A DNA molecular marker for identifying tobacco glandular hair density and its application

Abstract

The invention discloses a molecular marker for identifying tobacco glandular hair density and its application. The primers for identifying the molecular marker for tobacco glandular hair density include: upstream primer W12-seq-F: 5'-GACAGGGGAGATTATTCAAAA-3'; upstream primer W12-WT-F: 5'-CACCACGAGTGATCAACCACC-3'; public downstream primer W12-R: 5'-TTGAAAAACAGGGCTAGAGGG-3'. The primers of the present application can more accurately identify the glandular hair density of the tobacco plant to be tested, save the observation of the glandular hair on the leaf surface of the tobacco plant, and reduce the workload of phenotypic identification; the identification can be carried out in the early stage of tobacco plant development, and only the selected single plant is transplanted into the field, reducing the workload and planting cost of field planting; sequencing and agarose gel electrophoresis detection can be carried out simultaneously, and the two methods can be mutually verified to increase the accuracy of identification.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a molecular marker for identifying tobacco glandular hair density and an application thereof. Background Art

[0002] Tobacco is an important economic crop in my country. The surface of tobacco is densely covered with epidermal hairs, which can be divided into three types according to the morphological characteristics of the epidermal hairs and whether they have the ability to secrete: long-stalked glandular hairs, short-stalked glandular hairs and non-secretory epidermal hairs.

[0003] Tobacco glandular hairs have a strong secretory capacity and can synthesize and secrete a variety of secondary metabolites, playing an important role in tobacco plants' response to environmental stress. The glandular hair secretions can form a continuous and sticky film on the surface of tobacco plants, hindering the movement of insects, leading to their starvation and suffocation. In addition, the diterpenoids and sucrose esters in the glandular hair secretions are toxic to aphids; in terms of responding to pathogenic microorganisms, the resistance proteins Phylloplanins secreted by tobacco short-stalked glandular hairs can inhibit the germination of sporangia and conidia of tobacco downy mildew (Peronospora tabacina), thereby protecting tobacco from its invasion.

[0004] The aroma quality of flue-cured tobacco leaves is the most important trait of flue-cured tobacco, and the secretion of glandular hairs also has an important influence on the quality of tobacco leaves. The secretion of glandular hairs of flue-cured tobacco is mainly composed of cebetane-like diterpenoid compounds and sucrose esters, both of which are important aroma precursors. Cebetane-like compounds are mainly composed of α- and β-type cebetene triene monool (CBT-ol) and cebetene triene diol (CBT-diol) and their isomers. Most of these substances are degraded after modulation, and the main products are solanone and its derivatives. Solanone itself has a good aroma, and its conversion products such as solanol, solanifuran, and norsolanidion are also important aroma substances. Sucrose esters are related to the fullness of tobacco flavor. During the smoking process of cigarettes, sucrose esters are pyrolyzed to produce low-molecular volatile acids and esters, such as isobutyric acid, 3-methylbutyric acid and 3-methylvaleric acid, which can give cigarettes a unique aroma of oriental tobacco smoke; in addition, sugar esters also have moisturizing, moisture retention, and aroma retention.

[0005] As mentioned in the review, increasing the density of tobacco glandular hairs will effectively enhance the resistance of tobacco plants to environmental stress and improve the aroma quality of tobacco leaves. Therefore, increasing the density of glandular hairs is one of the important goals of tobacco breeding. However, since the density of tobacco glandular hairs is affected by many environmental factors such as cultivation methods and ecological conditions, and the glandular hairs are small in morphology and require microscopic observation for effective screening, the improvement of tobacco glandular hair traits by conventional breeding methods is greatly limited. The development of modern biotechnology provides a fast and effective method for the identification of tobacco glandular hair phenotypes - DNA molecular markers. By developing molecular markers that are closely linked to the density of glandular hairs, only a small amount of genomic DNA is needed to achieve accurate selection of the density of glandular hairs. Since any tissue of a tobacco plant has the same genetic composition, only a very small amount of tissue can be collected at any stage of tobacco plant development to complete the identification, avoiding destructive sampling of the selected individual plants and enabling early screening.

[0006] In the early stage, our research team found a mutant with significantly increased glandular hair density in the tobacco EMS mutagenesis population. Further research found that the mutant genome had a 7-base deletion, which was the reason for the multiple glandular hair phenotype of the single plant. Therefore, this mutant can be used as a donor parent with high glandular hair density. By developing molecular markers for this 7-base deletion mutation, the screening efficiency can be effectively improved, which has important application value for tobacco high glandular hair breeding. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a molecular marker for identifying tobacco glandular hair density and its application, which can effectively improve the screening efficiency and has important application value for tobacco high glandular hair breeding.

