CN115873983A - Molecular marker for improving utilization of nic1 locus for breeding low-nicotine tobacco and application thereof - Google Patents

Abstract

The invention discloses a molecular marker for improving the selection of low-nicotine tobacco by using the nic1 site and its application. The application is to detect the segregation population in the hybrid segregation progeny after using the main effect of tobacco nicotine synthesis to regulate the mutant site nic1 While retaining the nic1 locus in the genome DNA of a single plant of Nicotiana tabacum, the selection efficiency of the chromosomal region where the nic1 locus is located is restored to the genetic background of the recurrent parent by means of systematic selection or backcross breeding, thereby improving the adverse agronomic traits of the non-recurrent parent , to improve the genetic background selection efficiency of progeny tobacco plants, and accelerate the application of mutant nic1 loci in breeding low-nicotine tobacco varieties. The molecular marker described in the present invention can be used as an auxiliary molecular marker application for improving the selection efficiency of genetic background reversion of low-nicotine tobacco strains while utilizing the mutant nic1 site in tobacco quality breeding.

Description

Molecular markers and their application to improve the selection of low-nicotine tobacco by using the nic1 locus

technical field

The invention belongs to the field of biotechnology, and specifically relates to the application of co-dominant and dominant molecular markers for improving the recovery rate of genetic background of tobacco strains by utilizing tobacco nicotine synthesis main effect regulation mutation site nic1.

Background technique

Cultivated tobacco ( Nicotianatabacum L.) is an annual herb of Solanaceae and one of the important economic crops. Nicotine is an important characteristic alkaloid in cultivated tobacco. According to the genetic background of tobacco, the nicotine content in tobacco leaves accounts for 90-95% of the total alkaloid content in tobacco leaves. The level of nicotine content in tobacco leaves is also directly related to tobacco quality and commercial use. Compared with traditional cigarettes, the nicotine content in the upcoming VLN tobacco products (such as VLN ^® King, VLN ^® Menthol King) is reduced by about 95%, and it has the characteristics of low harm, low tar and no compensatory smoking. Tobacco companies and related research institutions at home and abroad are working to use the main regulatory gene locus of nicotine synthesis , NIC1 , to regulate the nicotine content of tobacco leaves, reduce the nicotine content in tobacco products, and reduce the harmfulness of tobacco to the human body. In addition, a large number of research institutions at home and abroad are using biotechnology methods such as changing tobacco cultivation measures, grafting with different Solanaceae crops, and gene editing to reduce the biosynthesis or nicotine transport process of tobacco to achieve the purpose of regulating the nicotine content of tobacco leaves. Because American tobacco products are blended tobacco, 22nd Century Tobacco is also working with North Carolina State University to develop low-nicotine tobacco varieties of burley, oriental tobacco and air-cured tobacco.

