CN115873983A - Molecular marker for improving utilization of nic1 locus for breeding low-nicotine tobacco and application thereof - Google Patents
Molecular marker for improving utilization of nic1 locus for breeding low-nicotine tobacco and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker for improving the breeding of low-nicotine tobacco by utilizing a nic1 locus and application thereof, wherein the application comprises the steps of utilizing tobacco nicotine to synthesize a main effect regulation mutant type locus nic1, detecting the genomic DNA of a tobacco single plant in a segregation population in a hybridization segregation progeny, keeping the nic1 locus, simultaneously improving the selection efficiency of a chromosome region where the nic1 locus is located to revert to a recurrent parent genetic background by a system breeding or backcross breeding means, thereby improving the poor agronomic characters of non-recurrent parents, improving the genetic background selection efficiency of progeny tobacco plants and accelerating the application of the mutant type nic1 locus in breeding of low-nicotine tobacco varieties. The molecular marker disclosed by the invention can be used as an auxiliary application of the molecular marker for improving the genetic background recovery selection efficiency of a low-nicotine tobacco strain while utilizing a mutant type nic1 locus in tobacco quality breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of co-dominant and dominant molecular markers for improving the recovery rate of a tobacco strain genetic background after mainly regulating and controlling mutation site nic1 by utilizing tobacco nicotine synthesis.
Background
Cultivation of tobacco (NicotianatabacumL.) is an annual herbaceous plant of the solanaceae family, which is also one of the important commercial crops. Nicotine (Nicotine) is an important characteristic alkaloid in tobacco cultivation. According to different genetic backgrounds of tobaccos, the content of nicotine in the tobacco leaves accounts for 90 to 95 percent of the total alkaloid content of the tobacco leaves. The content of nicotine in tobacco leaves is also directly related to the quality and commercial use of tobacco. In comparison with conventional cigarettes, a VLN tobacco product (e.g. VLN cigarette) to be delivered ® King、VLN ® Menthol King) has a nicotine content reduced by about 95%, and has the characteristics of low harm, low tar and no compensatory smoking. Tobacco companies and related research institutions at home and abroad are working on the utilization of the main regulatory gene locus for nicotine synthesisNIC1The nicotine content in tobacco leaves is regulated and controlled, and the nicotine content in tobacco products is reduced, so that the aim of reducing the harm of tobacco to human bodies is fulfilled. In addition, a great number of research institutions at home and abroad utilize biotechnology means mainly based on grafting and gene editing to reduce tobacco nicotine biosynthesis or nicotine transportation by changing tobacco cultivation measures so as to achieve the purpose of regulating and controlling the nicotine content in tobacco leaves. Since the american tobacco product is a blended tobacco, the 22 st century tobacco company is also working with north carolina state university to develop low nicotine tobacco varieties of burley, oriental, and sun cured tobacco.
Mutants with different contents of tobacco nicotine were originally found in cigars, and later tobacco breeders transferred these mutant sites into different lines of flue-cured tobacco and burley tobacco respectively by means of cross backcross breeding. In Burley tobacco, tobacco breeders breed four Burley tobacco nicotine synthetic Near Isogenic Lines (NILs) materials, named HA Burley21, hibarley 21, LIBurley 21 and LABurley21, respectively, by anther culture and chromosome doubling techniques. Classical genetic studies of these four materials found that tobacco NICOTINE content was regulated by two independently inherited dominant sites (designated nicotin 1 and nicotin 2, abbreviated as NIC1 and NIC 2) (Legg, p.d., collins, g.b.,1971. Heredity of percent total alkaloids in Nicotiana tabacum L.: genetic effects of two loci in Burley 21x LA Burley 21posts, can.J.Genet.Cytol.13,287-291.Legg, P.D., chaplin, J.F., collins, G.B.,1969. Introduction of percent total alkali metals in Nicotiana tabacum L.: post delivery derived from cross-correlations of low alkali lines with trough-current variations.J.Hered.60, 213-217). Wherein the NIC1 site has 2.4-fold more capacity to modulate nicotine synthesis than the NIC2 site. According to related reports, the NIC2 site in tobacco at least consists of 7 Ethylene Response Factor ERF (Ethylene Response Factor) transcription Factor genes, including NtERF189, ntERF115 and NtERF 179. These transcription factor genes are capable of binding to the promoter region of metabolic enzyme genes on the nicotine synthesis pathway (e.g., ntPMT2 and NtQPT 2) to transcriptionally activate downstream gene expression (Shoji, T., kajikawa, M., hashimoto, T.,2010. Customized transcription factor genes regulation in biosynthesis in bacteria co. Plant Cell 22,3390-3409, shoji, hashimoto, T.,2012.DNA-binding and transcription activity properties of bacteria NIC 2-circulation ERF189 and transcription factors in plant technology 29, 35-42). Among them, the