in Fujian province, China. However, it is highly susceptible to TMV. To enhance its resistance, introducing the N gene into CB-1 has become a key objective in its genetic improvement. For this purpose, we used 0970A as the donor parent and CB-1 as the recurrent parent, and incorporated the N gene through successive backcrossing. Nevertheless, as with other TMV resistance breeding programs in flue-cured tobacco, this effort also faces the challenge of minimizing the undesirable linkage drag associated with the N gene.

In this study, through genome sequencing and marker-assisted breeding techniques, we accurately determined the location and size of the N-fragment within the 0970A genome, along with the genes it encompasses, and significantly minimized the potential linkage drag when introducing the N gene from 0970A into the genetic background of CB-1. Our study successfully demonstrated the efficacy of this approach, presenting a compelling example of the viability of integrating genome sequencing and marker-assisted breeding to reduce or even completely eliminate linkage drag. The data and TMV-resistant lines acquired in this study will facilitate the efficient utilization of the N gene in TMV-resistance breeding programs and assist in circumventing linkage drag issues in tobacco cultivation.

2 Materials and methods

2.1 Plant materials

Four plant materials were utilized in this study, including N. glutinosa (the primary source of N gene), Coker 176 (a flue-cured tobacco variety possessing the N gene), 0970A (a flue-cured tobacco inbred line possessing the N gene inherited from Coker 176), and CB-1 (a TMV-susceptible elite flue-cured tobacco variety, and one of the parents of 0970A).

2.2 In vitro identification for TMV resistance

To enhance the efficiency of TMV resistance identification and to avoid the impact of TMV inoculation on the growth of tobacco plants, we employed an in vitro identification method (Yin et al., 2023) in this study. Diseased tobacco leaves were collected, ground, and filtered to prepare a TMV solution. At the seven-leaf stage of the tobacco seedlings, one well-grown leaf from each plant was selected. A toothbrush was used to apply the TMV solution to the surface of the leaf, which was then placed in a self-sealing bag and incubated in a growth chamber at 26 °C with a 12-hour light and 12-hour dark cycle. After 72 hours, the leaf conditions were observed. Leaves containing the N gene (resistant to TMV) would form immune spots, while those without the N gene (susceptible to TMV) would not form such spots (Yin et al., 2023).

2.3 Sequencing and assembly of 0970A genome

The genome of 0970A was sequenced and assembled by Berry Genomics (Beijing). Genomic DNA was extracted from approximately 300 mg of fresh leaf tissue using the CTAB method. DNA quality was assessed using a NanoDrop™ ND-2000 spectrophotometer (Thermo Fisher Scientific), a Qubit™ fluorometer (Invitrogen, Q33238), and the CHEF Mapper™ XA Chiller System (Bio-Rad, 1703672). A high-fidelity (HiFi) library was prepared with the SMRTbell^® Prep Kit 3.0 (Pacific Biosciences, 102-182-700) and sequenced on the PacBio Revio^® system (Pacific Biosciences, 102-090-600). The raw reads were processed with SMRT software (Ardui et al., 2018) to generate HiFi reads, which were then assembled using Hifiasm (Cheng et al., 2021). The resulting contigs were subsequently deduplicated using Purge dups (Guan et al., 2020). Contig quality and assembly completeness were assessed with Quast (Mikheenko et al., 2018) and BUSCO (Manni et al., 2021). All software was run using default parameters.

2.4 Identification, location, and annotation of N-contig

The N gene sequence (gene ID LOC107824161) was obtained from the public database GenBank. This sequence was then compared with all the contigs of 0970A through the BLAST comparison tool to identify the contig containing the N gene (termed N-contig). The collinearity between the N-contig and the CB-1 genome (unpublished) was analyzed to identify the location of the N-contig in the tobacco genome using Minimap2 software under default parameter -x asm20 (Li, 2018). Annotation of the N-contig was achieved by annotating all contigs. Initially, repetitive sequence elements within all contigs were identified using RepeatModeler v2.0.5 software (Flynn et al., 2020) and subsequently masked using RepeatMasker v4.1.2.p1–1 software (Tempel, 2012). Three strategies were then employed to predict protein-coding genes. The first strategy involved transcriptome-based prediction, utilizing the tobacco transcriptomes (NCBI accession numbers: SRR1199073, SRR1199122, SRR1199197, SRR1199202) and hisat2 v2.2.1–6 software (https://daehwankimlab.github.io/hisat2). The second strategy was homology-based prediction, leveraging the annotation sets from tomato (GCF_036512215.1), tobacco TN90 (GCA_000715135.1), and chili pepper (GCA_002878395.3). The third strategy was de novo prediction, employing GeneMark-ETP v1 software (Brüna et al., 2020) and AUGUSTUS v3.3.3 (Stanke et al., 2004). These results were integrated to generate an initial annotation file using Braker3 v3.0.8–0 software (Gabriel et al., 2024) and further refined using TSEBRA v1.2.5 software (Gabriel et al., 2021), resulting in the final protein annotation file.

