Molecular marker related to tobacco short petiole character and biological material and application thereof

Abstract

The invention discloses a biological material related to the character of a short leaf stalk of tobacco and application of the biological material. A short petiole mutant M46 is obtained through EMS mutagenesis, and a molecular marker SNP-4 which is completely co-separated from short petiole characters is developed in combination with whole genome re-sequencing. The SNP-4 is located at the 145975753bp of the 7th chromosome of the common tobacco NtaSR1 and has C / G single nucleotide polymorphism, and the genotype of the SNP-4 is completely associated with the phenotype of the short petiole. A near-isogenic line Z46 is constructed by using SNP-4 marker screening, and the near-isogenic line Z46 has a short petiole character and a genetic background close to that of Zhong tobacco 100. The SNP-4 marker disclosed by the invention can be used for molecular marker-assisted selection of the short leaf stalk character of the tobacco, and the breeding efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and more specifically, to molecular markers associated with tobacco short petiole traits, their related biological materials and applications in breeding. Background Art

[0002] The petiole is the part of the leaf midrib that grows on the main stem, which plays a role in mechanical support and material transport. In common tobacco, morphological variation in the presence or absence of petioles is common. Most cultivated tobacco types, such as flue-cured tobacco and burley tobacco, often have symmetrical leaves with widths of several centimeters on the sides of the petioles, which are called petiole-free tobacco; but some aromatic tobacco and cigar tobacco, especially the numerous local sun-cured tobacco germplasm resources, often have smooth petioles 1 to 5 cm long, and the leaves on the sides of the petioles degenerate and disappear toward the top, which is called petiole tobacco. The presence or absence of petioles is mainly determined by two pairs of genes with cumulative effects. The petiole trait is dominant or partially dominant to the petiole-free trait.

[0003] Previous studies have conducted genetic analysis on a long petiole mutant M50 and a short petiole mutant M46 obtained from flue-cured tobacco variety Zhongyan 100 by mutagenesis with ethyl methane sulfonate (EMS), and found that both long and short petiole traits are controlled by a dominant single gene, and the mutant gene was located on chromosomes using tobacco SSR (Simple sequence repeat) markers. Genetic analysis of the short petiole trait of sun-cured tobacco Kuban Sansheng revealed that the short petiole trait is controlled by a dominant single gene, and the gene was located on chromosomes using tobacco 430K SNP chips. Sequence comparison further revealed that the location of the Kuban Sansheng short petiole gene was close to the location of the short petiole mutant gene mentioned above, and it is speculated that the two are alleles.

[0004] Map-based cloning is a gene cloning method based on linkage inheritance and is widely used to identify genes for important agronomic traits of plants. In tobacco, this method has been used to clone a series of important genes, including the burley tobacco genes WS1A and WS1B and the TMV resistance genes NtsTOM2A and NttTOM2A. However, since this method requires the construction of a large isolation population and the development of a large number of molecular markers, it is time-consuming and labor-intensive, which limits its scope of application to a certain extent.

[0005] As mentioned above, the study used ethyl methanesulfonate (EMS) mutagenesis to obtain tobacco short petiole mutants. The BC1F1 segregation population prepared by the short petiole mutant M46 and the flue-cured tobacco variety Innovation No. 3 was used to preliminarily locate the mutant gene between the tobacco SSR marker 16th linkage group (LG 16) markers PT51801 and PT20196, and the genetic map positions were 37.06cM and 65.19cM, respectively. Due to the small mapping population used (86 strains) and the small number of available SSR markers (5), the positioning results are relatively rough and cannot be directly used for the positional cloning of mutant genes. In addition, it was found that in addition to a small petiole at the base of the leaves of the whole plant, M46 also had many other phenotypic differences with the control Zhongyan 100, and it is not clear whether it is related to the short petiole mutant gene. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention is based on high-density molecular marker technology, by expanding the target trait separation population and developing high-density SSR and SNP (Single nucleotide polymorphism) molecular markers in the initial positioning interval, the short petiole mutant gene is precisely positioned, and molecular markers closely linked to the short petiole mutant gene are developed. With the help of molecular marker-assisted selection, a short petiole near-isogenic line (NIL) is constructed, the validity of the molecular markers is verified, and the genetic effect of the short petiole mutant gene is accurately evaluated, thereby better providing valuable information for analyzing the molecular mechanism of tobacco petiole formation.