mutant nic2 site in the LA Burley21 line is caused by the loss of the chromosome fragment in which it is located (Kajikawa M., sierro N., kawaguchi H., et al.,2017.Genomic identities of the bacterial biosynthesis pathway in tobacao, plant Physiology 174, 999-1011.). On the other hand, recent studies have also found that other classes 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, which can be regulated in concert with a NIC2 site transcription factor (e.g., ntERF 189), can bind to the G-box and GCC-box transcription elements, respectively, in the promoters of key metabolic rate-limiting enzymes in the nicotine synthesis pathway (e.g., ntPMT, ntQPT) to maximize expression of these genes (Shoji T. And Hashimoto T.2011.Tobacco MYC2 derivatives-inducible genes and by way of the NIC 2-loci ERF genes, plant Cell physiol.52 (6): 1117-1130.). On the other hand, the decrease in tobacco alkaloid content caused by overexpression of the tobacco auxin response factor NtARF6 transcription factor is caused by antagonism between the jasmonic acid pathway and other phytohormone signal transduction pathways (e.g., ethylene, salicylic acid, abscisic acid). The jasmonic acid biosynthesis pathway is inhibited and simultaneously activated by ethylene, salicylic acid, abscisic acid and pathogen infection defense reaction, thereby antagonizing jasmonic acid-induced secondary metabolism regulation and reducing nicotine synthesis gene expression in tobacco (Hu et al, 2021. Transcriptional metabolism genes into the nicotine RESPONSE FACTOR 6-mediated expression of nicotine biosynthesis in tobaccos, plant Mol biol.107 (1-2): 21-36.). Furthermore, it has recently been found that the MYB transcription factor family gene NtMYB305a can be produced by binding to the AT-rich element in the GAG region on the tobacco NtPMT1a promoter. At the same time, ntMYB305a also regulates the expression of other nicotine synthesis genes in a similar manner, thereby positively regulating the biosynthesis of nicotine in tobacco (Bian et al, 2022.NtMYB305a bindings to the jasmonate-reactive GAG region of NtPMT1a promoter to regulated nicotine biosynthesis. Plant physiology.188 (1): 151-166.). Researchers at home and abroad have found a new ERF (NIC 2-like) gene cluster, whose composition is highly similar to that of NIC2 gene cluster, in bioinformatics means, in the genomes of flue-cured tobacco (Yunyan 87) and Burley (HABurley 21), respectively (Kajikawa M., sierro N., kawaguchi H., et al, 2017.Genomic instruments, in the evaluation of the microbial biosynthesis pathway in handbisco, plant Physiology 174,999-1011 Sui X.et al, 2019.Ethylene response factor NtERF91 porous regulation of microorganisms in handbisco (Nicora. L., biophys. 517-164, biological reaction 1.171, biological 1-biological 1. Biological of the family of academic systems). Recently, the NIC2-like gene cluster has been proved to be the NIC1 site 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. Urravel the family of NIC1-logic on a nucleotide biosynthesis regulation in tobacaco, biorxiv). A plurality of SNP (Single Nucleotide Polymorphism) molecular markers for detecting the NIC1 gene of burley tobacco are developed at the same time, and the detected markers and methods are applied for patents (PCT/US 2018/038679). On the other hand, the cause of the decrease in the expression level of nicotine synthesis gene in nic1 mutant has been substantially clarified. The chromosomal region of the mutant NIC1 gene cluster is flanked by two chromosomal segments (NIC1-S and NIC1-B) deletion, which eventually results in structural variation of the chromosome of the NIC1 gene cluster and thus affects the normal expression of the genes within the gene cluster (Sui X. Et al.,2020. Urravel the family of NIC1-loci biosynthesis in tobaco, biorxiv).
However, the development of low nicotine tobacco varieties using the mutant nic1 gene cluster as a common breeding tool still faces some problems. According to the related literature, mutant nic 1tobacco line (LA Burley 21) has many Agronomic disadvantages compared to normal tobacco, such as slow yellowing, difficult baking, low yield, poor quality of the tobacco leaves after baking (Chaplin, J.F., & Burk, L.G. (1983), agricultural, chemical, and book characteristics of fire-cured tobacco lines with differential levels of total alkali metals. Loop Science,75,133-136.Chaplin, J.F., & Weeks, W.W. (1976), association between local alkali metals and residues in fire-cured tobacco. C.C.C.Cruis, 16,416-418.; legg.D., P.D., col.B.1, C.10. C.1970). Therefore, in the process of breeding a low-nicotine tobacco variety by backcross by using the LAFC53 as a germplasm material, the nick 1 mutant locus is required to be retained to the maximum extent, and the chromosome segment of the recurrent parent in the system breeding or backcross generation is preferably retained. Therefore, the whole genome re-sequencing technology is utilized to compare recurrent parent genome (such as NC95 HA) and non-recurrent parent (LAFC 53) to search for difference in the peripheral region of the nic1 chromosome region, and a specific (codominant or dominant) molecular marker capable of distinguishing the difference of the amphipathic basic genome is developed, so that the method HAs important significance for breeding the tobacco variety with low nicotine content by adopting the molecular marker in an auxiliary way.
