Molecular markers related to cotton leaf shape control genes and their application

Abstract

The present invention belongs to the technical field of cotton leaf shape variety breeding, and relates to a molecular marker related to a cotton leaf shape control gene and its application. The molecular marker of the present invention includes an Indel molecular marker located in the promoter region of the cotton leaf shape control gene LBL1, which can distinguish the depth of cotton leaf fissures; further, it can also include two dCAPS markers, LBL1-3852 located in the 3'-UTR region of the cotton leaf shape control gene LBL1 and LBL2-4610 located in the 3'-UTR region of LBL2. These markers can be used in combination to distinguish four leaf shapes of cotton. This study deeply analyzed the polymorphic sites in the promoter region of the LBL1 and LBL2 alleles, designed specific molecular markers, and used the markers to perform early screening of various leaf shape varieties of cotton, providing a basis for the identification of different leaf shape genes and molecular assisted selection.

Description

Technical Field

[0001] The invention relates to the technical field of cotton leaf shape variety breeding, and in particular to a molecular marker related to a cotton leaf shape control gene and an application thereof. Background Art

[0002] Cotton is one of the important cash crops. It is not only a raw material for the textile industry, but also an important strategic material. It plays an important role in the development of the national economy. China is the largest producer and consumer of raw cotton. The release of the cotton genome sequence has greatly promoted the research on cotton molecular biology and provided a solid foundation for molecular design-assisted breeding. Further clarifying the function of genes and finding key genes that control important traits of cotton are the primary contents of cotton molecular biology at present.

[0003] Domestic and foreign scholars have identified the genetic diversity of upland cotton cultivars and found that the genetic background of the main upland cotton cultivars today is narrow, which poses a huge challenge to the breeding of new cultivars. To a certain extent, the level of genetic diversity of cotton germplasm determines the difficulty of breeding new varieties. Therefore, improving the genetic diversity of upland cotton cultivars is the primary task of breeding new cotton varieties. The construction of a mutant library is a favorable way to create new cotton germplasm resources. Mutants are ideal materials for discovering new genes and revealing gene functions. Functional genomics aims to identify gene functions in order to reveal the relationship between genes and traits. With the large-scale development of genome sequencing and the acquisition of a large amount of nucleic acid sequence information, the study of gene function only through mutants obtained by natural mutations is far from meeting the research needs of molecular biology.

[0004] The canopy structure of cotton is affected by external factors, but it is mainly closely related to its own biological characteristics such as leaf shape, leaf inclination, fruit branch length, and fruit branch angle. The leaf shape is mainly divided into four types: duck foot leaf, sub-chicken foot leaf, chicken foot leaf, and super chicken foot leaf. Chicken foot leaf cotton is widely planted in Australia, and the planting area in some cotton-growing areas is close to 50%. In the inland cotton-growing areas of northwest my country, chicken foot leaf cotton has high light energy utilization efficiency, and the lint yield has reached an ultra-high yield level (3500 kg·hm -2 The four main leaf shapes are shown in Table 1. Figure 1 , Figure 1 A is duckweed leaf, Figure 1 B is the leaves of the chicken claw, Figure 1 C is chicken claw leaf, Figure 1 D is super chicken claw leaf (willow leaf).

[0005] Some leaf shapes are extremely similar and difficult to distinguish accurately. At the same time, various leaf shapes can only become basically stable in the later stages, which brings certain difficulties to the early identification of germplasm and its breeding application.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In view of the defects of the prior art, the purpose of the present invention is to provide a molecular marker related to a cotton leaf shape control gene and its application, the molecular marker includes an InDel molecular marker related to the cotton leaf shape LBL1 gene used alone, or also includes a CAPS marker related to the cotton leaf shape LBL1 gene and a CAPS marker related to the cotton leaf shape LBL2 gene used in combination with the InDel molecular marker. The present invention uses these molecular markers to identify the leaf shape of cotton varieties.

