MdLOG gene promoter deletion fragment and its application in detecting salt tolerance in Malus plants

Abstract

The present invention discloses an MdLOG gene promoter deletion fragment and its application in detecting salt tolerance in apple plants. The present invention provides the application of the MdLOG gene promoter deletion fragment as a molecular marker for any of the following: detecting the salt tolerance of apple plant rootstocks, detecting the salt damage index of apple plants, detecting whether apple plants are salt-tolerant plants, and detecting whether apple plants can be used as salt-tolerant rootstocks. The MdLOG gene promoter deletion fragment is a DNA fragment represented by SEQ ID No. 3. The MdLOG gene promoter fragment deletion phenomenon is positively correlated with salt tolerance. The absence and presence of this fragment can be used to quickly screen salt-tolerant plants in offspring, which is of great significance in the field of plant salt tolerance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an MdLOG gene promoter deletion fragment and an application thereof in detecting salt tolerance of apple plants. Background Art

[0002] Rootstocks can influence tree development, fruit yield and quality, and resistance to environmental stress. Breeding salt-tolerant rootstocks can improve apple production yield and quality in salinized sites.

[0003] Cytokinins are important growth-promoting hormones. The cytokinin-activating enzyme gene, MdLOG, was cloned from the root DNA of the apple rootstock Malus balsamifera. The mRNA for this gene is 1963 bp long, consisting of seven exons and six introns. It encodes a 230-amino acid polypeptide containing a highly functionally conserved motif, PGGXGTXXE. LOG converts inactive cytokinin nucleotides into biologically active free base forms, catalyzing the hydrolysis of the N-glycosidic bond of the 5'-monophosphates of various pyrimidine and purine nucleotides to form ribose 5-phosphate and the corresponding free bases. Summary of the Invention

[0004] The purpose of the present invention is to provide an MdLOG gene promoter deletion fragment and an application thereof in detecting salt tolerance of apple plants.

[0005] In a first aspect, the present invention claims the use of a deletion fragment of the MdLOG gene promoter as a molecular marker in any of the following:

[0006] (A1) detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus, or preparing a product for detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus;

[0007] (A2) detecting or assisting in detecting the salt damage index of apple plants, or preparing a product for detecting or assisting in detecting the salt damage index of apple plants;

[0008] (A3) detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant;

[0009] (A4) detecting or assisting in detecting whether a tested apple plant can be used as a salt-tolerant rootstock, or preparing a product for detecting or assisting in detecting whether a tested apple plant can be used as a salt-tolerant rootstock;

[0010] The MdLOG gene promoter deletion fragment is a DNA fragment shown in SEQ ID No. 3. The MdLOG gene promoter deletion fragment is from position -1305 to position -1278 upstream of the A in the start codon ATG of the MdLOG gene in the apple plant genome; the A in the start codon ATG of the MdLOG gene is denoted as position +1.

[0011] Among them, the salt tolerance of the rootstock refers to the characteristics of the scion leaves when the same fruit tree scion is grafted onto different rootstocks and treated with a 200mM concentration of sodium chloride solution for 30 days, which is expressed by the salt damage index. The salt damage index is divided into five levels from 0 to 4: 0 is when the leaves are asymptomatic; 1 / 3 or less of the leaves are burnt yellow at the top or edges; 2 is when 1 / 3 to 2 / 3 (excluding the endpoints) of the leaves are brown at the top or edges; 3 is when 2 / 3 or more of the leaves are brown or dead (the whole plant has not died or withered yet); 4 is when the whole plant is dead or withered. Levels 0-1 are defined as salt-tolerant, and levels 2-4 are defined as salt-intolerant. The same below.

[0012] The salt tolerance refers to the characteristics of plant leaves after being treated with a 200mM sodium chloride solution for 30 days, and is expressed as a salt damage index. The salt damage index is divided into five levels from 0 to 4: Level 0 means that the leaves are asymptomatic; Level 1 means that 1 / 3 or less of the leaves are burnt yellow at the top or edges; Level 2 means that 1 / 3 to 2 / 3 (excluding the end points) of the leaves are brown at the top or edges; Level 3 means that 2 / 3 or more of the leaves are brown at the top or edges or dead (the whole plant has not died or withered yet); Level 4 means that the whole plant is dead or withered. Levels 0-1 are defined as salt tolerance, and levels 2-4 are defined as salt intolerant. The same applies below.