At present, although there are reports on the molecular marker development research of NiC1 site for synthesizing tobacco nicotine, the currently reported research results are all selected and developed based on the genetic prospect of NIC1 mutant site, namely, only deletion fragments (NIC1-S and NIC1-B) causing the expression level of genes in the NIC1 gene cluster to be reduced are selected for marker selection (Sui X.et al, 2020. Urravel the family of NIC1-deletion biosynthesis regulation in tobacaco, biorxiv). For example, markers linked to the nic1 gene cluster are reported as dCAPS markers and SSR markers developed based on SNP variation sites. There are some deficiencies in tobacco breeding practices and tobacco production: 1) There are serious limitations to the scope of use, namely the reported and patented detection of dCAPS markers identifying the nic1 gene, which is only applicable to burley tobacco types, is less extensive; the cultivated tobacco contains various types such as flue-cured tobacco, aromatic tobacco, cigar tobacco, air-cured tobacco and sun-cured tobacco and daylily tobacco besides burley tobacco types, and the various tobacco types or the varieties cannot be detected and identified by dCAPS markers. 2) The molecular markers currently developed are based solely on marker-assisted selection (i.e., foreground selection) of the nic1 mutant gene cluster itself, but not on the genetic background of recurrent or non-recurrent parents (e.g., LAFC 53). In the process of breeding or backcrossing the low-nicotine tobacco variety by using the LAFC53 as the non-recurrent parent, the nick-type site of the gene nic1 cannot be reserved to the maximum degree, and the chromosome segment of the recurrent parent in the backcross generation is reserved to the maximum degree, so that the defects of the mutant nic 1tobacco non-recurrent parent (LAFC 53) in agronomic characters (slow yellow fall, difficult baking, low yield and poor tobacco quality) are effectively improved.
The invention utilizes two near-isogenic system materials NC95 HA (NIC 1NIC1NIC2NIC 2) with different nicotine contents and a tobacco material LAFC53 (NIC 1NIC1NIC2NIC 2) with low nicotine content as experimental materials, and develops specific (codominant and dominant) molecular markers capable of distinguishing the difference of the amphiphilic basic genome by comparing the structural variation of the chromosome regions where the two near-isogenic systems are located at the NIC1 locus through a tobacco genome re-sequencing technology. By constructing the second generation of selfs (F) 2 ) And separating the population, and reserving seeds for the single strain after screening out individuals with the genotype of (nic 1nic1nic2nic 2) in the selected population. Specific utilization of the reserved single strain line in F3 generationThe marker is used for genetic background selection and strain agronomic character comparison test to screen out F 3 The low-nicotine tobacco strain with higher genetic background reversion and obviously improved agronomic characters in generations than a non-recurrent parent (LAFC 53) accelerates the utilization of molecular Marker Assisted Selection (MAS) in the breeding of the low-nicotine-content tobacco variety, thereby realizing the convenient, fast, stable and reliable cultivation of the low-nicotine-content and high-quality tobacco variety.
Disclosure of Invention
The first purpose of the invention is to provide a specific (dominant and co-dominant) molecular marker which effectively improves the genetic background selection efficiency of the offspring of the system breeding or backcross breeding after synthesizing the main effect regulation nic1 mutant type locus by using the tobacco nicotine; the second purpose is to improve the genetic background selection efficiency of backcross offspring after mainly and effectively regulating the nic1 mutant site through the synthesis with tobacco nicotine so as to improve the poor agronomic characters of non-recurrent parents (parents carrying the mutant nic1 site) and accelerate the application of the mutant nic1 site in breeding low-nicotine tobacco varieties.
The application of the co-dominant and dominant molecular markers which can improve the recovery rate of the genetic background of the tobacco strain after the main effect of tobacco nicotine synthesis is utilized to regulate and control the mutation site nic1 is used for detecting the selection efficiency of the chromosome region where the nic1 site is located to recover to the recurrent genetic background by a systematic breeding or backcross breeding means while the mutation type nic1 site is kept in the genome DNA of a single tobacco plant in a tobacco segregation population, and finally, the defect of the agronomic character introduced by the mutation type nic 1tobacco non-recurrent parent is effectively improved.
The molecular marker can detect chromosome structure variation generated in the peripheral region of a chromosome region where a tobacco nicotine synthesis major regulation mutant type site nic1 is located, and 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’。
the application of the codominant and dominant molecular marker provided by the invention comprises the following steps: and the genetic background recovery rate of the tobacco strain is improved.
The application of the invention is specifically as follows: the method has the advantages that the selection efficiency of the tobacco single plant genome DNA in the tobacco segregation population for reverting the chromosome region of the nick 1 locus to the recurrent genetic background is improved by a systematic breeding or backcross breeding means while the nick 1 locus is reserved, so that the undesirable agronomic characters (such as difficult yellowing, low yield, short and small plants and the like) of the non-recurrent parent (carrying the mutant nick 1 locus parent) are improved, the genetic background selection efficiency of the progeny tobacco plants is improved, and the application of the mutant nick 1 locus in breeding of low-nicotine tobacco varieties is accelerated.
The application of the invention is as follows: 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, detecting PCR amplification products, and analyzing according to the result of the amplification products.