2.5 Homology analysis of N-contig

The genomic DNA of CB-1, Coker 176, and N. glutinosa was extracted using the Plant Genomic DNA Kit (TIANGEN, Beijing) and the three DNA samples were subsequently sent to Berry Genomics (Beijing) for deep sequencing (at least 80 Gb each in depth) on the DNBSEQ-T7 platform. For each sample, adapter-trimmed reads were aligned to all contigs of 0970A using BWA-MEM2 v2.2.1 (Vasimuddin et al., 2019) under default parameters, with the exception of the -k option (minimum seed length), which was increased from 19 to 30 to enhance mapping stringency. The average depth of reads mapped to the N-contig was calculated every 10 kb using Samtools v1.19 (Li et al., 2009).

2.6 Creation of recombinant lines with reduced linkage drag of the N gene

To introduce the N gene from 0970A into CB-1 and disrupt and eliminate the linkage drag associated with the N gene, we crossed 0970A with CB-1 and performed consecutive backcrosses up to the BC₄F₁ generation. In each generation, we selected a small number of plants that were morphologically most similar to CB-1 but retained the N gene and backcrossed them with CB-1. In the BC₄F₁ generation, we established a large population. We then employed a molecular marker (P371) developed based on the sequencing data to screen TMV-resistant plants for chromosomal recombination events within the target region (the linkage drag of the N gene from N. glutinosa). Such recombination events would lead to the partial elimination of the linkage drag. For clarity, we designated TMV-resistant plants containing the target recombination events as “recombinant plants,” while those without such events were termed “non-recombinant plants.” The identified recombinant plants were self-pollinated to produce the corresponding BC₄F₂ lines. Within these BC₄F₂ lines, we used a linked molecular marker (NR5) developed based on the sequencing data to identify plants with a homozygous N-gene genotype. These plants were further self-pollinated to obtain the corresponding N-gene homozygous lines, which are the recombinant lines with reduced linkage drag wanted. The above procedure is illustrated in Figure 1.

Figure 1

Figure 1. Procedure of introducing the N gene from 0970A into CB-1 with reduced linkage drag. SPs, selected plants resistant to TMV and most similar to the recurrent parent CB-1 in morphology. The selection was performed according to the phenotypes of the plants. MAS, marker-assisted selection. RPs, recombinant plants with heterozygous genotype at the N locus (Nn). HRPs, recombinant plants with homozygous genotype (NN). HRLs, recombinant lines with homozygous genotype (NN).

2.7 Molecular analysis of recombinant lines

To precisely determine the locations of recombination events occurred within the target region, the “homology analysis of N-contig” approach described earlier was applied to the identified recombinant lines. Specifically, each recombinant line was subjected to deep sequencing (approximately 40 Gb in depth), and the resulting reads were mapped to all contigs of 0970A. In addition, a non-recombinant line was analyzed in the same manner for comparative purposes.

3 Results

3.1 Sequencing and assembly of 0970A genome

A total of 6,892,630 HiFi reads were obtained from sequencing the genome of 0970A, with an average read length of 17.3 kb, a read N50 of 17,254 bp, and an average sequencing depth of 29 times the genome. These HiFi reads were then assembled and redundancies were eliminated, resulting in 554 contigs. Among these contigs, the N₅₀ length was 81,799,233 bp, and the N₇₅ length was 53,933,392 bp. Notably, approximately 80 contigs comprehensively covered the entire genome. The assessment using BUSCO revealed that the assembled genome sequence exhibited an impressive 99% completeness, underscoring the exceptional quality of the sequencing and assembly process.

3.2 Identification, annotation, and chromosomal location of N-contig

Upon aligning the N gene sequence with all contigs of 0970A, it was determined that the N gene resided on contig PTG000094L, hereafter referred to as 094L (the detailed information of 094L, including full sequence and predicted genes, can be found at https://ngdc.cncb.ac.cn/genbase/search/gb/C_AA098042). The length of the N gene sequence used as the query was 7,345 bp, exhibiting a perfect match with the target sequence (E-value = 0, Score = 13,564). Notably, one end of contig 094L was identified as a telomere sequence with a repeat unit of TTTAGGG, suggesting that the contig represented a chromosomal terminus. This contig spanned 6.47 Mb in length, with the N gene located at 2.94 Mb from the non-telomere end. For convenience, we hereafter call the sequence of the contig 094L from the non-telomere end to the N gene and that from the telomere end to the N gene as the left side of N gene (LSN) and right side of N gene (RSN), respectively (Figure 2A).