[0008] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0009] A primer for identifying a molecular marker of tobacco glandular hair density, the primer comprising the following upstream primer and downstream primer:

[0010] Upstream primer W12-seq-F: 5′-GACAGGGGAGATTATTCAAAA-3′;

[0011] Upstream primer W12-WT-F: 5′-CACCACGAGTGATCAACCACC-3′;

[0012] Common downstream primer W12-R: 5′-TTGAAAAACAGGGCTAGAGGG-3′.

[0013] A kit comprising the above-mentioned molecular marker primers for identifying tobacco glandular hair density.

[0014] A method for identifying molecular markers of tobacco glandular hair density comprises the following steps:

[0015] (1) Primer synthesis: synthesize primers W12-seq-F, W12-WT-F, and W12-R;

[0016] (2) DNA extraction: Extracting genomic DNA from tobacco leaves;

[0017] (3) PCR amplification: Based on the above tobacco leaf genomic DNA, PCR amplification was performed using the primers synthesized in step (1), the primer combinations were W12-seq-F+W12-R and W12-WT-F+W12-R, and two sample DNAs with high and low glandular hair density and known gene sequences were added in advance as controls;

[0018] (4) Detection and analysis of amplification products:

[0019] Direct sequencing: The product amplified by the W12-seq-F+W12-R primer combination is sequenced directly. If the 7-bp at the 135-141bp position of the measured DNA fragment is missing, that is, it is the same as the sequence of SEQ ID NO.4, it is a high glandular hair density individual plant; if there is no deletion at the 135-141bp position of the measured DNA fragment, that is, it is the same as the sequence of SEQ ID NO.5, it is a low glandular hair density individual plant; if the sequencing peak graph is an overlapping peak, it is a low glandular hair density individual plant;

[0020] Agarose gel detection: The amplification product of the W12-WT-F + W12-R primer combination was subjected to agarose gel detection. If the W12-WT-F + W12-R primer combination could not amplify a band, it was a plant with high glandular hair density; if the W12-WT-F + W12-R primer combination could amplify a band, it was a plant with low glandular hair density.

[0021] Preferably, in the above technical solution, the PCR amplification reaction system in step (3) is: 1.0 μL of 50 ng / μL sample genomic DNA template, 10 μL of 2×EASY Taq mix, 0.4 μL of 10 μM upstream and downstream primers respectively, and ddH2O is added to 20 μL.

[0022] Preferably, in the above technical solution, the PCR amplification program in step (3) is: pre-denaturation at 95°C for 5 min, then 95°C for 30 s, 57°C for 30 s, 72°C for 30 s, for a total of 35 cycles, and finally 72°C for 10 min.

[0023] An application of the primer, the kit or the method in tobacco breeding.

[0024] An application of the primer, the kit or the method in screening tobacco plants with different glandular hair densities.

[0025] The above technical solution of the present invention has the following beneficial effects:

[0026] (1) The molecular marker primers provided by the present invention can more accurately identify the density of glandular hairs of the tobacco plants to be tested, thereby eliminating the need to observe the glandular hairs on the tobacco plant leaves and greatly reducing the workload of phenotypic identification;

[0027] (2) The molecular marker primers provided by the present invention can be used to identify tobacco plants in the early stages of their development, and only selected plants are transplanted into the field, greatly reducing the workload and cost of field planting;

[0028] (3) The molecular marker primers provided by the present invention can be used for sequencing and agarose gel electrophoresis detection at the same time. The two methods can verify each other, greatly increasing the accuracy of identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0030] Figure 1 This is the sequencing peak diagram of the amplification product of the W12-seq-F+W12-R primer combination;

[0031] Figure 2 Agarose gel detection of the amplification product of the W12-WT-F+W12-R primer combination;

[0032] Figure 3 This is an enlarged picture of the glandular hairs on tobacco leaves. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0034] Example 1 Construction of tobacco genetic segregation population

[0035] The individual plants carrying the 7-base deletion mutation were crossed with the low glandular hair density individual plants without deletion to obtain F1 plants. After self-pollination of F1 plants, F2 seeds were harvested, which was the genetic segregation population of tobacco glandular hair density. The F2 seeds were sown in seedling trays, and subsequent experiments were carried out when the tobacco plants grew to the six-leaf one-heart stage.