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.
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 invention is the non-recurrent parent genetic background with homozygous mutant nic 1.
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, the genetic background of the tobacco is heterozygote, namely the tobacco plant with the genetic background of recurrent parent and non-recurrent parent.
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 invention has recurrent parent or hybrid tobacco genetic background with recurrent parent and non-recurrent parent.
According to the invention, if the result of the PCR amplification product is a PCR-free amplification product, the tobacco plant with the non-recurrent parent genetic background of the homozygous mutant nic1 is obtained.
In order to simply and efficiently select a tobacco variety with low nicotine content and good agronomic characters, offspring material containing homozygous genotype nic1nic1 is selected in a targeted and specific manner, and chromosome segments carried by a chromosome region where a nic1 locus is located in a parent (carrying a nic1 mutant locus, such as LAFC 53) derived from low nicotine are replaced to the greatest extent. The invention provides a co-dominant and dominant molecular marker of Target1, target2, target3, target4, target5 and Target6 for improving the Tobacco strain genetic background recovery rate after the Tobacco nicotine is synthesized into the main effect regulation mutation site NIC1, the molecular marker adopts a method of Tobacco genome re-sequencing analysis (Tobacco wheelgenome sequencing), and a specific molecular marker capable of distinguishing the difference of amphiphilic gene groups is developed by comparing the structural variation of two near-isogenic materials NC95 HA (NIC 1NIC1NIC2NIC 2) and LAFC53 (NIC 1NIC1NIC2NIC 2) with different nicotine contents in the chromosome region where the mutation site NIC1 is located. The molecular marker can be used as an auxiliary means for breeding low-nicotine-content tobacco varieties, so that the molecular marker is used for assisting selection to improve genetic background selection efficiency of backcross breeding offspring and improve agronomic characters, and the breeding process of the low-nicotine-content tobacco varieties is accelerated.
The invention utilizes two near isogenic lines with different nicotine contents as the materials NC95 HA (high nicotine content: NIC1NIC2NIC 2) and LAFC53 (low nicotine content: NIC1NIC1NIC2NIC 2) as the parents, adopts a tobacco genome re-sequencing analysis method, and utilizes the chromosome Structure Variation (SV) existing in the chromosome flanking region of the mutant NIC 1to develop and design the specific molecular marker which can effectively distinguish chromosome segments from recurrent parents and non-recurrent parents (such as NC95 HA and LAFC 53). At the same time, through hybridization and selfing, the second generation of selfing (F) is screened out 2 ) In the population, 24 individual strains of genotype nic1nic1nic2nic2 are bagged and reserved. Extraction of F 2 And after the generation of genome DNA of each individual plant, screening genetic backgrounds of each strain by using the developed specific markers. On the other hand, 24 individuals were selfed into progeny (i.e., F) 3 ) And (3) performing field planting survey on the agronomic characters and statistical analysis of each strain to obtain the strain with obviously improved agronomic character(s) of the tobacco strain compared with the non-recurrent parent. The results show that the markers can improve the Selection efficiency of the Marker Assisted Selection (MAS) in the breeding of low-nicotine-content tobacco varieties.
The co-dominant and dominant molecular markers capable of improving the genetic background recovery rate of the tobacco strain after the mutation site nic1 is synthesized by utilizing the tobacco nicotine mainly and effectively have the characteristics of stability, reliability, simplicity, convenience, quickness and low cost, so that the molecular marker can be used as the molecular marker for improving the genetic background selection efficiency while utilizing the mutation type nic1 site in the cultivation of the tobacco variety with low nicotine content.
Drawings
FIG. 1 is a gel electrophoresis diagram of PCR amplification products of specific molecular markers in 2 nearly isogenic system materials, wherein the specific molecular markers are detected by chromosome structure variation existing in a chromosome flanking region where a tobacco nicotine synthesis major regulatory site mutant nic1 is located; wherein, A, co-dominant marker Target1; b, co-dominant marker Target2; c, co-dominant marker Target3; d, dominant marker Target4; e, a 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 respectively: 100bp,200bp,300bp,400bp,500bp;
FIG. 2 shows NC95 HA × LAFC 53F 2 Alkaloid content profiles for four genotypes A _ B _, A _ bb, aaB _, and aabb individuals in the population (where A: NIC1; B: NIC2; a: NIC1; B: NIC 2).
FIG. 3 shows that the codominant markers Target1, target2 and Target3 and the dominant markers Target4, target5 and Target6 are at 8 parts F 2 Statistical graphs of PCR amplification product bands in each individual strain of generations (genotype is nic1nic1nic2nic 2), wherein A represents a chromosome fragment derived from the recurrent parent NC95 HA, and B represents a chromosome fragment derived from the non-recurrent parent LAFC53 with a nic1 mutant site;
FIG. 4 shows the results of screening to F 2 F obtained from single plant 3 The statistical analysis chart of the agronomic characters of the tobacco-substitute strain and the reference variety LAFC53 comprises natural plant height, natural leaves, topping plant height, stem girth, waist leaf length, waist leaf width, effective leaf number and pitch.