Figure 2

Figure 2. Contig 094L and its collinearity with the Nt11 chromosome terminus of CB-1. (A) Schematic diagram of 094L showing the position of the N gene as well as the LSN and RSN regions. The green rectangular represents telomere. The red and blue arrowheads indicate the positions of markers P371 and NR5, respectively. (B) Collinearity comparison between 094L and the Nt11 terminus of CB-1. The identity scale is on the right. The red color indicates complete identity but in opposite direction. The vertical broken line indicates the position of the N gene.

A total of 167 genes were predicted on contig 094L, with 62 on the LSN and 104 on the RSN (Supplementary Table S1). Interestingly, within the LSN, three genes were also annotated to encode the TMV resistance protein N, namely, ptg94lG0000310.t1 (abbreviated as G31) located at 1.94 Mb, ptg94lG0000390.t1 (G39) at 2.26 Mb, and ptg94lG0000440.t1 (G44) at 2.43 Mb (Supplementary Table S1). However, they all were shorter than the full-length of the N gene sequence, measuring 6,419 bp (G31), 4,732 bp (G39), and 7,279 bp (G44), respectively, suggesting that they are only homologs of the N gene.

Since 094L was identified as the terminal sequence of a chromosome, we tried to locate the N gene on the chromosome by conducting collinearity analysis between 094L and the terminal sequences of all chromosomes of CB-1. The results revealed a significant collinearity between a segment of 094L and chromosome 11, referred to as Nt11 in accordance with the established chromosome numbering system of the K326 genome sequence (Figure 2B), suggesting that the N gene must be located on this chromosome. It is important to highlight that the collinearity between 094L and the corresponding CB-1 genome sequence is not consistent throughout. Specifically, the LSN exhibited a good collinearity with the CB-1 sequence, whereas the RSN displayed no collinearity with the CB-1 sequence (Figure 2B), implying that the RSN might not originate from N. tabacum.

3.3 Homology analysis of the sequences flanking the N gene

To precisely determine the homology of the sequences flanking the N gene, we performed deep sequencing of the genomes of CB-1, Coker 176, and N. glutinosa. We then mapped their reads to all contigs of 0970A and analyzed the mapping results for 094L (the reads of CB-1, Coker 176, and N. glutinosa mapped to 094L can be found at https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA030530). The reads from Coker 176 uniformly covered the entire 094L with an average depth of approximately 20 (Figure 3A), indicating that the whole 094L is homologous to Coker 176. This finding aligns with the fact that Coker 176 is the donor parent of the N gene in 0970A. In contrast, the reads from CB-1 were predominantly mapped to the LSN and sparsely to the RSN (Figure 3B), suggesting that the LSN is homologous to CB-1. This result is consistent with the collinearity analysis (Figure 2B). Notably, the reads from CB-1 were evenly distributed across the LSN with an average depth of about 45, except for a small region (spanning from 1.55 Mb to 1.70 Mb) where the depth was significantly lower (Figure 3B). This indicates a structural variation between CB-1 and 0970A in that region, which the collinearity analysis suggests is an insertion in 0970A (Figure 2B). Conversely, the reads from N. glutinosa were primarily mapped to the RSN and barely to the LSN (Figure 3C), suggesting that the RSN is homologous to N. glutinosa. It is noteworthy that the depth distribution of N. glutinosa reads on the RSN was highly uneven. Although there was an overall depth plateau at around 30, reflecting the average sequencing depth of the N. glutinosa genome, there were many locations with extremely high depths (over 150), especially in the region beyond 4.92 Mb, where these high depths appeared almost continuously. The extremely high depths likely resulted from the presence of multiple copies of sequences dispersed throughout the N. glutinosa genome. Since these reads of multiple copies could not be mapped to other contigs of 0970A, they were exclusively mapped to the RSN, leading to the observed high depths.

Figure 3

Figure 3. Distributions of deep sequencing reads of Coker 176 (A), CB-1 (B), and N. glutinosa (C) mapped to the contig 094L. The black arrow indicates the position of the N gene.

3.4 Obtainment of a recombinant plant and its progeny homozygous lines

The results presented above demonstrate that the linkage drag associated with the N gene from N. glutinosa in 0970A (as well as in Coker 176) is mainly restricted to the RSN of contig 094L. To facilitate removing the associated linkage drag while introducing the N gene from 0970A into CB-1, we developed a PCR-based molecular marker, designated as P371, located approximately 3.46 Mb from the N gene at the RSN end (close to the telomere) of contig 094L (Figure 2A). The P371 marker (forward primer: 5’-CTAAAAACGGGTGAAGGGGT-3’; reverse primer: 5’-ACATTCGGAGGTCAGGTCAAT-3’) is a dominant marker that produces a specific 162 bp amplification product in the donor parent 0970A but not in the recipient parent CB-1. Using the in vitro identification method, we identified 4,762 TMV-resistant plants (carrying the N gene) from a population of 10,013 BC₄F₁ plants. All resistant plants were subsequently genotyped using the P371 marker, and one plant was found to lack the specific amplification product (Supplementary Figure S1). This finding indicates a recombination event between the P371 marker and the N gene, suggesting that a portion of the linkage drag, including the telomere, has been replaced with the corresponding segment from CB-1 in this plant. Therefore, this is a desired recombinant plant.