[0013] In a second aspect, the present invention claims the use of a substance for detecting a deletion fragment of the MdLOG gene promoter in any of the following:

[0014] (A1) detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus, or preparing a product for detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus;

[0015] (A2) detecting or assisting in detecting the salt damage index of apple plants, or preparing a product for detecting or assisting in detecting the salt damage index of apple plants;

[0016] (A3) detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant;

[0017] (A4) detecting or assisting in detecting whether a tested apple plant can be used as a salt-tolerant rootstock, or preparing a product for detecting or assisting in detecting whether a tested apple plant can be used as a salt-tolerant rootstock;

[0018] The MdLOG gene promoter deletion fragment is a DNA fragment shown in SEQ ID No. 3. The MdLOG gene promoter deletion fragment is from position -1305 to position -1278 upstream of the A in the start codon ATG of the MdLOG gene in the apple plant genome; the A in the start codon ATG of the MdLOG gene is denoted as position +1.

[0019] Furthermore, the substance for detecting the MdLOG gene promoter deletion fragment may be a primer pair for amplifying the MdLOG gene promoter deletion fragment.

[0020] Furthermore, the upstream primer in the primer pair can be designed based on the sequence located upstream of the MdLOG gene promoter deletion fragment in the apple plant genome, and the downstream primer can be designed based on the sequence located downstream of the MdLOG gene promoter deletion fragment in the apple plant genome.

[0021] In a specific embodiment of the present invention, the primer pair consists of a single-stranded DNA molecule shown in SEQ ID No. 4 and a single-stranded DNA molecule shown in SEQ ID No. 5.

[0022] In a third aspect, the present invention claims protection for any of the following methods:

[0023] Method 1: A method for detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus, comprising the following steps (B1) and (B2):

[0024] (B1) detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter;

[0025] (B2) Based on the result of (B1), the salt tolerance of the rootstock of the tested Malus plant is determined as follows: the salt tolerance of the rootstock of the tested Malus plant whose genome does not contain the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is missing the "MdLOG gene promoter deletion fragment") is higher or candidate higher than the salt tolerance of the rootstock of the tested Malus plant whose genome contains the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is not missing the "MdLOG gene promoter deletion fragment");

[0026] The MdLOG gene promoter deletion fragment is a DNA fragment shown in SEQ ID No. 3. The MdLOG gene promoter deletion fragment is from position -1305 to position -1278 upstream of the A in the start codon ATG of the MdLOG gene in the apple plant genome; the A in the start codon ATG of the MdLOG gene is denoted as position +1.

[0027] Method 2: A method for detecting or assisting in detecting the salt damage index of apple plants, comprising the following steps (C1) and (C2):

[0028] (C1) detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter;

[0029] (C2) Based on the result of (C1), the salt damage index of the tested Malus plant is determined as follows: the salt damage index of the tested Malus plant whose genome does not contain the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is missing the "MdLOG gene promoter deletion fragment") is lower than, or is potentially lower than, the salt damage index of the tested Malus plant whose genome contains the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is not missing the "MdLOG gene promoter deletion fragment");

[0030] The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; the MdLOG gene promoter deletion fragment is from -1305 to -1278 upstream of the A in the start codon ATG of the MdLOG gene in the genome of the apple plant; the A in the start codon ATG of the MdLOG gene is recorded as the +1 position.