Detailed Description
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
The codominant and dominant molecular markers capable of improving the tobacco strain genetic background reversion rate after synthesizing the major regulatory mutation site nic1 by using the tobacco nicotine are numbered as Target1, target2, target3, target4, target5 and Target6, and the nucleotide sequences of PCR amplification products are respectively shown as 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 6 sites corresponding to the molecular markers are respectively as follows:
the 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’-CCTAATCCTCTTCGAATCTTAAATC-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’-TTTAAGTTCTTGTTTTTCTCCTTGA -3’(SEQ ID No.8)，
Target5R：5’-AATCAGTTCCTTCCTCACACTAAC-3’；
the Target6 sequence is:
Target6F：5’-AAATTCAGAGAGATTTTTGGAAAGT-3’(SEQ ID No.9)，Target6R：5’-ATAAAGAAGCAGAAATAGGGAAAAT-3’；
the invention relates to application of a specific molecular marker for detecting chromosome structure variation existing in a chromosome flanking region where a tobacco nicotine synthesis major regulatory site mutant nic1 is located, which is characterized in that when whether a mutant genotype nic1 site exists in a tobacco genome DNA is detected, the selection efficiency of tobacco genetic background in breeding offspring is improved by using system breeding and backcross breeding means, the agronomic characters of selected tobacco plants in the field are improved, and the application in breeding low-nicotine tobacco varieties is accelerated.
The application of the specific molecular marker for detecting the chromosome structure variation existing in the chromosome flanking region where the tobacco nicotine synthesis major regulatory site mutant nic1 is located is to respectively amplify the genomic DNA of tobacco to be detected by using a primer of a Target1 sequence, a primer of a Target2 sequence and a primer of a Target3 sequence, and detect a PCR amplification product, wherein if the PCR amplification product 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 recurrent parent NC95 HA is obtained; if the PCR amplification product simultaneously contains three nucleotide sequences shown as SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6, the PCR amplification product is the genetic background of the tobacco with the non-recurrent parent LAFC53; if the PCR amplification product simultaneously 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, namely the genetic background of the tobacco is the tobacco plant with the recurrent parent and non-recurrent parent heterozygous genetic background. If the primer of the Target4 sequence, the primer of the Target5 sequence and the primer of the Target6 sequence are used for respectively amplifying the genomic DNA of the tobacco to be detected, detecting a PCR amplification product, and if the PCR amplification product simultaneously contains three nucleotide sequences shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, the PCR amplification product is the tobacco genetic background of the homozygous type with the recurrent parent NC95 HA or the heterozygous type with the recurrent parent and the non-recurrent parent; if the PCR has no amplification product, the tobacco plant is the tobacco plant with homozygous mutant nic1 non-recurrent parent LAFC53 genetic background.
The invention is further illustrated by the following specific examples:
example 1
Two near-isogenic materials, namely a high-nicotine-content Tobacco strain NC95 HA (genotype NIC1NIC1NIC2NIC 2) and a low-nicotine-content Tobacco strain LAFC53 (genotype NIC1NIC1NIC2NIC 2), were subjected to deep re-sequencing by using a Tobacco whole genome re-sequencing analysis (Tobacco Wholegenome re-sequencing) method. And detecting and comparing whether the chromosome flanking region of the nic1 exists chromosome structure variation between the two parents through sequence read comparison, and acquiring related chromosome fragment sequence information according to the variation type. Visualization of the tobacco Whole genome re-sequencing analysis results was performed on IGV (Integrated Genomics Viewer) software, and the design of the relevant specific markers (primers) was done using the Primer3 on-line tool (https:// bioinfo. Ut. Ee/Primer3-0.4.0 /).
1. Experimental Material
Two near isogenic line materials, namely a high nicotine content tobacco strain NC95 HA (NIC 1NIC1NIC2NIC 2) is taken as a female parent, and a low nicotine content tobacco LAFC53 strain (NIC 1NIC1NIC2NIC 2) is taken as a male parent. Parent materials with high and low nicotine contents are planted in 2017, and F is obtained by hybridization 1 And (4) generation. First filial generation (F) was planted in 2018 winter 1 ) The nic1 and nic2 gene segregation population was obtained in the early 2019 (F) 2 Generation). Planting two parents in 2019, F 1 And F 2 Generation of material, and in F 2 Obtaining a single plant with the genotype of (nic 1nic1nic2nic 2) from generation materials, bagging and collecting seeds to obtain F 3 And (4) generation strain. Planting parent material with high and low nicotine content in 2021 year, and F 3 Generation line, obtaining parent and F 3 And (5) agricultural character survey data of generation fields.