To identify individuals with a homozygous genotype for the N gene, we developed an SSR marker, designated as NR5, located approximately 740 kb from the N gene on the LSN of contig 094L (Figure 2A). The NR5 marker (forward primer: 5’-GGTCTATGTGCACCCTACCCT-3’; reverse primer: 5’-TTGGTTACGGCACGTAATGTT-3’) produces a 200 bp amplification product in 0970A and a shorter amplification product in CB-1 (Supplementary Figure S2). Since NR5 is closely linked to the N gene, individuals exhibiting only the 0970A-specific band are likely to be homozygous for the N gene. Using the NR5 marker, we screened 30 progeny plants derived from self-pollination of the recombinant plant and identified 8 plants with a homozygous genotype for the N gene. These 8 plants were further confirmed to carry the N gene using the N gene-specific marker N1+N2 (forward primer N1: 5’-CGTCGACACATTATGCCATC-3’; reverse primer N2: 5’-GAGGGGTCTTACCCCATTGT-3’) (Lewis et al., 2005). Self-pollination of these plants yielded 8 recombinant lines with a homozygous genotype for the N gene. Using a similar approach, we also established 3 non-recombinant control lines (homozygous for the N gene) from a single non-recombinant plant.

3.5 Molecular characterization of recombinant lines

To precisely characterize the recombination events in the target region of the recombinant lines, we conducted deep sequencing analysis on 3 recombinant lines and 1 non-recombinant line (the reads of these lines mapped to 094L can be found at https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA030530). As expected, the non-recombinant line retained the complete 094L sequence (Figure 4A), whereas the 3 recombinant lines exhibited extensive replacement of the RSN sequence, with the crossover point located approximately at the position of 3 Mb, only ~60 kb away from the N gene (Figures 4B-D). This finding indicates that the residual linkage drag associated with the N gene in the recombinant lines is minimal.

Figure 4

Figure 4. Distributions of deep sequencing reads of a non-recombinant line (A) and three recombinant lines (B-D) mapped to the contig 094L. The black arrow indicates the position of the N gene.

Furthermore, based on SNP markers revealed by deep sequencing, we found that the 3 recombinant lines contained 97.85%-98.72% of the genetic components from the recipient parent CB-1 (Supplementary Table S2), indicating a high degree of genetic background restoration. However, the non-recombinant line had a comparatively lower restoration rate of the CB-1 genetic background, containing only 93.42% of the genetic components from CB-1 (Supplementary Table S2).

4 Discussion

This study demonstrates that the N gene, introgressed from N. glutinosa into N. tabacum, is localized to the distal region of chromosome Nt11 in the N. tabacum genome, approximately 3.53 Mb from the telomere (Figure 2A; Supplementary Table S1). Homology analysis confirmed that 0970A has fully retained the N gene and its flanking sequences from its parent, Coker 176 (Figure 3A), accurately reflecting the N-fragment in Coker 176. By mapping deep sequencing reads from CB-1 and N. glutinosa to contig 094L, we identified that the sequences flanking the N gene in 0970A/Coker 176 are predominantly derived from N. tabacum on the left side and N. glutinosa on the right side, with the boundary between these two species’ sequences lying at approximately 2.73 Mb, slightly upstream of the N gene’s position at 2.94 Mb (Figures 3B, C). This indicates that the N-fragment from N. glutinosa retained in 0970A/Coker 176 spans approximately 3.74 Mb (Figure 3C), marking the site of genetic exchange during the breeding of Coker 176.

This study identified that contig 094L not only carries the N gene but also contains three N-like genes within its LSN region (a segment derived from N. tabacum) (Supplementary Table S1). Given that R genes can evolve into new R genes through tandem duplication (Panchy et al., 2016), it is plausible that these N-like genes may have originated from the N gene. To explore this possibility, we used Liftoff software (Shumate and Salzberg, 2021) to project the gene annotations from contig 094L to the corresponding region in the CB-1 genome and performed comparative sequence analysis. The results revealed that all three N-like genes are also present in CB-1 (Supplementary Table S3), suggesting that they are native to N. tabacum rather than derived from the N gene. Interestingly, sequence alignment of the N gene itself identified a homologous sequence at the corresponding locus in CB-1, although with relatively low sequence identity (Supplementary Table S3). The biological significance of this observation remains unclear.