[0031] Method 3: A method for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, comprising the following steps (D1) and (D2):

[0032] (D1) detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter;

[0033] (D2) Based on the result of (D1), determining whether the tested Malus plant is a salt-tolerant plant is as follows: if the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is missing the "MdLOG gene promoter deletion fragment"), then the tested Malus plant is or is a candidate for being a salt-tolerant plant; if the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is not missing the "MdLOG gene promoter deletion fragment"), then the tested Malus plant is not or is not a candidate for being a salt-tolerant plant;

[0034] The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; the MdLOG gene promoter deletion fragment is from -1305 to -1278 upstream of the A in the start codon ATG of the MdLOG gene in the genome of the apple plant; the A in the start codon ATG of the MdLOG gene is recorded as the +1 position.

[0035] Method 4: A method for detecting or assisting in detecting whether a tested Malus plant can be used as a salt-tolerant rootstock, comprising the following steps (E1) and (E2):

[0036] (E1) detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter;

[0037] (E2) Based on the result of (E1), determining whether the tested Malus plant can be used as a salt-tolerant rootstock is as follows: if the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is missing the "MdLOG gene promoter deletion fragment"), then the tested Malus plant can or is a candidate for being used as a salt-tolerant rootstock; if the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is not missing the "MdLOG gene promoter deletion fragment"), then the tested Malus plant cannot or is a candidate for being used as a salt-tolerant rootstock;

[0038] The MdLOG gene promoter deletion fragment is a DNA fragment shown in SEQ ID No. 3. The MdLOG gene promoter deletion fragment is from position -1305 to position -1278 upstream of the A in the start codon ATG of the MdLOG gene in the apple plant genome; the A in the start codon ATG of the MdLOG gene is denoted as position +1.

[0039] In each of the above methods, the method for detecting whether the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment can be as follows (F1) or (F2):

[0040] (F1) direct sequencing (i.e., directly sequencing the genome of the tested Malus plant);

[0041] (F2) Using the genome of the test Malus plant as a template, perform PCR amplification with the primer pair described above, and determine whether the genome of the test Malus plant contains the MdLOG gene promoter deletion fragment based on the amplification results.

[0042] If the amplified product contains the MdLOG gene promoter deletion fragment, then the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment; if the amplified product does not contain the MdLOG gene promoter deletion fragment, then the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment.

[0043] When the tested Malus plant is a heterozygote, if the amplified product shows two bands after electrophoresis, the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment (that is, the MdLOG gene promoter sequence in the genome lacks the "MdLOG gene promoter deletion fragment"); if the amplified product shows one band after electrophoresis, the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment (that is, the MdLOG gene promoter sequence in the genome does not lack the "MdLOG gene promoter deletion fragment").

[0044] When the tested Malus plant is homozygous, the amplified product is a single band, regardless of whether the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment. If the amplified product is a relatively small band, the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is missing the "MdLOG gene promoter deletion fragment"); if the amplified product is a relatively large band, the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome is not missing the "MdLOG gene promoter deletion fragment").

[0045] Of course, no matter whether the tested Malus plant is a heterozygote or a homozygote, whether the MdLOG gene promoter deletion fragment is contained in the genome of the tested Malus plant can be determined by sequencing the amplified product after PCR amplification.

[0046] In the above aspects, the Malus plant to be tested may be a salt-tolerant Malus plant, a non-salt-tolerant Malus plant, or a hybrid of a salt-tolerant Malus plant and a non-salt-tolerant Malus plant.

[0047] In a specific embodiment of the present invention, the Malus plant to be tested is a hybrid offspring (such as F1 generation) of the salt-tolerant Malus plant Malus multiflorus and the non-salt-tolerant Malus plant M9.

[0048] In a fourth aspect, the present invention claims protection for any one of the following methods:

[0049] Method 5: A method for cultivating a salt-tolerant Malus plant or a salt-tolerant rootstock Malus plant, comprising the following steps: cultivating a Malus plant whose genome does not contain the aforementioned MdLOG gene promoter deletion fragment (i.e., the MdLOG gene promoter sequence in the genome lacks the "MdLOG gene promoter deletion fragment");

[0050] Method 6: A method for cultivating non-salt-tolerant Malus plants or cultivating Malus plants used as non-salt-tolerant rootstocks, comprising the following steps: cultivating Malus plants whose genome contains the MdLOG gene promoter deletion fragment mentioned above (i.e., the MdLOG gene promoter sequence in the genome does not lack the "MdLOG gene promoter deletion fragment").