[0007] Based on this, the present invention provides a molecular marker (SNP-4) related to the short petiole trait of tobacco, which can quickly and accurately identify plants with target traits in tobacco breeding and accelerate the breeding process. The specific contents include:

[0008] Molecular marker SNP-4: located at the 334th position of SEQ ID No. 1, with C / G polymorphism, can accurately distinguish short petioles (mutants) from no petioles (wild type).

[0009] Gene localization method: By constructing genetic populations and using high-density molecular marker technology, the short petiole mutation gene is precisely located.

[0010] Marker-assisted selection (MAS): Use SNP-4 to screen for the short petiole trait and improve breeding efficiency.

[0011] Biological materials: including the short petiole mutant M46, the near-isogenic line Z46 and related marker sequences.

[0012] The beneficial effects of the present invention are:

[0013] The present invention is based on high-density molecular marker technology, by expanding the target trait separation population and developing high-density SSR and SNP (Single nucleotide polymorphism) molecular markers in the initial positioning interval, the short petiole mutant gene is precisely positioned, and molecular markers closely linked to the short petiole mutant gene are developed. With the help of molecular marker-assisted selection, a short petiole near-isogenic line (NIL) is constructed, the validity of the molecular markers is verified, and the genetic effect of the short petiole mutant gene is accurately evaluated, thereby better providing valuable information for analyzing the molecular mechanism of tobacco petiole formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Comparison of the middle leaf morphology of Zhongyan 100 and the short petiole mutant M46.

[0015] Figure 2 Linked inheritance of tobacco SSR markers and short petiole mutation gene, A and B are the amplification status of tobacco SSR primers PT55279 and PT52719 in recessive individuals of M46, TN 90, and 39 BC1F1 segregating populations of hybrids of M46 and TN 90; Marker represents the DNA molecular weight standard pBR322 / MspI, the bands in A are 242bp, 238bp and 217bp from top to bottom, respectively, and the bands in B are 190bp, 180bp and 160bp from top to bottom, respectively; asterisks (*) indicate individuals with recombined SSR genotypes.

[0016] Figure 3 Precise positioning of the short petiole mutation gene, A is the initial positioning of the M46 mutation gene, which is a genetic map in cM. The molecular marker used is SSR. The positioning population size is 50. The blue numbers represent the recombinant plants detected by each marker; B and C are the fine positioning of the M46 mutation gene, which are physical maps in Mb. The molecular markers used are SSR and SNP, and the positioning population size is 3000.

[0017] Figure 4 Comparison of the middle leaf morphology of Zhongyan 100 and the short petiole near-isogenic line Z46. DETAILED DESCRIPTION

[0018] The present invention is based on high-density molecular marker technology. By expanding the target trait separation population and developing high-density SSR and SNP (Single nucleotide polymorphism) molecular markers within the initial positioning interval, the short petiole mutant gene is precisely positioned, and molecular markers closely linked to the short petiole mutant gene are developed. With the help of molecular marker-assisted selection, a short petiole near-isogenic line (NIL) is constructed, the validity of the molecular markers is verified, and the genetic effect of the short petiole mutant gene is accurately evaluated.

[0019] 1. Obtaining and phenotypic analysis of short petiole mutants

[0020] The short petiole mutant M46 was obtained by mutagenizing tobacco variety Zhongyan 100 with ethyl methanesulfonate. The main difference between M46 and Zhongyan 100 is that the lateral leaves at the base of the midrib are completely degenerated, forming smooth short petioles.

[0021] By hybridizing and backcrossing M46 with other wild-type cultivated tobacco varieties, a segregating population was constructed and genetic analysis was performed. The results showed that the short petiole trait was controlled by a single dominant nuclear gene. Using SSR molecular markers, the relevant genes were preliminarily located.