A previous study by Lewis et al. (2005) identified two distinct types of N. tabacum germplasm carrying the N gene: one with the N gene located on chromosome H and the other on an unidentified chromosome. The pioneering variety Holmes Samsoun, which introduced the N gene from N. glutinosa, belongs to the first type, as does Coker 176 (Lewis et al., 2005). Based on the sequence composition of contig 094L, we can infer that the N gene is positioned 3.53 Mb from the end of a chromosome in N. glutinosa, and the N-fragment was integrated into chromosome H in Holmes Samsoun through a simple or reciprocal translocation. This suggests that the initial N-fragment introgressed from N. glutinosa was likely substantial. Due to limited genetic lineage data, it remains unclear whether the second type of germplasm also originated from Holmes Samsoun. Although chromosome H was initially identified using monosomic material (Clausen and Cameron, 1944), its correspondence in the sequenced tobacco genome was previously unknown. This study clarifies that chromosome H corresponds to chromosome Nt11.

Lewis et al. (2005) identified 168 N. glutinosa-specific AFLP markers linked to the N gene in Holmes Samsoun. Their analysis of 15 N-gene-carrying N. tabacum varieties (8 of the first type and 7 of the second) revealed that all varieties retained some of these markers, albeit in significantly reduced numbers (3 to 17 markers) compared to Holmes Samsoun. This reduction indicates that linkage drag from N. glutinosa was substantially diminished after multiple backcross generations. Notably, linkage drag was less pronounced in the first type compared to the second. Among the varieties, Coker 176 and another first-type variety, Burley 21, exhibited the least linkage drag, retaining only 3 specific markers. Our study further reveals that the N-fragment in 0970A/Coker 176 spans only 3.74 Mb, with linkage drag primarily persisting on the right side of the N gene and nearly eliminated (only ~210 kb remaining) on the left side (Figure 2C). This suggests that the N-fragment in Coker 176 is among the smallest achievable through traditional breeding methods based on phenotypic selection, making Coker 176 an optimal donor parent for TMV-resistance breeding in tobacco.

However, a recent study by Liu et al. (2023) reported that the N-fragment from Coker 176 can increase chlorophyll content and leaf thickness, leading to slower yellowing in the field, greater difficulty in curing, and reduced yield and output value. This indicates that the N-fragment from Coker 176 may still carry undesirable effects. Our study predicts that the N-fragment in 0970A/Coker 176 harbors up to 117 genes from N. glutinosa, including the N gene (Supplementary Table S1). These genes encompass a wide range of functions, suggesting that the N-fragment in Coker 176 has the potential for negative impacts. Notably, within the contig 094L, the gene density on the N. glutinosa sequence (117/2.74 = 31.3 genes/Mb) is approximately 70% higher than that on the N. tabacum sequence (50/2.73 = 18.3 genes/Mb) (Supplementary Table S1). Given the high gene density in the N-fragment, minimizing potential linkage drag is critical to mitigating its adverse effects.

In this study, we introgressed the N gene from 0970A into CB-1 through backcross breeding, successfully eliminating 92.8% (3.47/3.74 × 100%) of the N-fragment from 0970A/Coker 176. This resulted in TMV-resistant near-isogenic lines of CB-1 with only ~270 kb (7.2%) of the N-fragment remaining. The residual segment contains 17 predicted genes, apart from the N gene (Supplementary Table S1). Interestingly, approximately 70% of these genes belong to the auxin-responsive protein SAUR (small auxin-up RNA) gene family, which is implicated in cell elongation and may also play roles in processes such as leaf senescence and cell division (Stortenbeker and Bemer, 2019). Additionally, SAUR genes are regulated by plant hormones such as ABA and JA, in addition to auxin (Stortenbeker and Bemer, 2019). Whether these genes in the residual N-fragment negatively impact flue-cured tobacco production remains to be investigated. The near-isogenic lines of CB-1 developed in this study provide an excellent resource to address this question.

To our knowledge, the TMV-resistant near-isogenic lines of CB-1 developed in this study represent the tobacco materials with the least residual N-fragment. These lines not only facilitate the breeding of TMV-resistant CB-1 without the undesirable effects of linkage drag but also serve as ideal N-gene donors for TMV resistance breeding in tobacco. Furthermore, this study provides a successful example of using genomic techniques combined with marker-assisted breeding to precisely analyze and minimize introgressed genomic fragments from wild species into cultivated species through distant hybridization.