Mutants with different nicotine content in tobacco were first discovered in cigars. Later, tobacco breeders transferred these mutation sites to different lines of flue-cured tobacco and Burley tobacco through cross-breeding and backcrossing. In Burley tobacco, tobacco breeders have bred four near isogenic lines (Near Isogenic Lines, NILs) for nicotine synthesis in Burley tobacco through anther culture and chromosome doubling techniques, named HA Burley 21, HI Burley 21. LI Burley 21 and LA Burley 21. Classical genetic studies on these four materials found that tobacco nicotine content is regulated by two independently inherited dominant loci (named NICOTINE 1 and NICOTINE 2, referred to as NIC1 and NIC2) (Legg, P.D., Collins, G.B., 1971.Inheritance of per cent total alkaloids in Nicotianatabacum L.:II.Genetic effects of two loci in Burley 21x LA Burley 21populations.Can.J.Genet.Cytol.13,287-291.Legg,P.D.,Chaplin,J.F.,Collins,G.B. , 1969. Inheritance of percent total alkaloids in Nicotiana tabacum L.: populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. J. Hered. 60, 213-217). Among them, the ability of NIC1 site to regulate nicotine synthesis is 2.4 times that of NIC2 site. According to related reports, the NIC2 locus in tobacco is composed of at least seven ethylene response factor ERF (Ethylene Response Factor) transcription factor genes, including NtERF189, NtERF115, NtERF179 and so on. These transcription factor genes can bind to the promoter regions of metabolic enzyme genes (such as NtPMT2 and NtQPT2) on the nicotine synthesis pathway to transcriptionally activate downstream gene expression (Shoji, T., Kajikawa, M., Hashimoto, T., 2010. Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell 22, 3390-3409; Shoji, T., Hashimoto, T., 2012. DNA-binding and transcriptional activation properties of tobacco NIC2-locus ERF189 and related transcription factors. Plant Biotechnology 29, 35-42. ). Among them, the mutant nic2 locus in the LA Burley 21 strain is caused by the loss of the chromosome segment where it is located (Kajikawa M., Sierro N., Kawaguchi H., et al., 2017. Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco, Plant Physiology 174, 999-1011.). On the other hand, recent studies have also found that other types of transcription factor (Transcription Factor) family genes are also involved in the transcriptional regulation of tobacco nicotine biosynthesis. For example, the basic Helix-Loop-Helix (bHLH) family transcription factor NtMYC2 can cooperate with the NIC2 site transcription factor (such as NtERF189), and the two can respectively bind to the key metabolic rate-limiting enzyme genes in the nicotine synthesis pathway (such as NtPMT, The G-box and GCC-box transcription elements in the NtQPT) promoter come up to maximize the activation of the expression of these genes (Shoji T. and Hashimoto T. 2011. Tobacco MYC2 regulates jasmonate-induciblenicotine biosynthesis genes directly and by way of the NIC2- locus ERF genes, Plant Cell Physiol. 52(6):1117–1130.). On the other hand, the reduction of tobacco alkaloid content caused by the overexpression of the tobacco auxin response factor NtARF6 transcription factor is due to the antagonism between the jasmonic acid pathway and other plant hormone signal transduction pathways (such as ethylene, salicylic acid, abscisic acid) caused by the effect. The inhibition of jasmonic acid biosynthetic pathway is accompanied by the activation of ethylene, salicylic acid, abscisic acid and the defense response to pathogen infection, thereby antagonizing the adjustment of secondary metabolism induced by jasmonic acid and reducing the expression of nicotine synthesis genes in tobacco ( Hu et al., 2021. Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.), PlantMol Biol. 107(1-2):21-36.). In addition, it was recently discovered that the MYB transcription factor family gene NtMYB305a can bind to the AT-rich element in the GAG region on the promoter of tobacco NtPMT1a. At the same time, NtMYB305a also regulates the expression of other nicotine synthesis genes in a similar way, thereby positively regulating the biosynthesis of nicotine in tobacco (Bian et al., 2022. NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1apromoter to regulate nicotine biosynthesis. Plant Physiol. 188(1):151-166.). Scholars at home and abroad have discovered a new ERF (NIC2-like) gene cluster in the genomes of flue-cured tobacco (Yunyan 87) and Burley tobacco (HABurely 21) through bioinformatics methods, and its composition is highly similar to the NIC2 gene cluster (Kajikawa M .,SierroN.,Kawaguchi H.,et al.,2017.Genomic insights into the evolution of thenicotine biosynthesis pathway in tobacco,Plant Physiology 174,999-1011;SuiX.et al.,2019.Ethylene response factor NtERF91 positively reculatesalo tabacum L.), Biochem Biophys ResCommun.517(1):164-171; Sui X. et al., 2020. Unravel the mystery of NIC1-locus onnicotine biosynthesis regulation in tobacco, Biorxiv). Recently, the above-mentioned NIC2-like gene cluster has been proved to be the NIC1 locus by means of genetic population mapping and gene function verification, and the functions of multiple genes in the cluster have been verified (Sui X. et al., 2020. Unravel the mystery of NIC1-locus onnicotine biosynthesis regulation in tobacco, Biorxiv). At the same time, a number of SNP (Single Nucleotide Polymorphism) molecular markers for the detection of Burley tobacco NIC1 gene were developed, and the markers and methods for detection were applied for a patent (PCT/US2018/038679). On the other hand, the reason for the decreased expression of nicotine synthesis genes in the nic1 mutant has also been basically clarified. Two chromosome fragments (nic1-S and nic1-B) were deleted on the flanks of the chromosomal region where the mutant nic1 gene cluster was located, which eventually led to structural variation in the chromosome where the NIC1 gene cluster was located, thereby affecting the normal expression of genes in the gene cluster (Sui X. et al., 2020. Unravel the mystery of NIC1-locus onnicotine biosynthesis regulation in tobacco, Biorxiv).

However, using the mutant nic1 gene cluster to develop tobacco varieties with low nicotine content as a common breeding method still faces some problems. According to relevant literature reports, the mutant nic1 tobacco line (LA Burley 21) has many disadvantages in agronomic traits compared with normal tobacco, such as slow yellowing, difficult curing, low yield, and poor quality of cured tobacco leaves (Chaplin, J.F., &Burk,L.G.(1983).Agronomic,chemical,and smoke characteristics of flue-curedtobacco lines with different levels of total alkaloids.Crop Science,75,133–136.Chaplin,J.F.,&Weeksther,W.W.(1976).Association between ids total alkaloids traits in flue-cured tobacco. Crop Science, 16, 416–418.; Legg, P.D., Collins, G.B., & Littion, C.C. (1970). Registration of LA Burley 21 tobacco germplasm. Crop Science, 10, 212.). Therefore, in the process of using LAFC53 as a germplasm material to breed low-nicotine tobacco varieties through backcrossing, it is necessary to retain the nic1 mutant locus to the greatest extent, and it is best to be able to transfer the recurrent parents in the systematic breeding or backcrossing generation. Chromosomal fragments are preserved. Therefore, using whole genome resequencing technology to compare the genomes of recurrent parents (such as NC95 HA) and non-recurrent parents (LAFC53) to find differences in the region around the chromosome region where nic1 is located, and develop specific (co-dominant or dominant) genes that can distinguish the differences between the genomes of the two parents. ) molecular marker, which is of great significance to the use of molecular marker-assisted breeding of tobacco varieties with low nicotine content.

So far, although there have been reports on the development of molecular markers for the NIC1 site of tobacco nicotine synthesis, the existing research results are all based on the genetic prospect selection of the nic1 mutant site, that is, only the selection results in gene expression in the nic1 gene cluster. The deletion fragments (nic1-S and nic1-B) that were downregulated were subjected to marker selection (Sui X. et al., 2020. Unravel the mystery of NIC1-locus on nicotine biosynthesis regulation intobacco, Biorxiv). For example, marker reports linked to the nic1 gene cluster are dCAPS markers and SSR markers developed based on SNP variant sites. There are some deficiencies in tobacco breeding practice and tobacco leaf production: 1) There are serious limitations in the scope of use, that is, the scope of use of the dCAPS marker for the detection and identification of the nic1 gene that has been reported and applied for a patent is relatively small, and it is only applicable to Burley tobacco In addition to the Burley tobacco type, cultivated tobacco includes flue-cured tobacco, oriental tobacco, cigar tobacco, air-cured tobacco, sun-cured tobacco, and other types of Nicotiana tabacum, and the above-mentioned many tobacco types or species cannot be detected by dCAPS markers. Identified. 2) The currently developed molecular markers are only based on marker-assisted selection (ie, foreground selection) for the nic1 mutant gene cluster itself, and cannot be selected for the genetic background of recurrent parents or non-recurrent parents (such as LAFC53). In the process of using LAFC53 as a non-recurrent parent to breed low-nicotine tobacco varieties through systematic breeding or backcrossing, it is impossible to preserve the nic1 mutant locus to the greatest extent and at the same time transfer the chromosomal fragments of the recurrent parent in the backcross generation It can be retained to the greatest extent, thereby effectively improving the shortcomings of the agronomic traits (slow yellow fall, difficult to cure, low yield, and poor quality of tobacco leaves) existing in the non-recurrent parent of the mutant nic1 tobacco (LAFC53).