[0008] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0009] The first aspect of the present invention provides a substance for detecting the polymorphism or genotype of a molecular marker related to a cotton leaf shape control gene, wherein the molecular marker comprises an InDel molecular marker located in the promoter region of the cotton leaf shape control gene LBL1, wherein the polymorphism of the InDel molecular marker is an insertion or deletion of the nucleotide sequence shown in sequence 1 between the 2331st and 2332nd positions of the nucleotide sequence shown in sequence 4; or the nucleotide sequence of the InDel molecular marker is as shown in sequence 6 or sequence 7; or the InDel molecular marker is located at position 61163862 on chromosome D01 of the upland cotton reference genome Ghir.HAU_TM-1 (V1), and the polymorphism thereof is an insertion or deletion of the nucleotide sequence shown in sequence 1 between the 61163862nd and 61163863rd positions; when the polymorphism of the InDel molecular marker is an insertion of the nucleotide sequence shown in sequence 1 or the nucleotide sequence of the InDel molecular marker is as shown in sequence 6, the InDel molecular marker is expressed as LBL1-2331-InDel. 133; when the InDel molecular marker is the nucleotide sequence shown in the deletion sequence 1 or the nucleotide sequence of the InDel molecular marker is as shown in sequence 7, the InDel molecular marker is represented as LBL1-2331-del 133; the substance includes a first primer pair for amplifying the InDel molecular marker.

[0010] The nucleotide sequence shown in Sequence 4 in the sequence table is the full length of the LBL1 leaf shape control gene of the duck-foot-leaved cotton material, which includes a promoter region formed by nucleotides 1-3000, a gene coding region formed by nucleotides 3001-3806, and a 3'-terminal UTR region formed by nucleotides 3807-4306. The InDel molecular marker is located exactly at position 2331 of the nucleotide sequence shown in Sequence 4. Specifically, the polymorphism of the InDel molecular marker is: there is no nucleotide at positions 2331 and 2332 of the nucleotide sequence shown in Sequence 4, which is represented as LBL1-2331-del 133, or there is and only the nucleotide sequence shown in Sequence 1 between positions 2331 and 2332 of the nucleotide sequence shown in Sequence 4, which is represented as LBL1-2331-In 133.

[0011] Of course, the polymorphism of the InDel molecular marker can also be described by the product sequence after amplification by the first primer pair. Further, the nucleotide sequences of the InDel molecular marker are shown in Sequence 6 and Sequence 7, respectively. When it is Sequence 6, the InDel molecular marker can also be called LBL1-2331-In 133, and when it is Sequence 7, the InDel molecular marker can also be called LBL1-2331-del 133.

[0012] If the situation of the InDel molecular marker is described by the cotton reference genome Ghir.HAU, the InDel molecular marker is located at position 61163862 on chromosome D01 of the cotton reference genome Ghir.HAU_TM-1 (V1), and its polymorphism is the insertion or deletion of the nucleotide sequence shown in sequence 1 between positions 61163862 and 61163863. Specifically, the polymorphism of the InDel molecular marker is: there is no nucleotide between positions 61163862 and 61163863, in which case it is named LBL1-2331-del 133, or there is and only the nucleotide sequence shown in sequence 1 between positions 61163862 and 61163863, in which case it is named LBL1-2331-In 133.

[0013] The first primer pair developed based on the InDel molecular marker can specifically be: the base sequence of the forward primer is shown as SEQ ID NO.2 in the sequence listing, and the base sequence of the reverse primer is shown as SEQ ID NO.3 in the sequence listing.

[0014] This molecular marker can be used for the preliminary identification of leaf shape of cotton materials, and it can distinguish the depth of leaf fissures. When the genotype is LBL1-2331-In 133: LBL1-2331-In 133, the leaf fissures of cotton are deep; when the genotype is LBL1-2331-del133: LBL1-2331-del 133, the leaf fissures of cotton are shallow.