5 Conclusion

In this study, we successfully addressed the challenge of linkage drag associated with the N gene in cultivated tobacco by combining advanced genome sequencing and marker-assisted breeding strategies. Through comprehensive genomic analysis, we pinpointed the N gene’s location and identified a 3.74 Mb introgressed fragment from N. glutinosa in the resistant line 0970A. By screening a large BC₄F₁ population, we obtained a recombinant plant with a drastically reduced N-fragment (~270 kb), significantly minimizing potential linkage drag while retaining robust TMV resistance. Our findings underscore the effectiveness of genomic tools in accelerating precision breeding and highlight the potential for applying similar approaches to other crop improvement programs. The developed TMV-resistant lines and molecular markers provide valuable resources for future tobacco breeding, paving the way for more efficient utilization of wild germplasm in enhancing crop resilience.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Author contributions

WG: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing – original draft, Writing – review & editing. WY: Data curation, Investigation, Writing – original draft. JiL: Data curation, Software, Writing – original draft. ZZ: Data curation, Investigation, Writing – original draft. NY: Data curation, Investigation, Writing – original draft. JuL: Data curation, Investigation, Writing – original draft. YC: Data curation, Investigation, Writing – original draft. LH: Investigation, Software, Writing – original draft. CL: Project administration, Supervision, Writing – review & editing. SC: Project administration, Supervision, Writing – review & editing. WW: Conceptualization, Writing – review & editing. SW: Conceptualization, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research and/or publication of this article. This work was supported by Fujian Corporation of China National Tobacco Corporation. The grant number is 2019350000240136.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors declare that this study received funding from Fujian Corporation of China National Tobacco Corporation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1666106/full#supplementary-material

References

Ardui, S., Ameur, A., Vermeesch, J. R., and Hestand, M. S. (2018). Single molecule real-time (smrt) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res. 46, 2159–2168. doi: 10.1093/nar/gky066

PubMed Abstract | Crossref Full Text | Google Scholar

Baker, B., Zambryski, P., Staskawicz, B., and Dinesh-Kumar, S. P. (1997). Signaling in plant-microbe interactions. Science 276, 726–733. doi: 10.1126/science.276.5313.726

PubMed Abstract | Crossref Full Text | Google Scholar

Bates, G. W. (1990). Asymmetric hybridization between Nicotiana tabacum and N. Repanda by donor recipient protoplast fusion: transfer of TMV resistance. Theor. Appl. Genet. 80, 481–487. doi: 10.1007/BF00226749

PubMed Abstract | Crossref Full Text | Google Scholar

Brüna, T., Lomsadze, A., and Borodovsky, M. (2020). Genemark-Ep+: Eukaryotic gene prediction with self-training in the space of genes and proteins. NAR Genomics Bioinf. 2, 1–14. doi: 10.1093/nargab/lqaa026

PubMed Abstract | Crossref Full Text | Google Scholar

Burk, L. G. (1967). An interspecific bridge-cross– Nicotiana repanda through N. Sylvestris to N. Tabacum. J. Hered. 58, 215–218. doi: 10.1093/jhered/58.4.215

Crossref Full Text | Google Scholar

Chaplin, J. F. and Mann, T. J. (1978). Evaluation of tobacco mosaic resistance factor transferred from burley to flue-cured tobacco. J. Hered. 69, 175–178. doi: 10.1093/jhered/69.3.175

Crossref Full Text | Google Scholar

Chaplin, J. F., Mann, T. J., and Apple, J. L. (1961). Some effects of the Nicotiana glutinosa type of mosaic resistance on agronomic characters of flue-cured tobacco. Tobacco Sci. 5, 80–83.

Google Scholar

Chaplin, J. F., Matzinger, D. F., and Mann, T. J. (1966). Influence of the homozygous and heterozygous mosaic-resistance factor on quantitative character of flue-cured tobacco. Tobacco Sci. 10, 81–84.

Google Scholar

Cheng, H., Concepcion, G. T., Feng, X., Zhang, H., and Li, H. (2021). Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat. Methods 18, 170–175. doi: 10.1038/s41592-020-01056-5

PubMed Abstract | Crossref Full Text | Google Scholar

Clausen, R. E. and Cameron, D. R. (1944). Inheritance in Nicotiana tabacum. Xviii. Monosomic analysis. Genetics 29, 447–477. doi: 10.1093/genetics/29.5.447

PubMed Abstract | Crossref Full Text | Google Scholar

Creager, A. (2022). Tobacco mosaic virus and the history of molecular biology. Annu. Rev. Virol. 9, 39–55. doi: 10.1146/annurev-virology-100520-014520

PubMed Abstract | Crossref Full Text | Google Scholar

Edwards, K. D., Fernandez-Pozo, N., Drake-Stowe, K., Humphry, M., Evans, A. D., Bombarely, A., et al. (2017). A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448. doi: 10.1186/s12864-017-3791-6

PubMed Abstract | Crossref Full Text | Google Scholar

Flynn, J. M., Hubley, R., and Goubert, C. (2020). Repeatmodeler2 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci. U. S. A. 117, 9451–9457. doi: 10.1073/pnas.1921046117/-/DCSupplemental