The present invention uses two near-isogenic line materials NC95 HA (NIC1NIC1NIC2NIC2) with different nicotine content and tobacco material LAFC53 (nic1nic1nic2nic2) with low nicotine content as experimental materials, and compares the two near-isogenic lines at the nic1 position through tobacco genome resequencing technology. Specific (co-dominant and dominant) molecular markers that can distinguish the differences between the genomes of the two parents are developed based on the structural variation in the chromosomal region where the point is located. By constructing self-crossing second-generation (F ₂ ) segregation population, after screening the individuals whose genotype is (nic1nic1nic2nic2) in the selection population, the single plant is kept as seed. For the single-plant lines that were kept in the F3 generation, the genetic background selection and the agronomic traits of the strains were selected using specific markers, and the _F3 generation was screened out. Low-nicotine tobacco strains, accelerating the use of marker assistant selection (MAS) in the breeding of low-nicotine content tobacco varieties, so as to achieve convenient, fast, stable and reliable breeding of high-quality tobacco with low nicotine content Variety.

Contents of the invention

The first object of the present invention is to provide a specificity (dominant and co-dominant) that can effectively improve the genetic background selection efficiency of systematic breeding or backcross breeding progeny after using tobacco nicotine synthesis as the main effect to regulate the nic1 mutant site Molecular markers; the second purpose is to improve the non-recurrent parent (parent carrying the mutant nic1 site) possessed by providing the main regulation nic1 mutant site with tobacco nicotine synthesis and improving the genetic background selection efficiency of backcross offspring Application of adverse agronomic traits, accelerated mutant nic1 loci in breeding low-nicotine tobacco varieties.

Using the application of co-dominant and dominant molecular markers that can increase the genetic background recovery rate of tobacco lines after the mutation site nic1, which is the main regulator of tobacco nicotine synthesis, detects the presence of mutant nic1 sites in the genomic DNA of individual tobacco plants in isolated populations of tobacco At the same time, through systematic breeding or backcross breeding, the selection efficiency of the chromosomal region where the nic1 locus is located is restored to the genetic background of the recurrent parent, and finally the shortcomings of the agronomic traits introduced by the non-recurrent parent of the mutant nic1 tobacco can be effectively improved.

A co-dominant and dominant molecular marker that uses the main regulatory mutation site nic1 for tobacco nicotine synthesis. The molecular marker of the present invention can detect the presence of nicotine synthesis in the region around the chromosome region where the main regulatory mutant site nic1 is located. The chromosomal structural variation produced, the specific co-dominant molecular markers are numbered Target1~Target3 and the dominant molecular markers are numbered Target4~Target6; the nucleotide sequences of the PCR amplification products are SEQ ID No.1 and SEQ ID No. .2, shown in SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7, SEQID No.8 and SEQ ID No.9; described specific The primer sequences of the six regions corresponding to the molecular markers are:

Target1 sequence is:

Target1F: 5'-GACTTATGGCAATTCAAAGATAAGA-3';

Target1R: 5'-CAGTTTCTGGAAATGTTTGTTAAGT-3';

The Target2 sequence is:

Target2F: 5'-AGTTCAACTATTGTTTTCTCGACAT-3';

Target2R: 5'-ATTTAGGCACTGTTATTACTTGTGG-3';

The Target3 sequence is:

Target3F: 5'-GCACCATCCAAACACAAGGTTAAAC-3';

Target3R: 5'-CCTAATCCCTCTTCGAATCTTAAATC-3';

The Target4 sequence is:

Target4F: 5'-TACTACTGTGCAGCAGATGATTTAG-3';

Target4R: 5'-TACCTTGCATATGTTCCTATATGGT-3';

The Target5 sequence is:

Target5F: 5'-TTTAAGTTCTTGTTTTTCTCTTGA-3';

Target5R: 5'-AATCAGTTCCTTCCTCACACTAAC-3';

The Target6 sequence is:

Target6F: 5'-AAATTCAGAGAGATTTTTGGAAAGT-3';

Target6R: 5'-ATAAAGAAGCAGAAAATAGGGAAAAT-3'.

The application of the co-dominant and dominant molecular markers of the present invention is to improve the recovery rate of the genetic background of tobacco lines.

The above-mentioned application of the present invention is specifically: detecting the genomic DNA of a single tobacco plant in a tobacco segregation population while retaining the nic1 site, and improving the selection efficiency of the chromosomal region where the nic1 site is located to return to the recurrent parental genetic background by means of systematic breeding or backcross breeding , so as to improve the unfavorable agronomic traits (such as hard yellowing, low yield, and short plants, etc.) of non-recurrent parents (parents carrying mutant nic1 sites), improve the genetic background selection efficiency of offspring tobacco plants, and accelerate the selection of mutant nic1 sites. Application of points in breeding low-nicotine tobacco varieties.