[0015] In order to further distinguish different leaf shapes, for example, to distinguish between duck-foot leaves, sub-chicken-claw leaves, chicken-claw leaves, and willow leaves in the early stages of cotton growth, the inventors developed two other molecular markers for use in combination with the above-mentioned InDel molecular markers.

[0016] The other two molecular markers are dCAPS marker LBL1-3852 located in the 3'-UTR region of cotton leaf shape control gene LBL1 and dCAPS marker LBL2-4610 located in the 3'-UTR region of cotton leaf shape control gene LBL2.

[0017] The polymorphism of the marker LBL1-3852 is the insertion or deletion of nucleotide G between the 3852nd and 3853rd positions of the nucleotide sequence shown in Sequence 4; or the nucleotide sequence of the marker LBL1-3852 is as shown in Sequence 8 or Sequence 9; or the marker LBL1-3852 is located at position 61165383 on the D01 chromosome of the cotton reference genome Ghir.HAU_TM-1 (V1), and its polymorphism is the insertion or deletion of nucleotide G between positions 61165383 and 61165384; when the polymorphism of the marker LBL1-3852 is the insertion of nucleotide G or the nucleotide sequence of the marker LBL1-3852 is as shown in Sequence 8, the marker LBL1-3852 is represented as LBL1-3852-In G; when the polymorphism of marker LBL1-3852 is a missing nucleotide G or the nucleotide sequence of marker LBL1-3852 is as shown in SEQ ID NO: 9, the marker LBL1-3852 is represented as LBL1-3852-del G;

[0018] The polymorphism of the marker LBL2-4610 is that the nucleotide sequence shown in sequence 30 is inserted or deleted between the 4610th and 4625th positions of the nucleotide sequence shown in sequence 5; or the nucleotide sequence of the marker LBL2-4610 is as shown in sequence 10 or sequence 11; or the marker LBL2-4610 is located at the 61039740-61039755th position on the D01 chromosome of the cotton reference genome Gbar.HAU_3-79, and its polymorphism is that the nucleotide sequence shown in sequence 30 is inserted or deleted between the 61039740th and 61039755th positions. The nucleotide sequence shown in sequence 30 is missing; when the polymorphism of marker LBL2-4610 is the nucleotide sequence shown in insertion sequence 30 or the nucleotide sequence of marker LBL2-4610 is as shown in sequence 10, the marker LBL2-4610 is represented as LBL2-4610-In14; when the polymorphism of marker LBL2-4610 is the nucleotide sequence shown in deletion sequence 30 or the nucleotide sequence of marker LBL2-4610 is as shown in sequence 11, the marker LBL2-4610 is represented as LBL2-4610-del14.

[0019] According to the above-mentioned dCAPS markers LBL1-3852 and LBL2-4610, the material of the first aspect of the present invention also includes: a second primer pair for amplifying the marker LBL1-3852 and a third primer pair for amplifying the marker LBL2-4610; further, the nucleotide sequence of the forward primer of the second primer pair is shown as SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.15; the nucleotide sequence of the forward primer of the third primer pair is shown as SEQ ID NO.16, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.17.

[0020] Furthermore, in order to simplify the leaf shape identification procedure, the substance may also include a first restriction endonuclease for enzymatically cleaving the amplification product of the second primer pair; and a second restriction endonuclease for enzymatically cleaving the amplification product of the third primer pair. Furthermore, the first restriction endonuclease is Sau3A I; and the second restriction endonuclease is BcoD I.

[0021] The second aspect of the present invention provides a kit, which includes the substance of the first aspect of the present invention and PCR auxiliary reagents and / or enzyme cleavage auxiliary reagents, such as Taq enzyme, buffer, etc., which can be specifically configured according to the reagents required for conventional PCR and enzyme cleavage.

[0022] The third aspect of the present invention provides the use of the substance described in the first aspect or the kit described in the second aspect in any of the following aspects:

[0023] (A) Application in identifying or breeding cotton varieties with different leaf crack depths;

[0024] (B) Application for identification or selection of cotton leaf shape varieties;

[0025] The depth of the cotton leaf fissures includes shallow leaf fissures and deep leaf fissures. The shallow leaf fissures correspond to duck-foot leaves or sub-chicken-claw leaves, and the deep leaf fissures correspond to chicken-claw leaves or willow leaves.