[0036] 48 individual plants were randomly selected from the above-mentioned isolated population, and the density of glandular hairs was identified using molecular marker primers. The detailed steps are as follows:

[0037] (1) Primer synthesis: According to the sequence information in Table 1, Beijing Liuhe BGI Genomics Co., Ltd. was commissioned to synthesize the primers.

[0038] Table 1 Primer sequence information

[0039]

[0040] Example 2 DNA extraction and amplification

[0041] (A) Take about 0.2 g of tobacco leaves, grind them into powder in liquid nitrogen, and transfer them to a 2 mL centrifuge tube;

[0042] (B) Tobacco genomic DNA was extracted using a high-efficiency plant genomic DNA extraction kit (DP350, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the specific steps were carried out according to the kit instructions;

[0043] (C) The extracted DNA was diluted to a concentration of 50 ng / μL for later use;

[0044] (3) The PCR amplification reaction system is: 1.0 μL of 50 ng / μL sample genomic DNA template, 10 μL of 2×EASY Taq mix, 0.4 μL of 10 μM forward and reverse primers (Example 1), and ddH2O is added to 20 μL;

[0045] (4) The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s, and finally 72°C for 10 min.

[0046] Example 3 Detection and analysis of amplification products

[0047] (1) Direct sequencing: The products amplified by the W12-seq-F+W12-R primer combination are directly sequenced.

[0048] If the 7-bp deletion at the 135-141bp position of the DNA fragment tested (such as Figure 1 As shown in A), it is a plant with high glandular hair density;

[0049] If there is no deletion at the position 135-141bp of the DNA fragment being tested (e.g. Figure 1 If the sequencing peak graph is overlapping peaks (as shown in Figure C), it is a single plant with low glandular hair density.

[0050] (2) Agarose gel detection: The amplification product of the W12-WT-F+W12-R primer combination was detected by agarose gel.

[0051] If the W12-WT-F+W12-R primer combination cannot amplify a band (such as Figure 2 If the W12-WT-F+W12-R primer combination can amplify a band (as shown in Figure 2 If the density of glandular hairs is low, the plant is single.

[0052] Example 4 Verification

[0053] The glandular hairs of the 48 identified plants were observed. For each plant, the third leaf was taken from the bottom to the top at the six-leaf and one-heart stage. The leaves were required to be basically the same size and develop normally. Microscopic observation was performed using a stereo microscope to determine the density of glandular hairs. The results are shown in Table 2 and Figure 3 shown.

[0054] Table 2 Observation of glandular hairs and identification of molecular markers in genetically segregating populations

[0055]

[0056]

[0057] The results showed that among the 48 individual plants observed, 13 were polyglandular hairy (e.g. Figure 3 ), 35 strains were of the oligoglandular type (as shown in A). Figure 3 The results are shown in Figure 2), wherein the sequences of the products amplified by the primer combination W12-seq-F+W12-R of the polyglandular hair type single plant are all the same as SEQ ID No: 5 in the sequence table, and the sequences of the products amplified by the primer combination W12-seq-F+W12-R of the polyglandular hair type single plant are the same as SEQ ID No: 4 in the sequence table, or the sequencing peak diagram is an overlapping peak; the primer combination W12-WT-F+W12-R of the polyglandular hair type single plant cannot amplify bands, while the primer combination W12-MT-F+W12-R can amplify bands, and the primer combination W12-WT-F+W12-R of the polyglandular hair type single plant can amplify bands (Table 2).