[0051] In a fifth aspect, the present invention claims protection for any of the following biological materials:

[0052] (G1) DNA fragment as shown in SEQ ID No. 3;

[0053] (G2) A primer pair, which is the primer pair according to claim 4.

[0054] Experiments conducted in the present invention demonstrate that, through PCR detection of the segregation of the MdLOG gene promoter fragment sequence in Malus multiflora, M9, and hybrid offspring, and combined with the salt tolerance phenotype of each offspring, it was found that the absence of the MdLOG gene promoter fragment (SEQ ID No. 3) is positively correlated with salt tolerance, and that the absence of the MdLOG gene promoter fragment (SEQ ID No. 3) enhances salt tolerance. Using the absence and presence of the MdLOG gene promoter fragment (SEQ ID No. 3) in the present invention, salt-tolerant plants can be rapidly screened in offspring, which is of great significance in the field of plant salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the location of the MdLOG gene in the apple genome.

[0056] Figure 2 Sequence alignment of insertion or deletion fragments in the MdLOG promoter. The top is M9; the bottom is Malus officinalis.

[0057] Figure 3 Agarose gel electrophoresis analysis of the separation of the MdLOG gene promoter fragment in apple offspring. M is a marker; lanes 1-10 represent 10 F1 plants. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0059] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0060] Apple rootstock M9: It has been disclosed in the literature "Yu Liang, Wang Fei. Establishment of rapid propagation technology of apple rootstock M_9 and anatomical study of the rooting process of test tube seedlings [J]. Journal of Northwest Forestry University, 2013, 28(4):106-110." and "Yi Feng et al., Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks. Plant Cell Environ. 2019; 42:424–436." The public can obtain it from China Agricultural University and can only be used for repeating the experiments of the present invention and may not be used for other purposes.

[0061] Malus occidentalis: disclosed in the literature "Zong Pengpeng, Qu Yanhua, Chai Peng, et al. Evaluation of salt and alkali tolerance of Malus occidentalis [J]. Journal of China Agricultural University, 2013, 18(3):96-100." and "Yi Feng et al., Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks. Plant Cell Environ. 2019; 42:424–436.", the public can obtain it from China Agricultural University, and it can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0062] Example 1: Obtaining the promoter deletion sequence of the cytokinin activating enzyme gene MdLOG

[0063] 1. MdLOG promoter sequence analysis

[0064] A search for the cytokinin biosynthesis activating enzyme gene MdLOG in the apple genome revealed that the MdLOG gene is located on chromosome 5. Figure 1 shown. Figure 1It shows that the basic structure of the genomic sequence of the MdLOG protein includes 7 exons and 6 introns. To facilitate the description of the gene structure, in the following examples, the position of A in the start codon ATG of the MdLOG gene in the genomic sequence of the MdLOG protein is denoted as +1, - represents the 5' direction of the start codon ATG of the MdLOG gene in the genomic sequence of the MdLOG protein, and + represents the 3' direction of the start codon ATG of the MdLOG gene in the genomic sequence of the MdLOG protein.

[0065] 2. Amplification of the MdLOG gene promoter in apple rootstock

[0066] Genomic DNA of salt-tolerant apple rootstock (Malus balsamifera) and non-salt-tolerant apple rootstock (M9) were extracted respectively, and PCR amplification was performed using them as templates and F1 and R1 as primers to obtain PCR amplification products of each rootstock.

[0067] F1: 5′-GACTTAACCCATCCACCGACA-3′,

[0068] R1: 5′-CATTTTGTTTATCTTCCACACACC-3′.

[0069] The nucleotide sequence of the PCR amplification product of the Malus multiflora rootstock is SEQ ID No. 1, named fragment 1 (part of the MdLOG gene promoter), which consists of positions 25,508,985 to 25,509,238 and positions 25,509,267 to 25,509,538 of chromosome 5 of the Golden Crown apple genome.