The above-mentioned application of the present invention is: respectively with the primer of Target1 sequence, the primer of Target2 sequence, the primer of Target3 sequence or the primer of Target4 sequence, the primer of Target5 sequence and the primer of Target6 sequence, respectively amplify the tobacco genome DNA to be detected, detect PCR The amplification product is analyzed according to the result of the amplification product.

In the present invention, if the result of the PCR amplification product contains three nucleotide sequences as shown in SEQ ID No.1, SEQ ID No.3 and SEQ ID No.5 at the same time, it is a tobacco genetic background with a homozygous recurrent parent.

In the present invention, if the results of the PCR amplification product contain three nucleotide sequences as shown in SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6 at the same time, it is a non-recurrent parent inheritance with homozygous mutant nic1 background.

In the present invention, if the PCR amplification product result contains 6 compounds as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 at the same time A nucleotide sequence, that is, the genetic background of tobacco is heterozygous, that is, tobacco plants with genetic backgrounds of recurrent parents and non-recurrent parents.

In the present invention, if the PCR amplification product results contain three nucleotide sequences as shown in SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 at the same time, that is, it has a recurrent parent or has both a recurrent parent and a non-recurrent parent. heterozygous tobacco genetic background.

In the present invention, if the result of the PCR amplification product is no PCR amplification product, it is a tobacco plant with the genetic background of the non-recurrent parent with the homozygous mutant nic1.

In order to simply and efficiently select tobacco varieties with low nicotine content and good agronomic traits, the progeny materials containing the homozygous genotype nic1nic1 are targeted and specifically selected, and at the same time, the parents with low nicotine content (carrying the nic1 mutant type) are replaced to the greatest extent. site, such as the chromosome segment carried by the chromosomal region where the nic1 site is located in LAFC53). The present invention provides a kind of co-dominant and dominant molecular markers Target1, Target2, Target3, Target4, Target5 and Target6, which utilize tobacco nicotine synthesis as the main effect to regulate the mutation site nic1 and increase the recovery rate of the genetic background of tobacco lines. The molecular markers adopt tobacco Genome resequencing analysis (Tobacco Whole Genome Resequencing) method, by comparing the structural variation in the chromosome region where the mutant nic1 locus is located, by comparing two near-isogenic line materials NC95 HA (NIC1NIC1NIC2NIC2) and LAFC53 (nic1nic1nic2nic2) with different nicotine contents. Specific molecular markers that can distinguish the differences between the genomes of the two parents. The molecular marker can be used as an auxiliary method for the selection of tobacco varieties with low nicotine content, so as to improve the efficiency of genetic background selection of backcross breeding progeny and improve the agronomic traits by using molecular marker-assisted selection, so as to accelerate the breeding process of tobacco varieties with low nicotine content.

The present invention utilizes two near-isogenic lines with different nicotine contents as materials NC95 HA (high nicotine content: NIC1NIC1 NIC2NIC2) and LAFC53 (low nicotine content: nic1nic1 nic2nic2) as parents, adopts tobacco genome resequencing analysis method, utilizes The Structural Variation (SV) in the flanking region of the chromosome where nic1 is located was developed to design specific molecular markers that can effectively distinguish chromosome segments from recurrent parents and non-recurrent parents (such as NC95 HA and LAFC53). At the same time, 24 individual plants of genotype nic1nic1nic2nic2 in the self-crossing second generation (F ₂ ) population were screened out through hybridization and selfing, and bagged for seedling retention. The genetic background of each strain was screened using the developed specific markers after extracting the genomic DNA of each individual plant of the _F2 generation. On the other hand, 24 self-bred progenies of a single plant (ie F ₃ ) were planted in the field to investigate the agronomic traits of each line and statistical analysis, and the agronomic traits (one or more) of the tobacco lines were compared with those of the non-recurrent parents. Strains with significantly improved ratio. The results showed that the use of these markers could improve the selection efficiency of marker-assisted selection (MAS) in the selection of low-nicotine content tobacco varieties.

The co-dominant and dominant molecular markers of the present invention that utilize tobacco nicotine synthesis to regulate the mutation site nic1, which can increase the genetic background recovery rate of tobacco lines, have the characteristics of stability, reliability, simplicity, speed and low cost. Molecular markers can be used as molecular markers to improve the selection efficiency of genetic background while utilizing mutant nic1 loci in the cultivation of low-nicotine content tobacco varieties.

Description of drawings

Figure 1 is the gel electrophoresis of the PCR amplification product in two near-isogenic line materials of the specific molecular markers detected with the chromosome structure variation existing in the chromosome flanking region of the tobacco nicotine synthesis main regulatory site mutant nic1; , A, codominant marker Target1; B, codominant marker Target2; C, codominant marker Target3; D, dominant marker Target4; E, dominant marker Target5; F, dominant marker Target6; LA, low nicotine Content parent LAFC53; HA, high nicotine content parent NC95 HA; M, 1000bp DNA ladder, the length fragments are: 100bp, 200bp, 300bp, 400bp, 500bp;

Figure 2 is the alkaloid content map of four genotypes A_B_, A_bb, aaB_, and aabb individuals in the _F2 population of NC95 HA × LAFC53 (wherein, A: NIC1; B: NIC2; a: nic1; b: nic2 ).

Fig. 3 is the statistical chart of the PCR amplification product bands of the co-dominant markers Target1, Target2 and Target3 and the dominant markers Target4, Target5 and Target6 in each individual plant of 8 F2 _generations (genotype is nic1nic1 nic2nic2), wherein, A represents the chromosome segment derived from the recurrent parent NC95 HA, B represents the chromosome segment derived from the non-recurrent parent LAFC53 with the nic1 mutant site;

Figure 4 is a statistical analysis chart of the agronomic traits of the F ₃ generation tobacco lines obtained from the F ₂ generation single plant and the control variety LAFC53, including natural plant height, natural leaves, topping plant height, stem circumference, waist leaves Length, waist leaf width, effective number of leaves, pitch.