[0026] The cotton leaf shapes include duck palm leaf, sub-chicken claw leaf, chicken claw leaf, and willow leaf;

[0027] A material for detecting the polymorphism or genotype of the InDel molecular marker is used to identify or breed cotton varieties with deep or shallow leaf fissures. When the genotype of the InDel molecular marker is LBL1-2331-In 133: LBL1-2331-In 133, the leaf fissures of the cotton are deep; when the genotype of the InDel molecular marker is LBL1-2331-del 133: LBL1-2331-del133, the leaf fissures of the cotton are shallow.

[0028] The cotton varieties with different leaf shapes are identified or bred by using a material for detecting the polymorphism or genotype of the InDel molecular marker and a material for detecting the polymorphism or genotype of the markers LBL1-3852 and LBL2-4610, wherein:

[0029] When the genotype of the marker LBL1-3852 is LBL1-3852-In G: LBL1-3852-In G, the leaf shape of the cotton is sub-chicken claw leaf, chicken claw leaf or willow leaf; when the genotype of the marker LBL1-3852 is LBL1-3852-delG: LBL1-3852-In G, the leaf shape of the cotton is duck foot leaf;

[0030] When the genotype of the marker LBL2-4610 is LBL2-4610-In14: LBL2-4610-In14, the leaf shape of the cotton is sub-chicken claw leaf or chicken claw leaf; when the genotype of the marker LBL2-4610 is LBL2-4610-del 14: LBL2-4610-del 14, the leaf shape of the cotton is duck foot leaf or willow leaf.

[0031] The genotype of the InDel molecular marker is LBL1-2332-In 133: LBL1-2332-In 133 means that the InDel molecular marker on the allele of the homologous chromosome is inserted into the nucleotide sequence shown in Sequence 1 between the 2331st and 2332nd positions of the nucleotide sequence shown in Sequence 4, that is, it is homozygous; the genotype of other molecular markers also represents the type or polymorphism of two molecular markers on the allele of the homologous chromosome. I will not repeat them one by one here.

[0032] The fourth aspect of the present invention provides a method for identifying cotton leaf shape using the substance of the first aspect of the present invention or the kit of the second aspect, comprising the following steps:

[0033] S1. Extracting genomic DNA of cotton to be tested;

[0034] S2, using the genomic DNA as a template, using the first primer pair to perform PCR amplification, and determining the leaf fissure depth of the cotton to be tested by identification;

[0035] The identification is to perform electrophoresis or sequencing on the PCR product. When the PCR product is 437bp, the cotton to be tested has shallow leaf lobes, and the shallow leaf lobes are duck-foot leaves or sub-chicken-claw leaves; when the PCR product is 570bp, the cotton to be tested has deep leaf lobes, and the deep leaf lobes are chicken-claw leaves or willow leaves.

[0036] Furthermore, the PCR reaction system in step S2 was: 1.0 μL of cotton genomic DNA at about 200 ng / μL, 10 μL of 2× Taq Plus Master Mix II (Dye Plus), 7 μL of ddH2O, 1.0 μL of forward primer GH1-P-F, 1.0 μL of reverse primer GH1-PR, a total of 20 μL;

[0037] The PCR program was as follows: pre-denaturation at 95°C for 3 minutes; 95°C for 15 seconds (denaturation), 55°C for 15 seconds, extension at 72°C for 20 seconds, 37 cycles; and 72°C for 5 minutes.

[0038] On the basis of steps S1 and S2, in order to further identify the cotton leaf shape, the method further includes:

[0039] S3, using the genomic DNA of the cotton to be tested as a template, and using the second primer pair to perform PCR amplification to obtain an amplification product containing the marker LBL1-3852; and / or

[0040] The third primer pair is used for PCR amplification to obtain an amplification product containing the marker LBL2-4610.