[0070] The nucleotide sequence of the PCR amplification product of the M9 rootstock is SEQ ID No. 2, named fragment 2 (a portion of the MdLOG gene promoter). The fragment is located at positions 25,508,985 to 25,509,538 of chromosome 5 of the Golden Delicious apple genome.

[0071] The amplified product SEQ ID No. 2 of M9 was compared with the amplified product SEQ ID No. 1 of Malus multiflora. The sequence alignment results with differences are shown as follows: Figure 2 As shown, it can be seen that there is a deletion fragment 5'-GACTTGCCCGACGAGACTTGCCCGACAA-3' (SEQ ID No. 3) in the MdLOG gene promoter of Malus multiflora, i.e., at positions -1305 to -1278 upstream of ATG, recorded as "MdLOG gene promoter deletion fragment", which is located at positions 25,509,239 to 25,509,266 of chromosome 5 in the Golden Crown apple genome.

[0072] Therefore, the MdLOG gene promoter deletion fragment shown in SEQ ID No. 3 may be a marker for salt tolerance in apple plants. This fragment is located from positions -1305 to -1278 upstream of the A in the start codon ATG of the MdLOG gene in the apple genome, with the A in the start codon ATG of the MdLOG gene being designated as position +1. This fragment also represents nucleotides 25,509,239 to 25,509,266 on chromosome 5 of the Golden Delicious apple genome (SEQ ID No. 3). Apple rootstocks that do not contain the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (i.e., have a fragment deletion in the MdLOG gene promoter) have higher salt tolerance than apple plants that contain the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (i.e., have no fragment deletion in the MdLOG gene promoter).

[0073] Example 2: Application of the MdLOG gene promoter deletion fragment in detecting salt tolerance of apple rootstocks I. Detection of the MdLOG gene promoter deletion fragment

[0074] 1. Obtaining genomic DNA of hybrid offspring with different salt tolerance

[0075] The hybridization was carried out with Malus multiflora as the female parent and M9 as the male parent to obtain the F1 hybrid offspring.

[0076] The leaves of 10 F1 generation hybrid offspring plants (plant numbers 1-10) with different salt tolerance were collected and genomic DNA was extracted.

[0077] 2. Detection of MdLOG gene promoter deletion fragment

[0078] Design and synthesize the following primers: (the upstream primer is designed about 150 bp upstream of the "MdLOG gene promoter deletion fragment", and the downstream primer is designed downstream of the deletion fragment)

[0079] MdLOG-F: 5′-ACGCTTGAGCATGGAATGGA-3′ (SEQ ID No. 4);

[0080] MdLOG-R: 5′-AGATATGTACATGTGTTTGCGT-3′ (SEQ ID No. 5).

[0081] PCR amplification was performed using the genomic DNA obtained in step 1 as a template and MdLOG-F and MdLOG-R as primers. The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2 to obtain various PCR amplification products.

[0082] Table 1. PCR amplification system (15 μl)

[0083] PCR reaction components Amount added to each system 2.5×Buffer V 6.0 μl MdLOG-F and MdLOG-R (5 μM each) 1.0 μl Taq DNA polymerase (5 U / μl) 0.1 μl DNA 1.0 μl <![CDATA[ddH2O]]> Make up to 15 μl

[0084] Table 2. PCR reaction procedure

[0085]

[0086] Each PCR amplification product was subjected to agarose gel electrophoresis using an electrophoresis apparatus, and the obtained gel was visualized and detected using a gel imaging system.

[0087] The results are as follows Figure 3 As shown, 5 of the 10 F1 generations (lanes 1, 5, 6, 7, and 8) were able to amplify two bands, and another 5 (lanes 2, 3, 4, 9, and 10) were able to amplify one band, indicating that the samples that could amplify two bands did not contain the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (i.e., there was a fragment deletion in the MdLOG gene promoter), and the samples that could amplify one band contained the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (i.e., there was no fragment deletion in the MdLOG gene promoter).