[0058] 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. Sequence Listing <110> Henan China Tobacco Industry Co., Ltd. Technology Development Branch <120> A DNA molecular marker for identifying tobacco glandular hair density and its application <130> WPC221262 <160> 5 <170> SIPOSequenceList 1.0 <210> 1 <211> 21 <212> DNA <213> Private Sequence(Artificial Sequence) <400> 1 gacaggggag attattcaaa <210> 2 <211> 21 <212> DNA <213> Private Sequence(Artificial Sequence) <400> 2 snowflake gatcaaccac c <210> 3 <211> 21 <212> DNA <213> Private Sequence(Artificial Sequence) <400> 3 ttgaaaaca gggctagagg g <210> 4 <211> 498 <212> DNA <213> Private Sequence(Artificial Sequence) <400> 4 gacaggggag attattcaaa aaagaagaag aaaagaaatc attattatta caagtacaat tagccatgag ccgaagaat ggaaatggtg agcttgatct aaagctgaat ttgtcaccac 180. cacgagtgat caacaagtgg aatcaccgag caggtcgttg acggcgtcgc cgacgagttc atgcgtgtcg tccgatgatt cctccgccgt tcaacgttat tccgacagcc cagaaacaat tacttcaatg gtgttggttg gttgtccacg gtgcctcatg tatgttatgt tagcagaagt 300 taatgatcca agatgcccta aatgcaagag tacttgtttg cttgatgtta tgtatgagaa 360 gaacaagact actaggagga atatcagtta gactatagtc aagaatctat cacattctgc 420 tattactagg tttatcaaca atcttgcccc ccaccttgga aaaaatgaaa attaaaccc 480 tctagccctg tttttcaa 498 <210> 5 <211> 505 <212> DNA <213> Artificial Sequence <400> 5 gacaggggag attattcaaa aaagaagaag aaaagaaatc atatttatta caagtacaat 60 tagccatgag ccgaagaaat ggaaatggtg agcttgatct aaagctgaat ttgtcaccac 120 cacgagtgat caaccaccca caagtggaat caccgagcag gtcgttgacg gcgtcgccga 180 cgagttcatg cgtgtcgtcc gatgattcct ccgccgttca acgttattcc gacagcccag 240 aaacaattac ttcaatggtg ttggttggtt gtccacggtg cctcatgtat gttatgttag 300 cagaagttaa tgatccaaga tgccctaaat gcaagagtac ttgtttgctt gatgttatgt 360 atgagaagaa caagactact aggaggaata tcagttagac tatagtcaag aatctatcac attctgctat tactaggttt atcaacaatc ttgcccccca ccttggaaaa aatgaaaaat taaccctct agccctgttt ttcaa

Claims

1. A primer for identifying molecular markers of tobacco glandular hair density, characterized in that: The primers include the following upstream primers and downstream primers: Upstream primer W12-seq-F: 5′-GACAGGGGAGATTATTCAAAA-3′; Upstream primer W12-WT-F: 5′-CACCACGAGTGATCAACCACC-3′; Common downstream primer W12-R: 5′-TTGAAAAACAGGGCTAGAGGG-3′.

2. A kit comprising the primers for molecular markers for identifying tobacco glandular hair density according to claim 1.

3. A method for identifying molecular markers for tobacco glandular hair density, characterized in that: The following steps are involved: (1) Primer combination: synthesizing the primers W12-seq-F, W12-WT-F and W12-R as described in claim 1; (2) DNA extraction: Extracting genomic DNA from tobacco leaves; (3) PCR amplification: Based on the above tobacco leaf genomic DNA, PCR amplification was performed using the primers synthesized in step (1). The primer combinations were W12-seq-F+W12-R and W12-WT-F+W12-R, and two sample DNAs with high and low glandular hair density and known gene sequences were added in advance as controls; (4) Detection and analysis of amplification products: Direct sequencing: The product amplified by the W12-seq-F+W12-R primer combination was sequenced directly. If the 7-bp position of the DNA fragment 135-141 bp was missing, that is, it was the same as SEQ ID NO. 4, it was a high glandular hair density plant; if there was no missing position of the DNA fragment 135-141 bp, that is, it was the same as SEQ ID NO. 5, it was a low glandular hair density plant; if the sequencing peak graph was overlapping peaks, it was a low glandular hair density plant; Agarose gel detection: The amplification product of the W12-WT-F + W12-R primer combination was subjected to agarose gel detection. If the W12-WT-F + W12-R primer combination could not amplify a band, it was a plant with high glandular hair density; if the W12-WT-F + W12-R primer combination could amplify a band, it was a plant with low glandular hair density.

4. The method for identifying molecular markers for tobacco glandular hair density according to claim 3, characterized in that: The PCR amplification reaction system in step (3) is: 1.0 μL of 50 ng / μL sample genomic DNA template, 10 μL of 2× EASY Taq mix, 0.4 μL of each 10 μM upstream and downstream primers, and ddH2O to make up to 20 μL.

5. The method for identifying molecular markers for tobacco glandular hair density according to claim 3, characterized in that: The PCR amplification program in step (3) was as follows: pre-denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, and finally 72 °C for 10 min.

6. Use of the primer according to claim 1, the kit according to claim 2 or the method according to any one of claims 3 to 5 in screening tobacco plants with different glandular hair densities.

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