Detailed ways

Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased.

The numbering of the co-dominant and dominant molecular markers that can improve the genetic background recovery rate of tobacco lines after utilizing the main effect of tobacco nicotine synthesis to regulate the mutation site nic1 according to the present invention is Target1, Target2, Target3 Target4, Target5 and Target6, and its PCR The nucleotide sequences of the amplification products are respectively SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7, Shown in SEQ ID No.8 and SEQ ID No.9.

The primer sequences of the six sites corresponding to the molecular markers are:

Target1 sequence is:

Target1F: 5'-GACTTATGGCAATTCAAAGATAAGA-3' (SEQ ID No.1),

Target1R: 5'-CAGTTTCTGGAAATGTTTGTTAAGT-3' (SEQ ID No.2);

The Target2 sequence is:

Target2F: 5'-AGTTCAACTATTGTTTTCTCGACAT-3' (SEQ ID No.3),

Target2R: 5'-ATTTAGGCACTGTTATTACTTGTGG-3' (SEQ ID No.4);

The Target3 sequence is:

Target3F: 5'-GCACCATCCAAACACAAGGTTAAAC-3' (SEQ ID No.5),

Target3R: 5'-CCTAATCCCTCTTCGAATCTTAAATC-3' (SEQ ID No.6);

The Target4 sequence is:

Target4F: 5'-TACTACTGTGCAGCAGATGATTTAG-3' (SEQ ID No.7),

Target4R: 5'-TACCTTGCATATGTTCCTATATGGT-3';

The Target5 sequence is:

Target5F: 5'-TTTAAGTTCTTGTTTTTCTCTTGA-3' (SEQ ID No.8),

Target5R: 5'-AATCAGTTCCTTCCTCACACTAAC-3';

The Target6 sequence is:

Target6F: 5'-AAATTCAGAGAGAGATTTTTGGAAAGT-3' (SEQ ID No.9), Target6R: 5'-ATAAAGAAGCAGAAATAGGGAAAAT-3';

The application of the specific molecular marker for the detection of chromosome structural variation existing in the chromosome flanking region of the main regulatory site mutant nic1 of tobacco nicotine synthesis according to the present invention is characterized in that it detects whether there is a mutant genotype nic1 in the tobacco genomic DNA At the same time, through the use of systematic breeding and backcross breeding methods to improve the selection efficiency of tobacco genetic background in the breeding progeny, improve the field agronomic characteristics of the selected tobacco plants, thereby accelerating the application of breeding low-nicotine tobacco varieties.

The application of the specific molecular markers for the detection of chromosomal structural variation in the flanking region of the chromosome where the main regulatory site mutant nic1 is located in tobacco nicotine synthesis is to use the primers of the Target1 sequence, the primers of the Target2 sequence and the primers of the Target3 sequence respectively. Amplify the tobacco genomic DNA to be detected, and detect the PCR amplification product, if the PCR amplification product contains three nucleotide sequences as shown in SEQ ID No.1, SEQ ID No.3 and SEQ ID No.5, namely It is the tobacco genetic background with the recurrent parent NC95 HA; if the PCR amplification product contains three nucleotide sequences as shown in SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6 at the same time, it is a non- The genetic background of the recurrent parent LAFC53 tobacco; if the PCR amplification product contains both SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. The 6 nucleotide sequences shown in 6, that is, the genetic background of the tobacco is a tobacco plant with a heterozygous genetic background of the recurrent parent and the non-recurrent parent. If the primers of the Target4 sequence, the primers of the Target5 sequence and the primers of the Target6 sequence amplify the tobacco genome DNA to be detected respectively, and detect the PCR amplification product, if the PCR amplification product simultaneously contains such as SEQ ID No.7, SEQ ID No. 8 and the three nucleotide sequences shown in SEQ ID No.9, that is, the tobacco genetic background with the homozygous type of the recurrent parent NC95 HA or the heterozygous type with the recurrent parent and the non-recurrent parent; if there is no amplification product in PCR , which is a tobacco plant with the genetic background of the non-recurrent parent LAFC53 of the homozygous mutant nic1.

The present invention will be further described below with specific implementation case:

Example 1

Using Tobacco Whole Genome Resequencing analysis (Tobacco Whole Genome Resequencing) method, deep resequencing was performed on two near-isogenic materials, the high nicotine content tobacco line NC95 HA (genotype NIC1NIC1NIC2NIC2) and the low nicotine content tobacco line LAFC53 (genotype nic1nic1nic2nic2) . Sequence alignment is used to detect and compare whether there is a chromosomal structural variation between the two parents in the flanking region of the chromosome where nic1 is located, and obtain relevant chromosomal fragment sequence information according to the variation type. The visualization of tobacco whole genome resequencing analysis results was performed on IGV (Integrative Genomics Viewer) software, and the design of relevant specific markers (primers) was completed using the Primer3 online tool (https://bioinfo.ut.ee/primer3-0.4.0/ ).