[0022] 2. Confirm and optimize positioning

[0023] Polymorphism analysis: The location of the short petiole mutation gene was further clarified by screening high-density SSR genetic continuous map markers. The segregating population formed by hybridization between M46 and other wild-type cultivated tobacco varieties showed high polymorphism and was used for fine positioning.

[0024] Interval optimization: Recessive individual plants (without petioles) and SSR markers were used to further locate the short petiole gene and narrow the target gene interval.

[0025] 3. Accurately locate target genes and develop SSR and SNP markers

[0026] The genetic map was converted into a physical map: DNA fragments were recovered from the polyacrylamide gel, and the secondary PCR products were sequenced and compared with the common tobacco genome to confirm the location interval of the short petiole gene.

[0027] Whole genome resequencing: Whole genome resequencing was performed on other wild-type tobacco varieties and the M46 mutant, and high-quality SSR and SNP variations within the position interval were extracted after comparison. Based on the SSR variation, primers were designed and polymorphisms were detected by polyacrylamide gel electrophoresis, and multiple SSR markers were successfully developed.

[0028] Precise positioning: Recessive plants (plants without petioles) were screened in the backcross population of other wild-type tobacco varieties and the M46 mutant, and the short petiole gene interval was further narrowed by changing the number of recombinant plants.

[0029] SNP marker development: Extract SNP variation within the narrowed interval, design primers and verify by agarose electrophoresis and sequencing, and develop multiple SNP markers. These markers are used for further analysis, and finally the short petiole gene is located and isolated.

[0030] SNP marker verification: SNP-4 is located at the 334th position of sequence 1 (SEQ ID No. 1) and has C / G polymorphism. The relationship between genotype and phenotype is as follows: CC (wild type): no short petiole, offspring do not separate; GG (mutant): short petiole, offspring do not separate; CG (heterozygous): short petiole, offspring separate into CC and GG types.

[0031] 4. Marker-Assisted Selection (MAS) and Construction of Z46

[0032] The SNP-4 marker was used to perform continuous backcrossing on M46, and finally the short petiole near-isogenic line Z46 was constructed.

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1 Acquisition of short petiole mutants

[0035] In our previous study (Chen Guo. Genetic analysis and gene localization of petiole mutants of common tobacco [D]. Beijing: Chinese Academy of Agricultural Sciences, 2017), we used the following method to prepare the offspring of the wild-type cultivated tobacco variety Zhongyan 100 (ZY) chemically induced by ethyl methanesulfonate (EMS): prepare an EMS solution of appropriate concentration (usually between 0.2% and 1%, and the optimal concentration is determined based on preliminary experiments), soak Zhongyan 100 seeds in the EMS solution, and the treatment time is generally 8-12 hours, and keep a constant temperature (such as 25°C). During the mutagenesis process, the seeds need to be shaken slightly to ensure that the solution contacts the seeds evenly. After the mutagenesis treatment, the seeds are immediately rinsed with a large amount of clean water for multiple times (at least 10 times) to remove residual EMS. The washed seeds are sown conventionally to cultivate M1 generation plants. Among the M1 generation plants, the leaf morphology is observed, and individual plants with obvious petiole mutations are selected. The selected mutant plants are self-pollinated to obtain the M2 generation to ensure that the mutant traits can be stably inherited. After multiple generations of self-pollination, a homozygous short petiole mutant M46 was identified. Compared with the wild-type tobacco variety Zhongyan 100, the leaves on the flanks of the midrib base of this mutant degenerate toward the top and disappear, resulting in the midrib being exposed and forming a smooth short petiole ( Figure 1 ).

[0036] By hybridizing and backcrossing the M46 mutant with another wild-type cultivated tobacco variety, Gexin 3 (G3), the F2 (hybrid combination: G3 / M46) and BC1F1 (hybrid combination: (G3 / M46) / G3) populations with M46 short petiole trait were obtained. The chi-square test showed that the M46 short petiole trait was controlled by a dominant nuclear gene. The gene was located on the chromosome using tobacco SSR molecular markers and was preliminarily located between markers PT51801 and PT20196 on the 16th linkage group (LG 16) of tobacco SSR markers, with genetic map positions of 37.06cM and 65.19cM, respectively.