2. Parent and F 2 Isolated population nicotine content determination
Transplanting the test material to a field after seedling formation, wherein the row spacing is 100cm multiplied by 50cm; conventional cultivation and field management are adopted, F 2 And (4) topping after the whole group buds, and harvesting and baking each single fresh tobacco leaf after topping for 2 weeks. Grinding each leaf after baking and measuring the nicotine content. For the obtained parents and F 2 The nicotine content data of each individual leaf of the colony is analyzed, and the genotype data analysis basis is as follows: performing data statistics according to the banding patterns of the individual plants, namely, the individual plants of which two sites are consistent with the banding patterns of the NC95 HA parent are marked as 'A _' or 'B _'; the single plant bands with two sites consistent with the LAFC53 parent band type are marked as "aa" or "bb", and the single plant bands with unclear bands or no amplified bands are marked as "U".
3. Specific marker design
Before topping, collecting parents and F in the field 2 Each single fresh leaf of the group refers to the extraction of the whole genome DNA of the tested material by adopting a conventional CTAB method or a plant tissue DNA extraction kit, and the method can refer to the existing literature or the instruction in the kit. PCR reaction system preparation, product amplification and 2% agarose gel electrophoresis detection of the amplified products were carried out according to the method provided by Sui et al (Sui X., huang Y., et al.2011, molecular analysis of an experimental plant, sabia parviflora and its additives by DNA screening technique. Plant medicine 2011,77 (5): 492-496.).
The PCR reaction system is as follows: 30-50 ng/. Mu.L DNA, 1.0. Mu. Mol/L each of forward and reverse primers, 1.5mmol/L dNTPs, 2. Mu.L 10 XPCR Buffer (Mg) 2+ plus), 0.75-1.0U Q5 Hi-Fi DNA polymerase (c) ((ii)
High-Fidelity DNA Polymerase, NEB), add double distilled water to 20. Mu.L.
The PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30sec, renaturation at 55 ℃ for 30sec, extension at 72 ℃ for 30sec,30 cycles, and extension at 72 ℃ for 5min.
The gel electrophoresis detection is to adopt 2 percent agarose gel and 1 xTBE electrophoresis buffer solution to carry out electrophoresis for 45 minutes at constant voltage of 100V for separation, and finally develop by an ethidium bromide method.
4. Parent verification for developing specific marker based on nic1 locus chromosome structure variation
In the seedling stage, primers designed according to the result of analysis of the re-sequencing data are used for carrying out PCR amplification on 2 parts of materials of the high-nicotine-content parent NC95 HA (NIC 1NIC1NIC2NIC 2) and the low-nicotine parent LAFC53 (NIC 1NIC1NIC2NIC 2), and specific markers (dominant or co-dominant) capable of distinguishing chromosome segments of chromosome regions of mutant NIC1 sites of the two parents are screened and verified. The results of the screening are shown in FIG. 1: the codominant specific markers Target1, target2 and Target3 can distinguish chromosome fragments of two parents around a chromosome region where a nic1 locus is located, namely, the band types of the markers Target1, target2 and Target3 in a high-nicotine-content parent NC95 HA are completely consistent, and only one specific band of 776bp (a sequence is shown as SEQ ID No. 1), 716bp (a sequence is shown as SEQ ID No. 3) and 1458bp (a sequence is shown as SEQ ID No. 5) appears; the band patterns in the parent LAFC53 with low nicotine content are completely consistent, and only one specific band with 457bp (the sequence is shown as SEQ ID NO. 2), 396bp (the sequence is shown as SEQ ID NO. 4) and 420bp (the sequence is shown as SEQ ID NO. 6) appears. Wherein the lengths of the sequences shown by SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5 are 776bp, 716bp and 1458bp respectively, and are the specific PCR amplification nucleotide sequences of codominant specific markers Target1, target2 and Target3 in the tobacco parent NC95 HA with high nicotine content respectively; the lengths of the sequences shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 are 457bp, 369bp and 420bp respectively, and the sequences are respectively specific PCR amplification nucleotide sequences of codominant specific markers Target1, target2 and Target3 in the low-nicotine-content tobacco parent LAFC53 containing a nic1 mutant site.
The dominant specific markers Target4, target5 and Target6 can also distinguish chromosome segments of two parents around the chromosome region where the nic1 locus is located, namely, the band types of the markers Target4, target5 and Target6 in the high-nicotine-content parent NC95 HA are completely consistent, and only one specific band of 1051bp (the sequence is shown as SEQ ID NO. 7), 675bp (the sequence is shown as SEQ ID NO. 8) and 1317bp (the sequence is shown as SEQ ID NO. 9) appears; whereas the corresponding specific band was not successfully amplified in the low nicotine content parent LAFC 53. Wherein the lengths of the sequences shown by SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 are 1051bp, 675bp and 1317bp respectively, and are the specific PCR amplification nucleotide sequences of dominant specific markers Target4, target5 and Target6 in the tobacco parent NC95 HA with high nicotine content respectively; while dominant specific markers Target4, target5 and Target6 failed to amplify specific PCR bands in the low nicotine content tobacco parent LAFC53 containing a nic1 mutant site.