[0088] 2. Salt tolerance phenotypic detection

[0089] Each of the 10 F1 generation plants with plant numbers 1-10 obtained above was used as a rootstock to graft Fuji scion. After the 10 rootstocks with plant numbers 1-10 were grafted onto the scion, and the characteristics of the scion leaves were expressed by the salt damage index after being treated with a 200mM concentration of sodium chloride solution for 30 days. The salt damage index is divided into five levels from 0 to 4: 0 is when the leaves are asymptomatic; 1 / 3 or less of the leaves are burnt yellow at the top or edge; 2 is when the leaves are brown at the top or edge from 1 / 3 to 2 / 3 (excluding the end points); 3 is when the leaves are brown at the top or edge or dead (the whole plant has not died or withered yet); 4 is when the whole plant is dead or withered. Levels 0-1 are defined as salt-tolerant, and levels 2-4 are defined as salt-intolerant. The corresponding plant phenotypic data are shown in Table 3 below. The phenotype of each individual plant has 3 biological replicates. The biological replicate plants are asexually propagated by cuttings and rooting of the branches of the individual plant, so the genome sequences between the biological replicates are completely consistent.

[0090] Table 3. Phenotypes of 10 rootstocks grafted with scions three years later

[0091] Strain No. Marking situation Salt damage index Salt damage index Salt damage index mean 1 2 0 1 0 0.33b 2 1 3 2 3 2.67a 3 1 3 3 3 3.00a 4 1 3 4 2 3.00a 5 2 1 0 1 0.67b 6 2 1 1 0 0.67b 7 2 0 0 1 0.33b 8 2 1 1 1 1.00b 9 1 2 2 3 2.33a 10 1 2 2 2 2.00a

[0092] In the table, the label "2" indicates that the MdLOG gene promoter has a deletion (i.e., the genome does not contain the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3); the label "1" indicates that the MdLOG gene promoter does not have a deletion (i.e., the genome contains the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3). Columns with different lowercase letters in the same column are significantly different at the P < 0.05 level (Duncan method).

[0093] It can be seen that the salt damage index values ​​of the rootstock and scion plants of the apple plant whose genome does not contain the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (that is, the MdLOG gene promoter has a fragment deletion phenomenon) are lower than those of the rootstock and scion plants of the apple plant whose genome contains the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 (that is, the MdLOG gene promoter has no fragment deletion phenomenon).

[0094] Therefore, the "MdLOG gene promoter deletion fragment" shown in SEQ ID No. 3 can be used as a marker for detecting the salt tolerance of apple plant rootstocks, and can be used to detect or assist in detecting the salt tolerance of apple plant rootstocks, detect or assist in detecting the salt tolerance status of apple plants (salt damage index), detect or assist in detecting whether the apple plant to be tested is a salt-tolerant plant, and detect or assist in detecting whether the apple plant to be tested is used as a salt-tolerant rootstock.

[0095] The salt tolerance of the rootstock of the tested apple plant whose genome does not contain the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has a fragment deletion phenomenon) is higher than that of the tested apple plant whose genome contains the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has no fragment deletion phenomenon).

[0096] The salt damage index of the tested apple plants whose genome does not contain the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has a fragment deletion phenomenon) is lower than that of the tested apple plants whose genome contains the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has no fragment deletion phenomenon).

[0097] If the genome of the tested Malus plant does not contain the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has a fragment deletion phenomenon), then the tested Malus plant is or is a candidate for being a salt-tolerant plant, or the tested Malus plant can or is a candidate for being able to be used as a salt-tolerant rootstock; if the genome of the tested Malus plant contains the "MdLOG gene promoter deletion fragment" (i.e., the MdLOG gene promoter has no fragment deletion phenomenon), then the tested Malus plant is not or is not a candidate for being a salt-tolerant plant, or the tested Malus plant cannot or is a candidate for being unable to be used as a salt-tolerant rootstock.