1. Experimental materials

Two near-isogenic tobacco line NC95 HA (NIC1NIC1NIC2NIC2) with high nicotine content were used as female parent, and tobacco LAFC53 line with low nicotine content (nic1nic1nic2nic2) was used as male parent. In 2017, high and low nicotine content parental materials were planted and crossed to obtain the F ₁ generation. The hybrid generation (F ₁ ) was planted in the winter of 2018, and the nic1 and nic2 gene segregation population (F ₂ generation) was obtained in early 2019. In 2019, two parents, F ₁ and F ₂ generation materials were planted, and a single plant with genotype (nic1nic1nic2nic2) was obtained from the F ₂ generation materials, and the F ₃ generation lines were obtained by bagging. In 2021, high and low nicotine content parent materials, as well as the F ₃ generation lines will be planted, and the field survey data of the parents and F ₃ generation agronomic traits will be obtained.

2. Determination of nicotine content in parents and _F2 segregation populations

The test materials were transplanted to the field after seedlings, with a row-to-plant spacing of 100cm×50cm; using conventional cultivation and field management, the _F2 population was topped after budding as a whole, and after two weeks of topping, the fresh tobacco leaves of each individual plant were harvested and roasted. After harvesting and roasting, the leaves of each plant were ground separately and the nicotine content was determined. The obtained nicotine content data of each leaf of each individual plant of the parent and _F2 populations were analyzed, and the genotype data analysis was based on the following: according to the band type of each individual plant, the data statistics were carried out, that is, the band type of the two loci and the NC95 HA parent The consistent individual plants are marked as "A_" or "B_"; the single bands of the two loci consistent with the band pattern of the LAFC53 parent are marked as "aa" or "bb", and the bands are not clear or have no amplified bands Record it as "U".

3. Specific marker design

Before topping, collect the fresh leaves of each individual plant of the parents and the _F2 population in the field. The whole genome DNA of the test materials can be extracted by conventional CTAB method or plant tissue DNA extraction kit. The method can refer to the existing literature or Instructions in the kit. PCR reaction system preparation, product amplification and 2% agarose gel electrophoresis detection of amplified products, refer to Sui et al. (Sui X., Huang Y., et al.2011, Molecular authentication of an ethnomedicinalplant Sabia parviflora and its adults by DNA barcoding technique.PlantaMedica 2011,77(5):492-496.) The method provided.

High-Fidelity DNA Polymerase，NEB)，加双蒸水至20μL。The PCR reaction system is: 30-50ng/μL DNA, 1.0μmol/L forward and reverse primers, 1.5mmol/LdNTPs, 2μL 10×PCR Buffer (Mg ²⁺ plus), 0.75～1.0U Q5 high-fidelity DNA polymerase (

High-Fidelity DNA Polymerase, NEB), add double distilled water to 20μL.

The PCR amplification program is: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, and after 30 cycles, extension at 72°C for 5 min.

The gel electrophoresis detection refers to the use of 2% agarose gel, 1×TBE electrophoresis buffer, separation at 100V constant voltage electrophoresis for 45 minutes, and finally developing with ethidium bromide.

4. Parental verification of developing specific markers based on nic1 locus chromosome structural variation

At the seedling stage, according to the method described in Example 1, the primers designed by the analysis results of the resequencing data were used to perform PCR amplification on 2 parts of the parent NC95 HA (NIC1NIC1NIC2NIC2) with medium and high nicotine content and the parent LAFC53 (nic1nic1nic2nic2) with low nicotine content. Screening verification can The specific marker (dominant or co-dominant) of the chromosomal segment in the chromosomal region where the mutant nic1 locus is located distinguishes between the two parents. The results of the screening are shown in Figure 1: the co-dominant specific markers Target1, Target2 and Target3 can distinguish the chromosomal segments around the chromosome region where the nic1 locus is located between the two parents, that is, the markers Target1, Target2 and Target3 in the high nicotine content parent NC95 The band patterns in HA are completely consistent, only a specific band of 776bp (sequence such as SEQ ID NO.1), 716bp (sequence such as SEQ ID NO.3) and 1458bp (sequence such as SEQ ID NO.5) appears; The band pattern in the nicotine content parent LAFC53 is completely consistent, only one specific band of 457bp (sequence such as SEQ ID NO.2), 396bp (sequence such as SEQ ID NO.4) and 420bp (sequence such as SEQ ID NO.6) appears bring. Among them, the lengths of the sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 are 776bp, 716bp and 1458bp, respectively, which are co-dominant specific markers Target1, Target2 and Target3 in tobacco with high nicotine content. The specific PCR amplified nucleotide sequence in the parental NC95 HA; the sequence lengths shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 are 457bp, 369bp and 420bp, respectively, which are co-dominant specific Specific PCR-amplified nucleotide sequences of sex markers Target1, Target2 and Target3 in low-nicotine content tobacco parent LAFC53 containing the nic1 mutant locus.

Dominant specific markers Target4, Target5 and Target6 can also distinguish the chromosome segments around the chromosome region where the nic1 locus is located between the two parents, that is, the band patterns of the markers Target4, Target5 and Target6 in the high-nicotine content parent NC95 HA are completely consistent, only A specific band of 1051bp (sequence such as SEQ ID NO.7), 675bp (sequence such as SEQ ID NO.8) and 1317bp (sequence such as SEQ ID NO.9) appeared; while the low nicotine content parent LAFC53 could not be successfully amplified Corresponding specific bands were added. Among them, the lengths of the sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 are 1051bp, 675bp and 1317bp respectively, which are respectively dominant specific markers Target4, Target5 and Target6 in tobacco parents with high nicotine content Specific PCR amplified nucleotide sequences in NC95 HA; while dominant specific markers Target4, Target5 and Target6 could not amplify specific PCR bands in LAFC53, a low-nicotine content tobacco parent containing the nic1 mutant site.