The results show that the markers Target1, target2 and Target3 can respectively distinguish chromosome segments of the chromosome regions of the two parents at the nick 1 site of the mutant type, and the markers are codominant markers. If the PCR amplification product simultaneously contains three nucleotide sequences shown as SEQ ID No.1 (776 bp), SEQ ID No.3 (716 bp) and SEQ ID No.5 (1458 bp), the three nucleotide sequences are the NC95 HA chromosome background segments with recurrent parent; if the PCR amplification product simultaneously contains three nucleotide sequences shown as SEQ ID No.2 (457 bp), SEQ ID No.4 (396 bp) and SEQ ID No.6 (420 bp), the three nucleotide sequences are chromosome fragments with non-recurrent parent LAFC53 chromosome background; if the PCR amplification product simultaneously contains six nucleotide sequences shown as 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, the hybrid chromosome fragment with recurrent parent and non-recurrent parent is obtained.
Meanwhile, markers Target4, target5 and Target6 can also distinguish the chromosome segment sources of the two parents in the chromosome region where the mutant nic1 locus is located, and the markers are dominant markers. If the PCR amplification product simultaneously contains three nucleotide sequences shown as SEQ ID No.7 (1051 bp), SEQ ID No.8 (675 bp) and SEQ ID No.9 (1317 bp), the three nucleotide sequences are chromosome source fragments at least in the recurrent parent NC95 HA; if the markers Target4, target5 and Target6 can not successfully amplify the corresponding specific bands, the specific bands are chromosome segments with the region where the nic1 genotype in the non-recurrent parent LAFC53 is located.
Example 2
Specific molecular markers for detecting chromosome structure variation existing in the chromosome flanking region where the tobacco nicotine synthesis major regulatory site mutant nic1 is located are used for improving molecular marker-assisted selection, improving genetic background selection efficiency of backcross breeding offspring, improving agronomic characters and improving the agronomic characters in F 3 And (5) verifying strains.
Data analysis
Tobacco genomic DNA extraction and purification was carried out as described in example 1，F 2 And (4) carrying out nicotine content determination and NIC1 and NIC2 locus marker typing analysis after topping of individual plants of the population. Secondly, specific molecular markers Target1, target2, target3, target4, target5 and Target6 obtained by analyzing chromosome structure variation existing in the chromosome flanking region where the tobacco nicotine synthesis major regulatory site mutant nic1 is located and screened by a resequencing method are utilized to perform F pair 2 23 individuals in the population were genotyped. Finally, performing data statistics on the belt types of the selected 8 single plants, namely, the single plant consistent with the belt type of the NC95 HA parent is marked as 'A'; the individual bands that are identical to the LAFC53 parental band pattern are marked "B".
F 2 The results of the nicotine content of the individual plants in the population are as follows: among 377 individuals, there were 224 individuals with genotype A _ B _, 36 individuals with genotype A _ bb, 94 individuals with genotype aaB _, and 23 individuals with genotype aabb. To F 2 Genotype analysis was performed on 377 individuals as shown in FIG. 2.
Example 3
Specific molecular markers detected by chromosome structure variation existing in chromosome flanking regions where tobacco nicotine synthesis major regulatory site mutant nic1 is located are utilized to improve F in filial generations of parent NC95 HA and parent LAFC53 3 The field agronomic characters of the generation low nicotine content strain.
1. Experimental Material
The used markers are specific molecular markers for detecting the structural variation of the chromosome in the flanking regions of the chromosome where six nic1 s of Target1, target2, target3, target4, target5 and Target6 are located. The plant material is HA NC95 (genotype NIC1NIC1NIC2NIC 2), LAFC53 (genotype NIC1NIC1NIC2NIC 2), F NC95 (genotype NIC1NIC2NIC 2), or F NIC2 2 8 individuals with genotype nic1nic1nic2nic2 in segregating population (NC 95 HA. Times.LAFC 53) and corresponding F 3 And 8 strains are generated.
2. Data processing
First, the tobacco material was subjected to extraction and purification of tobacco genomic DNA by the method described in example 1. Secondly, the tobacco individual plant is subjected to genotype analysis by utilizing a specific molecular marker for detecting the chromosome structure variation existing in the chromosome flanking region where the tobacco nicotine synthesis major regulatory site mutant nic1 is located. Finally, carrying out data analysis on the banding patterns of each individual plant, namely, simultaneously appearing nucleotide sequences shown as SEQ ID No.1, SEQ ID No.3 and SEQ ID No.5 in the PCR amplification product, namely the specific PCR amplification nucleotide sequence of the NC95 HA genetic background of the tobacco recurrent parent with high nicotine content; if the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6 appear in the PCR amplification product at the same time, the hybrid chromosome fragment with recurrent parent and non-recurrent parent (carrying nic1 locus mutant) is obtained; if the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.2 and SEQ ID NO.4 are simultaneously present in the PCR amplification product, the specific PCR amplification nucleotide sequence with the genetic background of the non-recurrent parent LAFC53 containing the nic1 mutant site is obtained.