[0098] The salt tolerance of the rootstock of the apple plant whose genome does not contain the "MdLOG gene promoter deletion fragment" (i.e., there is a fragment deletion in the MdLOG gene promoter) is higher than that of the apple plant whose genome contains the "MdLOG gene promoter deletion fragment" (i.e., there is no fragment deletion in the MdLOG gene promoter). This is reflected as follows: the salt damage index of the rootstock and scion of the apple plant whose genome does not contain the "MdLOG gene promoter deletion fragment" (i.e., there is a fragment deletion in the MdLOG gene promoter) is lower than that of the rootstock and scion of the apple plant whose genome contains the "MdLOG gene promoter deletion fragment" (i.e., there is no fragment deletion in the MdLOG gene promoter).

[0099] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. <110> China Agricultural University <120> MdLOG gene promoter deletion fragment and its application in detecting salt tolerance in Malus plants <130> GNCLN221224 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 526 <212> DNA <213> Artificial sequence <400> 1 gacttaaccc atccacgaca gtccttgata aacgagaagt ccgaagttac ttggggcgca 60 agtaatcgac ctaccctcta gttttattc tattatta tgattaccat agaacgcttg 120 agcatggaat ggattctgat ggaaagccctc aaggcctaca cctaaggccc cacaaaggca 180 cctctcaaag ttactctgt ccttcttt cagccttgcc cgacgagcct tgcccgaaga 240 gtcttgcccg acaaccccg acagtgaagc agaagcccga cagcagccct CAccggcgc 300 haacctccag gagagctgtg tgctcgtcgg ccaggaaaca tccccgacgg aaattcctga 360 cgcaacaca atgtacatat ctatttatgt gttaaaaaaa atgctattta caaatctatt 420 tttacttga atatatacct ttcaatctc aaccatcaa tttataaaa ttgaatagaa 480 taaaaaaata taactaata aaggtgtgt ggagatac aaaatg 526 <210> 2 <211> 554 <212> DNA <213> Artificial Sequence <400> 2 gacttaaccc atccacgaca gtccttgata aacgagaagt ccgaagttac ttggggcgca 60 agtaatcgac ctaccctcta gttttattc tattatta tgattaccat agaacgcttg 120 agcatggaat ggattctgat ggaaagccctc aaggcctaca cctaaggccc cacaaaggca 180 cctctcaaag ttactctgt ccttcttt cagccttgcc cgacgagcct tgcccgaaga 240 gtcttgcccg acagacttg cccgacgaga cttgcccgac aacgcccgac agtgaagcag 300 aagcccgaca gcagccctca accggcgcca acctccagga gagctgtgtg ctcgtcggcc 360 aggaaacatc cccgacggaa attcctgacg siaacaat gtacatatct atttatgtgt 420 taaaaaaaat gctattaca aatctatttt tactttgaat ataccttt caatcttca 480 ccatcaattt taaaaatt gatgaata aaaaaata aactaaa aggtgtgtgg 540 Agathaace etg 554 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <400> 3 gacttgcccg acgagacttg cccgacaa 28 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 acgcttgagc 20 <210> 5 <211> 23 <212> DNA <213> Artificial sequence <400> 5 agatatgtac attgtgtttg cgt 23

Claims

1. Application of the MdLOG gene promoter deletion fragment as a molecular marker in any of the following: (A1) Detecting or assisting in detecting the salt tolerance of rootstocks of plants of the genus Apple, or preparing products for detecting or assisting in detecting the salt tolerance of rootstocks of plants of the genus Apple; (A2) Detecting or assisting in detecting the salt damage index of apple plants, or preparing products for detecting or assisting in detecting the salt damage index of apple plants; (A3) Detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant; (A4) Detecting or assisting in detecting whether a plant of the genus Malus to be tested can be used as a salt-tolerant rootstock, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested can be used as a salt-tolerant rootstock; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