[0041] At this time, the genotype of the corresponding marker can be determined by sequencing the amplification products containing the respective markers. For example, when the sequence of the amplification product containing the marker LBL1-3852 is only the nucleotide sequence shown in sequence 8, the genotype of the marker is LBL1-3852-In G: LBL1-3852-In G, and the leaf shape of the cotton material to be tested is sub-chicken claw leaf, chicken claw leaf or willow leaf; when the sequence of the amplification product containing the marker LBL1-3852 is the nucleotide sequence shown in sequence 9, the genotype of the marker is LBL1-3852-del G: LBL1-3852-del G, and the leaf shape of the cotton material to be tested is duck palm leaf; when the sequence of the amplification product containing the marker LBL2-4610 is the nucleotide sequence shown in sequence 10, the genotype of the marker is LBL2-4610-In14: LBL2-4610-In 14, the leaf shape of the cotton material to be tested is sub-chicken claw leaf or chicken claw leaf; when the sequence of the amplification product containing the marker LBL2-4610 is the nucleotide sequence shown in sequence 11, the genotype of the marker is LBL2-4610-del 14: LBL2-4610-del 14, and the leaf shape of the cotton material to be tested is duck palm leaf or willow leaf.

[0042] Furthermore, the PCR amplification conditions in step S3 are: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 20 seconds, 37 cycles; and 72°C for 5 minutes to complete the product extension.

[0043] The PCR reaction system was: 1.0 μL of cotton genomic DNA (about 200 ng / μL), 10 μL of 2×Taq Plus MasterMix II (Dye Plus), 7 μL of ddH2O, 1.0 μL of forward primer, and 1.0 μL of reverse primer, for a total of 20 μL.

[0044] In order to reduce costs or simplify the identification procedure, the method may further include:

[0045] S4, treating the amplification product containing the marker LBL1-3852 with the first restriction endonuclease, and verifying the genotype of the marker LBL1-3852 by digesting the product with the enzyme;

[0046] Further, when the enzyme cleavage product is 55 bp and 243 bp, the leaf shape of the cotton to be tested is sub-chicken claw leaf, chicken claw leaf or super chicken claw leaf, and the genotype of the marker LBL1-3852 is LBL1-3852-In G: LBL1-3852-In G (also known as Hap2); when the enzyme cleavage product is 298 bp, the leaf shape of the cotton to be tested is duck claw leaf, and the haploid type is the genotype of the marker LBL1-3852 is LBL1-3852-delG: LBL1-3852-In G (also known as Hap1); and / or

[0047] The amplification product containing the marker LBL2-4610 is treated with a second restriction endonuclease, and the genotype of the marker LBL2-4610 is verified by the enzyme digestion product.

[0048] When the size of the enzyme cleavage product is 173bp and 194bp, the leaf shape of the cotton to be tested is sub-chicken claw leaf shape or chicken claw leaf shape, and the genotype of the marker LBL2-4610 is LBL2-4610-In14: LBL2-4610-In14 (also known as Hap3); when the size of the enzyme cleavage product is 368bp, the leaf shape of the cotton to be tested is duck foot leaf shape or super chicken claw leaf shape, and the genotype of the marker LBL2-4610 is LBL2-4610-del 14: LBL2-4610-del 14 (also known as Hap4).

[0049] In order to further simplify the method for identifying leaf shape, the following method can be used to select the identification step: when the identification result in step S2 is shallow leaf lobation, the second primer pair and the first restriction endonuclease are used to further detect the genotype of the marker LBL1-3852, or the third primer pair and the second restriction endonuclease are used to further detect the genotype of the marker LBL2-4610, thereby distinguishing the cotton of the duck palm leaf variety from the cotton of the sub-chicken claw leaf variety;

[0050] When the identification result of step S2 is deep leaf fissure, the third primer pair and the second restriction endonuclease are used to further detect the genotype of marker LBL2-4610, thereby distinguishing the cotton of the chicken-claw leaf variety from the cotton of the willow leaf variety.