As can be seen from fig. 3: in 8 candidate F to be tested 3 Among the tobacco-substitute strains, the strain NC95 HA × LAFC 53F 3 The-72 is a strain with the most NC95 HA parent genetic background while ensuring the prospect of having low nicotine character (nic 1nic1nic2nic 2), the agronomic characters of the strain are not obviously different from the parent NC95 HA, and the main agronomic characters are greatly improved; while strain NC95 HA × LAFC 53F 3 112 is a strain which is closest to the genetic background of the parent LAFC53 under the condition of ensuring that the strain has a low nicotine character (nic 1nic1nic2nic 2), has a stem circumference slightly higher than that of the parent LAFC53 and has an effective leaf number lower than that of the parent LAFC53, has no obvious difference between other agronomic characters (such as natural plant height, natural leaf number and topping plant height) and the parent LAFC53, and has poor overall agronomic characters; and others F 3 Strains (e.g. NC95 HA. Times. LAFC 53F 3 -112、NC95 HA ×LAFC53F 3 -12、NC95 HA×LAFC53 F 3 -151 and NC95 HA × LAFC 53F 3 308) the chromosomal region around its nic1 locus was replaced (homozygous or heterozygous) to a different extent by the chromosomal fragment of the parent (NC 95 HA), and its overall agronomic performance was also improved to a different extent compared to the parent LAFC53, as shown in figure 3. These candidate lines can be further accelerated by accelerating line homozygosis through systematic breeding or by backcrossing with the parent NC95 HA 3 The genetic background selection efficiency of different individual plants in the candidate plant line is improved continuously, and finally the molecular assisted selection is used for breeding the tobacco variety with low nicotine content.
And (4) conclusion: the six specific (dominant or co-dominant) markers can be used for simply, quickly and stably detecting the chromosome fragment replacement condition around the chromosome where the tobacco nic1 locus is located, clearly identifying the homozygous or heterozygous condition of chromosome fragments from different parents, and meanwhile, selecting the tobacco intermediate material with low nicotine content, which has high genetic background recovery rate and greatly improved various agronomic characters, in a targeted and specific manner, so that the breeding efficiency of the tobacco variety with low nicotine content can be greatly improved.
The foregoing is only a part of the specific embodiments of the present invention, and the specific contents or common general knowledge in the schemes are not described herein too much (including but not limited to the shorthand, abbreviation, units commonly used in the art). It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and it is obvious for those skilled in the art that all the technical solutions obtained by using the equivalent substitution or the equivalent change fall within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents 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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| CN202211469357.5A CN115873983A (en) | 2022-11-22 | 2022-11-22 | Molecular marker for improving utilization of nic1 locus for breeding low-nicotine tobacco and application thereof |
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Citations (4)
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|---|---|---|---|---|
| US20160374387A1 (en) * | 2015-06-26 | 2016-12-29 | Altria Client Services Llc | Compositions and Methods for Producing Tobacco Plants and Products Having Altered Alkaloid Levels |
| CN107365777A (en) * | 2017-09-08 | 2017-11-21 | 云南省烟草农业科学研究院 | One grows tobacco nicotine content controlling gene NtCLC b and its cloning process and application |
| CN111836539A (en) * | 2018-01-12 | 2020-10-27 | 奥驰亚客户服务有限公司 | Compositions and methods for producing tobacco plants and products with altered levels of alkaloids |
| CN115279177A (en) * | 2020-01-08 | 2022-11-01 | 北卡罗来纳州立大学 | Genetic method for realizing ultra-low nicotine content in tobacco |
-
2022
- 2022-11-22 CN CN202211469357.5A patent/CN115873983A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160374387A1 (en) * | 2015-06-26 | 2016-12-29 | Altria Client Services Llc | Compositions and Methods for Producing Tobacco Plants and Products Having Altered Alkaloid Levels |
| CN107365777A (en) * | 2017-09-08 | 2017-11-21 | 云南省烟草农业科学研究院 | One grows tobacco nicotine content controlling gene NtCLC b and its cloning process and application |
| CN111836539A (en) * | 2018-01-12 | 2020-10-27 | 奥驰亚客户服务有限公司 | Compositions and methods for producing tobacco plants and products with altered levels of alkaloids |
| CN115279177A (en) * | 2020-01-08 | 2022-11-01 | 北卡罗来纳州立大学 | Genetic method for realizing ultra-low nicotine content in tobacco |
Non-Patent Citations (4)
| Title |
|---|
| QIULIN QIN等: "NIC1 cloning and gene editing generates low-nicotine tobacco plants", PLANT BIOTECHNOLOGY JOURNAL, vol. 19, 1 September 2021 (2021-09-01), pages 2150 - 2152 * |
| RAMSEY S. LEWIS等: "Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes", FRONTIERS IN PLANT SCIENCE, vol. 11, 3 April 2020 (2020-04-03), pages 368 * |
| SUI XUEYI等: "Unravel the Mystery of NIC1-locus on Nicotine Biosynthesis Regulation in Tobacco", BIORXIV, 4 July 2020 (2020-07-04), pages 1 - 44 * |
| 史宏志等: "低/超低烟碱含量烟叶生产途径及对烟叶化学成分和质量的影响", 中国烟草学报, vol. 24, 31 December 2018 (2018-12-31), pages 102 - 111 * |
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