2. Use of a substance for detecting a deletion fragment of the MdLOG gene promoter in any of the following: (A1) Detecting or assisting in detecting the salt tolerance of rootstocks of plants of the genus Apple, or preparing products for detecting or assisting in detecting the salt tolerance of rootstocks of plants of the genus Apple; (A2) Detecting or assisting in detecting the salt damage index of apple plants, or preparing products for detecting or assisting in detecting the salt damage index of apple plants; (A3) Detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant; (A4) Detecting or assisting in detecting whether a plant of the genus Malus to be tested can be used as a salt-tolerant rootstock, or preparing a product for detecting or assisting in detecting whether a plant of the genus Malus to be tested can be used as a salt-tolerant rootstock; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

3. The use according to claim 2, characterized in that: The substance used to detect the MdLOG gene promoter deletion fragment is a primer pair used to amplify the MdLOG gene promoter deletion fragment.

4. The use according to claim 3, characterized in that: The upstream primer in the primer pair is designed based on the sequence located upstream of the MdLOG gene promoter deletion fragment in the apple plant genome, and the downstream primer is designed based on the sequence located downstream of the MdLOG gene promoter deletion fragment in the apple plant genome.

5. The use according to claim 4, characterized in that: The primer pair consists of a single-stranded DNA molecule shown in SEQ ID No. 4 and a single-stranded DNA molecule shown in SEQ ID No.

5.

6. A method for detecting or assisting in detecting the salt tolerance of a rootstock of a plant of the genus Malus, comprising the following steps (B1) and (B2): (B1) Detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter; (B2) Based on the result of (B1), the salt tolerance of the rootstock of the tested Malus plant is determined as follows: the salt tolerance of the rootstock of the tested Malus plant whose genome does not contain the MdLOG gene promoter deletion fragment is higher or is a candidate higher than the salt tolerance of the rootstock of the tested Malus plant whose genome contains the MdLOG gene promoter deletion fragment; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant to be tested is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

7. A method for detecting or assisting in detecting the salt damage index of a plant of the genus Malus, comprising the following steps (C1) and (C2): (C1) Detect whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter; (C2) Based on the result of (C1), the salt damage index of the tested Malus plant is determined as follows: the salt damage index of the tested Malus plant whose genome does not contain the MdLOG gene promoter deletion fragment is lower than, or is a candidate lower than, the salt damage index of the tested Malus plant whose genome contains the MdLOG gene promoter deletion fragment; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant to be tested is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

8. A method for detecting or assisting in detecting whether a plant of the genus Malus to be tested is a salt-tolerant plant, comprising the following steps (D1) and (D2): (D1) Detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter; (D2) Based on the result of (D1), determining whether the tested Malus plant is a salt-tolerant plant is as follows: if the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment, then the tested Malus plant is or is a candidate for being a salt-tolerant plant; if the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment, then the tested Malus plant is not or is not a candidate for being a salt-tolerant plant; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant to be tested is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

9. A method for detecting or assisting in detecting whether a tested Malus plant can be used as a salt-tolerant rootstock, comprising the following steps (E1) and (E2): (E1) Detecting whether the genome of the tested Malus plant contains a deletion fragment of the MdLOG gene promoter; (E2) Based on the result of (E1), determining whether the tested Malus plant can be used as a salt-tolerant rootstock is as follows: if the genome of the tested Malus plant does not contain the MdLOG gene promoter deletion fragment, then the tested Malus plant can or is a candidate for being used as a salt-tolerant rootstock; if the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment, then the tested Malus plant cannot or is a candidate for being used as a salt-tolerant rootstock; The MdLOG gene promoter deletion fragment is the DNA fragment shown in SEQ ID No. 3; The Malus plant to be tested is: (1) Malus occidentalis; (2) M9; or (3) a hybrid of Malus occidentalis and M9.

10. The method according to any one of claims 6 to 9, characterized in that: The method for detecting whether the genome of the tested Malus plant contains the MdLOG gene promoter deletion fragment is as follows (F1) or (F2): (F1) direct sequencing; (F2) Using the genome of the test Malus plant as a template, perform PCR amplification using the primer pair described in any one of claims 3 to 5, and determine whether the genome of the test Malus plant contains the MdLOG gene promoter deletion fragment based on the amplification results.

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