[0037] Example 2 Reconfirmation of the initial location of the short petiole mutation gene

[0038] In order to further clarify the accuracy of the short petiole mutation gene positioning results and prepare for the subsequent fine gene positioning, based on the high-density SSR genetic linkage map of tobacco (Nicotiana tabacum L.) obtained from large scale microsatellite marker development [J]. Theoretical and Applied Genetics, 2011, 123: 219-230), all SSR markers in the above initial positioning interval and its flanking segments were selected to screen for polymorphisms between M46, Gexin No. 3 and another petioleless burley tobacco variety TN 90. The results showed that the polymorphism between M46 and TN 90 was higher than that between M46 and Gexin No. 3. Among the 28 SSR markers screened, there were 18 polymorphic markers between M46 and TN 90, and 12 between M46 and Gexin No. 3. Since fine positioning requires the development of more polymorphic markers in a smaller interval, it is more conducive to fine positioning to use M46 and TN 90 to prepare a segregating population. Therefore, the hybrid F1, F2 and BC1F1 populations prepared by M46 and TN 90 were first phenotypically identified and genetically analyzed. As shown in Table 1, the short petiole trait still showed a single gene dominant inheritance, indicating that the recessive individual plants (i.e., petiole-less individual plants) in the segregating population can be used to finely locate the gene.

[0039] Table 1 Segregation and statistics of petiole phenotypes in each generation of hybridization between M46 and TN 90

[0040] generations Hybrid combination Total number of plants Number of plants with petioles Number of plants without petiole Separation ratio Chi-square value F1 TN 90×M46 15 15 0 -- -- F2 TN 90×M46 212 162 50 3.24：1 0.226 BC1F1 (TN 90×M46)×TN 90 199 103 96 1.07：1 0.246

[0041] Note: “--” indicates missing data.

[0042] Based on the genotype identification and linkage genetic analysis of 50 recessive plants in the BC1F1 population, the 8 SSR markers were linked to the short petiole trait, and different numbers of recombinant plants (such as Figure 2 According to the differences in recombinant plants and the decreasing trend, the short petiole mutation gene was further located between markers PT52719 and PT55279, with a genetic distance of 2.46 cM ( Figure 3 A).

[0043] Example 3 Fine positioning of short petiole mutation gene and development and screening of SNP markers

[0044] In order to precisely locate the short petiole gene of the M46 mutation, it is first necessary to convert the genetic map into a physical map. To this end, the linked DNA specific bands of PT52719 and PT55279 were recovered from the polyacrylamide gel ( Figure 2 ), and then a secondary PCR was performed. The product was sequenced and compared with the genome of NtaSR1 of common tobacco. The results showed that the short petiole gene was located between 143.27Mb and 150.17Mb on chromosome 7 (Chr 7) of NtaSR1 of common tobacco, with an interval size of 6.9Mb ( Figure 3 B).

[0045] In order to further narrow the above positioning interval, referring to previous research methods (Gong et al., 2020: Fine mapping of QTLs for resistance to Phytophthora nicotianae in flue-cured tobacco using a high-density genetic map.; Li et al., 2023: Genome-widesignatures of the geographic expansion and breeding of soybean.), the TN 90 and M46 mutants were whole-genome resequencing based on the second-generation sequencing technology. After a series of processing, the sequencing data were compared with the common tobacco NtaSR1 genome to obtain a large number of high-quality SSR and SNP variations that differed between TN 90 and M46. The SSR variation obtained by whole genome resequencing between TN 90 and M46 within the above 6.9Mb positioning interval was extracted. Referring to the previous research method (Wu et al., 2020: A two-step mutation process in the double WS1 homologs drives the evolution of burley tobacco, a special chlorophyll-deficient mutant with abnormal chloroplast development.), primers were designed and polyacrylamide gel electrophoresis was used to detect its length polymorphism between TN 90 and M46. Six SSR markers were successfully developed, as shown in Table 2. These markers were used to screen 3000 recessive plants in the BC1F1 segregation population generated by backcrossing TN 90 and M46. Based on the differences in the detected recombinant plants and the decreasing trend, the short petiole gene was further located between markers SSR-3 and SSR-4, with an interval size of 1.6Mb ( Figure 3 B).