PubMed Abstract | Crossref Full Text | Google Scholar

Gabriel, L., Brůna, T., and Hoff, K. J. (2024). Braker3: fully automated genome annotation using RNA-seq and protein evidence with genemark-etp, augustus and tsebra. Genome Res. 34, 769–777. doi: 10.1101/2023.06.10.544449

PubMed Abstract | Crossref Full Text | Google Scholar

Gabriel, L., Hoff, K. J., and Brůna, T. (2021). Tsebra: transcript selector for braker. BMC Bioinf. 22, 566. doi: 10.1186/s12859-021-04482-0

PubMed Abstract | Crossref Full Text | Google Scholar

Gerstel, D. U. (1943). Inheritance in Nicotiana tabacum. Xvii. Cytogenetical analysis of glutinosa-type resistance to mosaic disease. Genetics 28, 533–536. doi: 10.1093/genetics/28.6.533

PubMed Abstract | Crossref Full Text | Google Scholar

Gerstel, D. U. (1945a). Inheritance in Nicotiana tabacum. Xx. The addition of Nicotiana glutinosa chromosomes to tobacco. J. Hered. 36, 197–206. doi: 10.1093/oxfordjournals.jhered.a105500

Crossref Full Text | Google Scholar

Gerstel, D. U. (1945b). Inheritance in Nicotiana tabacum. Xix. Identification of the tabacum chromosome replaced by one from N. glutinosa in mosaic-resistant Holmes Samsoun tobacco. Genetics 30, 448–454. doi: 10.1093/genetics/30.5.448

PubMed Abstract | Crossref Full Text | Google Scholar

Gerstel, D. U. (1946). Inheritance in Nicotiana tabacum. Xxi. The mechanism of chromosome substitution. Genetics 31, 421–427. doi: 10.1093/genetics/31.4.421

PubMed Abstract | Crossref Full Text | Google Scholar

Guan, D., McCarthy, S. A., Wood, J., Howe, K., Wang, Y., and Durbin, R.. (2020). Identifying and removing haplotypic duplication in primary genome assemblies. Bioinformatics 36, 2896–2898. doi: 10.1093/bioinformatics/btaa025

PubMed Abstract | Crossref Full Text | Google Scholar

Gwynn, G. R. B. K. (1986). Genetic resistance to tobacco mosaic virus, cyst nematodes, root-knot nematodes, and wildfire from Nicotiana repanda incorporated into N. tabacum. Plant Dis. 70, 958–962. doi: 10.1094/PD-70-958

Crossref Full Text | Google Scholar

Holmes, F. O. (1937). Genes affecting response of Nicotiana tabacum hybrids to tobacco mosaic virus. Science 85, 104–105. doi: 10.1126/science.85.2195.104

PubMed Abstract | Crossref Full Text | Google Scholar

Holmes, F. O. (1938). Inheritance of resistance to tobacco mosaic disease in tobacco. Phytopathology 28, 553–561.

Google Scholar

Huang, X., Huang, S., Han, B., and Li, J. (2022). The integrated genomics of crop domestication and breeding. Cell 185, 2828–2839. doi: 10.1016/j.cell.2022.04.036

PubMed Abstract | Crossref Full Text | Google Scholar

Legg, P. D., Collins, G. B., and XXXC, L. C. (1979). Effects of the N mosaic-resistance factor on agronomic and chemical traits in burley tobacco. Crop Sci. 19, 455–457. doi: 10.2135/cropsci1979.0011183X001900040007x

Crossref Full Text | Google Scholar

Lewis, R. S., Linger, L. R., Wolff, M. F., and Wernsman, E. A. (2007). The negative influence of N-mediated TMV resistance on yield in tobacco: linkage drag versus pleiotropy. Theor. Appl. Genet. 115, 169–178. doi: 10.1007/s00122-007-0552-y

PubMed Abstract | Crossref Full Text | Google Scholar

Lewis, R. S., Milla, S. R., and XXXS, L. J. (2005). Molecular and genetic characterization of Nicotiana glutinosa l. Chromosome segments in tobacco mosaic virus-resistant tobacco accessions. Crop Sci. 45, 2355–2362. doi: 10.2135/cropsci2005.0121

Crossref Full Text | Google Scholar

Li, H. (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100. doi: 10.1093/bioinformatics/bty191

PubMed Abstract | Crossref Full Text | Google Scholar

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., and Homer, N.. (2009). The sequence alignment/map format and samtools. Bioinformatics 25, 2078–2079. doi: 10.1093/bioinformatics/btp352

PubMed Abstract | Crossref Full Text | Google Scholar

Liu, Y., Yu, H. Q., Yuan, C., Zeng, J. M., and Huang, C. J. (2023). Linkage drag field phenotype analysis of flue-cured tobacco with TMV resistant N introgression fragments. Mol. Plant Breed. 21, 7105–7112. doi: 10.13271/j.mpb.021.007105