The above results indicated that the markers Target1, Target2 and Target3 could distinguish the chromosome segments of the two parents in the chromosome region where the mutant nic1 locus was located, and the markers were co-dominant markers. If the PCR amplification product contains three nucleotide sequences shown in SEQ ID No.1 (776bp), SEQ ID No.3 (716bp) and SEQ ID No.5 (1458bp) at the same time, it is the recurrent parent NC95 HA chromosome background fragment; if the PCR amplification product contains three nucleotide sequences as shown in SEQ ID No.2 (457bp), SEQ ID No.4 (396bp) and SEQ ID No.6 (420bp) at the same time, it is Chromosomal fragments with the chromosomal background of the non-recurrent parent LAFC53; if the PCR amplification product contains both SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No. The six nucleotide sequences shown in ID No.6 are heterozygous chromosome fragments with recurrent parents and non-recurrent parents.

At the same time, the markers Target4, Target5 and Target6 can also distinguish the source of the chromosomal fragment in the chromosome region where the mutant nic1 locus is located between the two parents, and these markers are dominant markers. If the PCR amplification product contains three nucleotide sequences as shown in SEQ ID No.7 (1051bp), SEQ ID No.8 (675bp) and SEQ ID No.9 (1317bp) at the same time, it means that it has at least the recurrent parent NC95 Chromosomal fragments in HA; if markers Target4, Target5, and Target6 cannot successfully amplify the corresponding specific bands, it is a chromosome fragment with the region of the nic1 genotype in the non-recurrent parent LAFC53.

Example 2

Improve the use of specific molecular markers for the detection of chromosomal structural variation in the flanking region of the chromosome where the main regulatory site mutant of tobacco nicotine synthesis, nic1, is used in molecular marker-assisted selection to improve the efficiency of genetic background selection in backcross breeding progeny and improve agronomic traits and Validation in _F3 strains.

data analysis

Tobacco genomic DNA was extracted and purified according to the method described in Example 1, and the nicotine content and NIC1 and NIC2 locus marker typing analysis were performed after topping the individual plants of the _F2 population. Secondly, the specific molecular markers Target1, Target2, Target3, Target4, Target5 and Target6 obtained from the analysis of the chromosome structure variation in the flanking region of the chromosome of the main regulatory site mutant of tobacco nicotine synthesis, nic1, which were screened by resequencing 23 individual plants in the F ₂ population were genotyped. Finally, the band patterns of the 8 selected individual plants were counted, that is, the individual plants with the same band pattern as the NC95HA parent were recorded as "A"; the individual plants with the same band pattern as the LAFC53 parent were marked as "B" .

The results of the nicotine content in a single plant of the _F2 population were: among the 377 single plants, there were 224 single plants with genotype A_B_, 36 single plants with genotype A_bb, and 94 single plants with genotype aaB_ There are 23 individual plants with genotype aabb. The genotype analysis of 377 individual plants of the F ₂ generation is shown in Figure 2.

Example 3

Improving the field agronomic traits of the F ₃ generation low nicotine content lines in the offspring of parental NC95 HA and parental LAFC53 hybrids using specific molecular markers detected with chromosome structural variation in the flanking region of the chromosome where the major regulatory site mutant of tobacco nicotine synthesis, nic1, is located .

1. Experimental materials

The markers used are Target1, Target2, Target3, Target4, Target5 and Target6 specific molecular markers for the detection of chromosome structural variation existing in the flanking region of the chromosome where nic1 is located. The plant materials are HA NC95 (genotype NIC1NIC1NIC2NIC2), LAFC53 (genotype nic1nic1nic2nic2), 8 individual plants of genotype nic1nic1nic2nic2 in F ₂ segregation population (NC95 HA×LAFC53), and the corresponding 8 plants of F ₃ generation Tie.

2. Data processing

First, according to the method described in Example 1, the tobacco genomic DNA was extracted and purified from the tobacco material. Secondly, the genotype analysis of the above individual tobacco plants was carried out by using the specific molecular markers detected with the chromosomal structural variation in the chromosome flanking region of the tobacco nicotine synthesis main regulatory site mutant nic1. Finally, data analysis is carried out on the band pattern of each individual plant, that is, the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 appear in the PCR amplification product at the same time. Specific PCR amplification nucleotide sequence of high nicotine content tobacco recurrent parent NC95 HA genetic background; if the PCR amplification product appears simultaneously such as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO .4 and the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 are heterozygous chromosome fragments with recurrent parents and non-recurrent parents (carrying nic1 site mutation); if the PCR amplification product The simultaneous appearance of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.2 and SEQ ID NO.4 is the specific PCR amplification of the genetic background of the non-recurrent parent LAFC53 containing the nic1 mutant site Nucleotide sequence.

It can be seen from Figure 3 that among the 8 candidate F ₃ generation tobacco lines to be tested, the line NC95 HA×LAFC53 F ₃ -72 has the most NC95 HA while ensuring the prospect of low nicotine traits (nic1nic1nic2nic2) The agronomic traits of the strains with the parental genetic background are not significantly different from those of the parent NC95 HA, and the main agronomic traits have been greatly improved; while the strain NC95 HA × LAFC53 F ₃ -112 is guaranteed to have the prospect of low nicotine traits (nic1nic1nic2nic2) At the same time, the line with the closest genetic background to the parent LAFC53 had stem girth slightly higher than the parent LAFC53 and effective leaf number lower than the parent LAFC53, and other agronomic traits (such as natural plant height, natural leaf number, topping plant height) had no significant difference with the parent LAFC53, and the overall agronomic traits were poor; while other F ₃ lines (such as NC95 HA×LAFC53 F ₃ -112, NC95 HA×LAFC53F ₃ -12, NC95 HA×LAFC53 F ₃ -151 and NC95 HA×LAFC53 F ₃ -308) the chromosomal region around the nic1 locus was replaced (homozygous or heterozygous) by the parental (NC95 HA) chromosome segment to varying degrees, and its overall agronomic traits were also different to varying degrees compared with the parental LAFC53 improved, as shown in Figure 3. These candidate lines can further speed up the genetic background selection efficiency of different individual plants in the _F3 candidate lines through systematic breeding to accelerate line homozygosity or through backcrossing with the parental NC95 HA, and finally make its agronomic traits continuously improved, and finally Molecular-Assisted Selection Breeding of Tobacco Varieties with Low Nicotine Content.