[0046] In the above 1.6Mb positioning interval, the SNP variation obtained by whole genome resequencing between TN 90 and M46 was extracted, primers were designed and the polymorphism between TN 90 and M46 was obtained by agarose gel electrophoresis and conventional sequencing, and 8 SNP markers were successfully developed, as shown in Table 2. These markers were used to screen the recombinant plants detected by the above SSR markers. According to the differences in the recombinant plants and the trend of reduction, the short petiole gene was finally located between markers SNP-3 and SNP-6, with an interval size of 210 Kb, and co-segregated with markers SNP-4 and SNP-5 ( Figure 3 C).

[0047] Table 2 SSR and SNP markers used for fine mapping of short petiole mutation genes

[0048]

[0049] Note: “--” indicates missing data.

[0050] In the aforementioned 210Kb M46 mutant gene localization interval, there are SNP markers SNP-4 and SNP-5. The two markers did not detect single plants with recombinant marker genotypes in 3000 BC1F1 recessive single plants (i.e. wild type), indicating that the two markers and the M46 mutant gene are genetically co-segregated and can be used as molecular markers for the M46 short petiole mutant trait in breeding, which can accelerate the relevant breeding process. In order to further expand its scope of use, we tested the effectiveness of the two markers in the main cultivated tobacco types. The results showed that in the wild-type germplasm resources of major flue-cured tobacco, burley tobacco, oriental tobacco, and cigar tobacco cultivated tobacco such as Zhongyan 100, TN 90, BasmaXanthi, and Havana10 (as shown in Table 3), the genotypes detected by SNP-4 were all wild types (i.e. CC) and no mutants (i.e. GG), while the genotypes detected by SNP-5 included both wild types (i.e. CC) and mutants (i.e. TT). This indicates that the mutation in SNP-4 should be unique to the M46 mutant and does not exist in any wild-type cultivated tobacco germplasm.

[0051] Table 3 Cultivated tobacco germplasm resources and their types used for SNP-4 genotype screening

[0052]

[0053]

[0054] Finally, we found a region, which is sequence 1; in the chromosome genome of the M46 mutant, the nucleotide sequence of sequence 1 at position 334 is G; in the chromosome genome of wild-type cultivated tobacco germplasm resources such as Zhongyan 100, TN 90, Basma Xanthi, and Havana10, the nucleotide sequence of sequence 1 at position 334 is C.

[0055] That is, SNP-4 has three genotypes, namely CC, GG and CG. Genotype CC is the homozygous type of SNP-4 being C, genotype GG is the homozygous type of SNP-4 being G, and genotype CG is the heterozygous type of SNP-4 being CG; the homozygous type of SNP-4 being C is the homozygous type in which the 334th nucleotide of sequence 1 is C, as shown in SEQ ID No.1, the homozygous type of SNP-4 being G is the homozygous type in which the 334th nucleotide of sequence 1 is G, as shown in SEQ ID No.2; the heterozygous type of SNP-4 being CG is the heterozygous type in which the 334th nucleotide of sequence 1 is C and G.

[0056] The heterozygous sequences are as follows:

[0057]

[0058] CC type sequence, as shown in SEQ ID No.1:

[0059]

[0060]

[0061] GG type sequence, as shown in SEQ ID No.2:

[0062]

[0063] Example 4 Molecular marker verification

[0064] The SNP marker (SNP-4) can be effectively used for auxiliary selection of the short petiole mutant trait of M46 in tobacco breeding. Specifically, there is a C / G polymorphic site at the 334th position of the 797 bp nucleotide sequence (sequence 1). When the marker is used to screen the genotype of the offspring produced by the cross between the M46 short petiole mutant and any wild-type cultivated tobacco variety, the single plant with the SNP-4 genotype of CC has a phenotype of no short petiole, and the self-pollination offspring does not separate; the single plant with the SNP-4 genotype of GG has a phenotype of short petiole, and the self-pollination offspring does not separate; the single plant with the SNP-4 genotype of CG has a phenotype of short petiole, but its self-pollination offspring can separate the single plant with the SNP-4 genotype of CC and the phenotype of no short petiole and the single plant with the SNP-4 genotype of GG and the phenotype of short petiole.