[0051] The cotton material to be tested in the method of the present invention is preferably an inbred homozygous material. The accuracy rate of leaf shape identification of heterozygous cotton materials using the method of the present invention is low.

[0052] Beneficial effects of the present invention:

[0053] The molecular markers related to the cotton leaf shape control gene provided by the present invention include an Indel molecular marker (with 133bp tandem repeat) located in the promoter region of the cotton leaf shape control gene LBL1, which can distinguish the depth of cotton leaf cracks; further, it can also include two dCAPS markers, LBL1-3852 (with 1bp insertion / deletion) located in the 3'-UTR region of the cotton leaf shape control gene LBL1 and LBL2-4610 (with 14bp insertion / deletion) located in the 3'-UTR region of LBL2. These markers can be used in combination to distinguish four leaf shapes of cotton. The molecular markers of the cotton leaf shape control genes LBL1 and LBL2 of the present invention are both located on chromosome D01 of cotton. This study deeply analyzes the polymorphic sites in the promoter region of the LBL1 and LBL2 alleles, designs specific molecular markers, and uses the markers to perform early screening of various cotton leaf shape varieties, providing a basis for the identification of different leaf shape genes and molecular assisted selection, and realizing molecular assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 There are four main leaf shapes: A is duck's palm leaf, B is sub-chicken's claw leaf, C is chicken's claw leaf, and D is super chicken's claw leaf (willow leaf).

[0055] Figure 2 These are the sequence comparison results of the four main leaf shapes. A is the comparison result of the promoter region of the LBL1 gene; B is the partial sequence comparison result of the 3' UTR region; C is the partial sequence comparison result of the 3' UTR region of the LBL2 gene.

[0056] Figure 3 This is the electrophoresis diagram of PCR products when the leaf crack depth of some cotton to be tested is identified based on InDel molecular markers;

[0057] Figure 4 This is the electrophoresis diagram of the PCR product after enzyme digestion when leaf shape identification is performed based on dCAPS molecular markers. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a basis for further improvement or application by those of ordinary skill in the art, and do not constitute specific limitations of the present invention in any way.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified and can be performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from conventional commercial channels unless otherwise specified.

[0060] The terms "insertion" and "deletion" used in the present invention can also be replaced by "presence" or "absence". "Insertion" of a sequence or a nucleotide between two sites means that there is and only the sequence or the nucleotide between the two nucleotide sites. "Deletion" of a sequence or a nucleotide between two sites means that there is neither the sequence nor any other nucleotide sequence between the two nucleotide sites, that is, there is no nucleotide sequence or single nucleotide between the two nucleotide sites.

[0061] The source of the cotton varieties used in the present invention:

[0062] The resource materials used for detecting cotton leaf shape in Table 1 are obtained from the Germplasm Resource Bank of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0063] The DNA extraction involved in the following examples was all carried out using the CTAB method.

[0064] The leaf shapes of the cotton to be tested were duck-foot leaf, sub-chicken-claw leaf, chicken-claw leaf and super-chicken-claw leaf by naked eye observation. The specific determination of leaf shape can be found in the attached Figure 1 , or refer to the literature: He Daofang, Cloning and functional analysis of cotton leaf margin notch depth regulatory gene LBL1, Anhui Agricultural University, 2018; and RJ Andres etc., Modifications to a LATE MERISTEMIDENTITY1 gene are responsible for the major leaf shapes of Upland cotton (Gossypium hirsutum L.), "Proceedings of the National Academy of Sciences of the United States of America", 2017.

[0065] Example 1 Obtaining InDel and dCAPS Molecular Markers Related to Cotton Leaf Shape Control Genes

[0066] 1. Cloning of candidate genes

[0067] Cotton genomic DNA was extracted using the CTAB method from Duckfoot leaves, Sub-chicken claw leaves, and Chicken claw leaves.