Conclusion: Using the above six specific (dominant or co-dominant) markers can easily, quickly and stably detect the replacement of chromosome segments around the chromosome where the tobacco nic1 locus is located, and can clearly identify chromosome segments from different parents Homozygous or heterozygous conditions, and targeted and specific selection of tobacco intermediate materials with low nicotine content that have a high genetic background recovery rate and greatly improved various agronomic traits can greatly increase the yield of tobacco varieties with low nicotine content. Breeding efficiency.

The above are only some specific embodiments of the present invention, and the specific content or general knowledge known in the scheme is not described here (including but not limited to abbreviations, abbreviations, units commonly used in the field). It should be pointed out that the above embodiments do not limit the present invention in any way, and for those skilled in the art, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention. The scope of protection required by this application shall be based on the content of the claims, and the specific implementation methods and other records in the specification may be used to interpret the content of the claims.

Claims (10)

1. A codominant and dominant molecular marker for synthesizing a major regulatory mutation site nic1 by using tobacco nicotine is characterized in that the molecular marker can detect the chromosome structure variation generated in the surrounding area of a chromosome region where the major regulatory mutation site nic1 is located in the tobacco nicotine synthesis, the specific codominant molecular marker is numbered from Target 1to Target3, and the dominant molecular marker is numbered from Target4 to Target6; the nucleotide sequences of PCR amplification products are respectively shown in SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No. 9; the primer sequences of the 6 regions corresponding to the specific molecular markers are respectively as follows:

the Target1 sequence is:

Target1F：5’-GACTTATGGCAATTCAAAGATAAGA -3’；

Target1R：5’-CAGTTTCTGGAAATGTTTGTTAAGT-3’；

the Target2 sequence is:

Target2F：5’-AGTTCAACTATTGTTTTCTCGACAT-3’；

Target2R：5’-ATTTAGGCACTGTTATTACTTGTGG-3’；

the Target3 sequence is:

Target3F：5’-GCACCATCCAAACACAAGGTTAAAC-3’；

Target3R：5’-CCTAATCCTCTTCGAATCTTAAATC-3’；

the Target4 sequence is:

Target4F：5’-TACTACTGTGCAGCAGATGATTTAG-3’；

Target4R：5’-TACCTTGCATATGTTCCTATATGGT-3’；

the Target5 sequence is:

Target5F：5’-TTTAAGTTCTTGTTTTTCTCCTTGA -3’；

Target5R：5’-AATCAGTTCCTTCCTCACACTAAC-3’；

the Target6 sequence is:

Target6F：5’-AAATTCAGAGAGATTTTTGGAAAGT-3’；

Target6R：5’-ATAAAGAAGCAGAAATAGGGAAAAT-3’。

2. the use of the co-dominant and dominant molecular markers of claim 1, wherein the use is: and the genetic background recovery rate of the tobacco strain is improved.

3. The application according to claim 2, characterized in that it is specifically: the method has the advantages that the tobacco single plant genome DNA in the tobacco segregation population is detected, the selection efficiency of the recurrent genetic background in the chromosome region where the nic1 locus is located is improved by means of system breeding or backcross breeding, so that the poor agronomic characters of non-recurrent parents are improved, the genetic background selection efficiency of the progeny tobacco plants is improved, and the application of the mutant type nic1 locus in breeding low-nicotine tobacco varieties is accelerated.

4. The application according to claim 2, characterized in that it is: respectively amplifying the genomic DNA of the tobacco to be detected by using a primer of a Target1 sequence, a primer of a Target2 sequence, a primer of a Target3 sequence or a primer of a Target4 sequence, a primer of a Target5 sequence and a primer of a Target6 sequence, and detecting a PCR amplification product; analyzing the PCR amplification product.

5. The use of claim 4, wherein the PCR amplification product is: if the PCR amplification product result simultaneously contains three nucleotide sequences shown as SEQ ID No.1, SEQ ID No.3 and SEQ ID No.5, the tobacco genetic background with homozygous recurrent parents is obtained.

6. The use of claim 4, wherein the PCR amplification product is: if the PCR amplification product result contains three nucleotide sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6 at the same time, the result is the non-recurrent parent genetic background with homozygous mutant nic 1.

7. The use of claim 4, wherein the PCR amplification product is: if the result of the PCR amplification product contains 6 nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 at the same time, namely the genetic background of the tobacco is a heterozygote type, namely the tobacco plant with the genetic backgrounds of recurrent parent and non-recurrent parent.

8. The use according to claim 4, wherein the PCR amplification product is: if the PCR amplification product result contains three nucleotide sequences shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 at the same time, the hybrid tobacco genetic background with recurrent parent or both recurrent parent and non-recurrent parent is obtained.

9. The use of claim 4, wherein the PCR amplification product is: if the result of the PCR amplification product is no PCR amplification product, the tobacco plant with the non-recurrent parent genetic background of the homozygous mutant nic1 is obtained.

10. Use according to claim 3, wherein the undesirable agronomic traits comprise difficult yellowing, low yield and short plant.

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