[0065] In order to further verify the validity of the SNP-4 marker, a hybrid combination was prepared with Zhongyan 100 (ZY) and the M46 mutant to obtain F1, which was then backcrossed with Zhongyan 100 for 5 generations to BC5F1, and then self-pollinated to obtain the BC5F2 segregation population, and then some individual plants were selected from them for further self-pollination to obtain the corresponding BC5F3 family, and the short petiole near-isogenic line with the genetic background of Zhongyan 100 was constructed. During this period, SNP-4 was used for detection in each generation, and the validity of the SNP marker was verified by judging the phenotype of the detected individual plants with or without short petioles during the flowering period in the field. The results are shown in Table 3.

[0066] Table 3. Genotypes of main stem and SNP-4 locus of 75 tobacco inbred lines

[0067]

[0068]

[0069]

[0070] The results showed that the individual plants with SNP-4 genotype CC all had a phenotype of no short petiole, and the self-pollination offspring did not separate; the individual plants with SNP-4 genotype GG all had a phenotype of short petiole, and the self-pollination offspring did not separate; the individual plants with SNP-4 genotype CG all had a phenotype of short petiole, but their self-pollination offspring could separate out individual plants with genotype CC and phenotype all without short petiole, and individual plants with genotype GG and phenotype all with short petiole. It can be seen that this molecular marker can be used for the breeding of tobacco with or without short petiole.

[0071] Finally, the marker was used in combination with phenotypic identification to obtain the short petiole near-isogenic line Z46 under the genetic background of Zhongyan 100 in BC5F3. Except for the short petiole at the base of the leaf, the leaf morphology of Z46 is basically similar to that of Zhongyan 100; although the leaf length (including petiole) is slightly shorter, the leaf width is slightly wider, and the leaf surface is slightly wrinkled, the difference is not obvious ( Figure 4 ). These analyses showed that the short petiole mutant gene mainly affects the formation of the petiole at the base of the leaf blade and has little effect on other traits.

[0072] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A molecular marker SNP-4 associated with the short petiole trait of tobacco, characterized in that: At the 334th position of its nucleotide sequence, there is a single nucleotide polymorphism of C / G.

2. The molecular marker SNP-4 associated with the short petiole trait of tobacco according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID No.

1.

3. A tobacco gene chip, characterized in that: It includes the tobacco SNP-4 molecular marker as described in claim 1.

4. The method for obtaining the molecular marker SNP-4 according to claim 1 or 2, characterized in that: include: Starting from the short petiole mutant M46 induced by ethyl methanesulfonate, a trait segregating population was constructed; Preliminary positioning was performed using genetic maps, and further fine positioning was performed using physical maps and whole-genome resequencing; SNP markers were developed within the target interval and their linkage with the short petiole trait was verified.

5. A method for detecting the short petiole trait of tobacco, comprising: Extract tobacco DNA; Amplifying the region containing the molecular marker SNP-4 site; Detect the genotype of the polymorphic site (CC, CG, GG).

6. A tobacco short petiole breeding method, characterized in that: The method comprises using the molecular marker SNP-4 described in claim 1 or 2 to perform marker-assisted selection.

7. A tobacco biomaterial, characterized in that: The invention comprises a tobacco biological material, characterized in that it comprises a short petiole mutant M46 and its near isogenic line Z46, and is combined with the molecular marker SNP-4 described in claim 1 or 2 which is completely co-segregated with the short petiole trait, and is used for molecular marker-assisted selection breeding of the short petiole trait.

8. Use of the molecular marker SNP-4 marker associated with the short petiole trait of tobacco according to claim 1 or 2 in molecular marker-assisted selection for the short petiole trait of tobacco.

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