Crossref Full Text | Google Scholar

Mallah, G. S. (1943). Inheritance in Nicotiana tabacum. Xvi. Structural differences among the chromosomes of a selected group of varieties. Genetics 28, 525–532. doi: 10.1093/genetics/28.6.525

PubMed Abstract | Crossref Full Text | Google Scholar

Manni, M., Berkeley, M. R., Seppey, M., Simao, F. A., and Zdobnov, E. M. (2021). Busco update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 38, 4647–4654. doi: 10.1093/molbev/msab199

PubMed Abstract | Crossref Full Text | Google Scholar

Marathe, R., Anandalakshmi, R., Liu, Y., and SP., D. (2002). The tobacco mosaic virus resistance gene, N. Mol. Plant Pathol. 3, 167–172. doi: 10.1046/j.1364-3703.2002.00110.x

PubMed Abstract | Crossref Full Text | Google Scholar

Mikheenko, A., Prjibelski, A., Saveliev, V., Antipov, D., and Gurevich, A. (2018). Versatile genome assembly evaluation with quast-lg. Bioinformatics 34, i142–i150. doi: 10.1093/bioinformatics/bty266

PubMed Abstract | Crossref Full Text | Google Scholar

Nifong, J. M. (2008). Assessment of the diversity and disease resistance properties within a collection of nicotiana rustica (North Carolina: North Carolina State University).

Google Scholar

Panchy, N., Lehti-Shiu, M., and Shiu, S.-H. (2016). Evolution of gene duplication in plants. Plant Physiol. 171, 2294–2316. doi: 10.1104/pp.16.00523

PubMed Abstract | Crossref Full Text | Google Scholar

Shumate, A. and Salzberg, S. (2021). Liftoff: accurate mapping of gene annotations. Bioinformatics 37, 1639–1643. doi: 10.1093/bioinformatics/btaa1016

PubMed Abstract | Crossref Full Text | Google Scholar

Sierro, N., Auberson, M., Dulize, R., and Ivanov, N. V. (2024). Chromosome-level genome assemblies of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana tomentosiformis. Sci. Data 11, 135. doi: 10.1038/s41597-024-02965-2

PubMed Abstract | Crossref Full Text | Google Scholar

Sierro, N., Battey, J. N., Ouadi, S., Bakaher, N., Bovet, L., Willig, A., et al. (2014). The tobacco genome sequence and its comparison with those of tomato and potato. Nature Commun. 5, 3833. doi: 10.1038/ncomms4833

PubMed Abstract | Crossref Full Text | Google Scholar

Stanke, M., Steinkamp, R., and Waack, S. (2004). Augustus: a web server for gene finding in eukaryotes. Nucleic Acids Res. 2, lqaa26. doi: 10.1093/nargab/lqaa026

PubMed Abstract | Crossref Full Text | Google Scholar

Stortenbeker, N. and Bemer, M. (2019). The saur gene family: the plant’s toolbox for adaptation of growth and development. J. Exp. Bot. 70, 17–27. doi: 10.1093/jxb/ery332

PubMed Abstract | Crossref Full Text | Google Scholar

Tempel, S. (2012). Using and understanding repeatmasker. Methods Mol. Biol. 859, 29–51. doi: 10.1007/978-1-61779-603-6_2

PubMed Abstract | Crossref Full Text | Google Scholar

Vasimuddin, M. d., Misra, S., and Li, H. (2019). “Efficient architecture-aware acceleration of bwa-mem for multicore systems,” in 2019 Ieee International Parallel and Distributed Processing Symposium (Ipdps) (IPDPS, Rio de Janeiro, Brazil). 314–324. doi: 10.1109/IPDPS.2019.00041

Crossref Full Text | Google Scholar

Wang, X., Shen, Z., Li, C., Bai, Y., Li, Y., Zhang, W., et al. (2024). Fine mapping and identification of two nttom2a homeologs responsible for tobacco mosaic virus replication in tobacco (Nicotiana tabacum L.). BMC Plant Biol. 24, 67. doi: 10.1186/s12870-024-04744-y

PubMed Abstract | Crossref Full Text | Google Scholar

Whitham, S., Dinesh-Kumar, S. P., Choi, D., Hehl, R., Corr, C., and Baker, B. (1994). The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor. Cell 78, 1101–1115. doi: 10.1016/0092-8674(94)90283-6

PubMed Abstract | Crossref Full Text | Google Scholar

Yin, N. N., Yu, W., Wu, S. X., Cheng, Y. Z., Lai, J. X., Chen, S. H., et al. (2023). A rapid identification method for TMV resistance in vitro and its application in tobacco breeding. Mol. Plant Breed. 21, 8144–8150. doi: 10.13271/j.mpb.021.008144

Crossref Full Text | Google Scholar