[0068] The genomic DNA of cotton materials with super-chicken claw leaves was used as a template, and the full-length LBL1 and LBL2 genes, as well as 3000 bp sequences of the promoter regions of the two genes and 500 bp sequences of the 3'-end UTR regions were obtained by referring to the cotton reference genome sequences Ghir.HAU_TM-1 (V1) and Gbar.HAU_3-79 (V1). The version number of the cotton reference genome assembly Ghir.HAU_TM-1 is V1, of which the two genes LBL1 and LBL2 are Ghir_D01G021810 (LBL1); Ghir_D01G021830 (LBL2). The version number of the cotton reference genome assembly Gbar.HAU_3-79 is V1, of which the two genes LBL1 and LBL2 are Gbar_D01G021920 (LBL1); Gbar_D01G021940 (LBL2), downloaded from https: / / cottonfgd.net / . Primer Premier5.0 (https: / / www .PremierBiosoft.com) software was used to design specific primers for fragment amplification. The primer sequences for amplifying different regions are as follows. The full-length gene was cloned in four typical leaf-shaped cotton materials: duck-foot leaf, sub-chicken-claw leaf, chicken-claw leaf, and super-chicken-claw leaf according to the following operation.

[0069] 1. The primer pair used for PCR amplification of the LBL1 gene is:

[0070] F1:5'-ATGGATTGGGATGGCACCATT-3' (Sequence 18 in the sequence list)

[0071] R1: 5'-CTACTTTCGATGATCTCTCCGC-3' (sequence 19 in the sequence listing);

[0072] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 60 seconds, 34 cycles; and 72°C for 5 minutes to complete the product extension.

[0073] 2. The primer pair used for PCR amplification of the LBL1 gene promoter region is:

[0074] F2: 5'-agggagatccgcttccgtgt-3' (sequence 20 in the sequence list)

[0075] R2: 5'-ttctttgaatgaagaaagcgagtat-3' (sequence 21 in the sequence listing);

[0076] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 180 seconds, 34 cycles; and 72°C for 5 minutes to complete the product extension.

[0077] 3. The primer pair used for PCR amplification of the 3′ UTR region of the LBL1 gene is:

[0078] F3: 5'-ctcgattcggagctcgaaca-3' (sequence 22 in the sequence list)

[0079] R3: 5'-gtattgaacagcttaacaagaataat-3' (sequence 23 in the sequence listing);

[0080] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 30 seconds, 34 cycles; and then 72°C for 5 minutes to complete the product extension.

[0081] 4. The primer pair used for PCR amplification of LBL2 gene is:

[0082] F4: 5'-CCCCCTTTCAGTCTCCCCCT-3' (Sequence 24 in the sequence listing)

[0083] R4: 5'-ATGCAAAAAAAAGTAGTTCAAGCT-3' (Sequence 25 in the sequence listing)

[0084] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 60 seconds, 34 cycles; and 72°C for 5 minutes to complete the product extension.

[0085] 5. The primer pair used for PCR amplification of the promoter region of the LBL2 gene is:

[0086] F5: 5'-atacttacttctttacaatatagtccaa-3' (sequence number 26 in the sequence listing)

[0087] R5: 5'-gggggggttgcaggggtt-3' (sequence 27 in the sequence listing);

[0088] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 180 seconds, 34 cycles; and 72°C for 5 minutes to complete the product extension.

[0089] 6. The primer pair used for PCR amplification of the 3′ UTR region of the LBL2 gene is:

[0090] F6: 5'-gaataaggctgaatttttgtttttttt-3' (sequence 28 in the sequence listing)

[0091] R6: 5'-gattatttctttcttcgtctttacaaaga-3' (sequence number 29 in the sequence listing);

[0092] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 30 seconds, 34 cycles; and then 72°C for 5 minutes to complete the product extension.

[0093] The reaction system (20 μL) for PCR amplification reaction using the above six primer pairs contains: 1.0 μl of cotton genomic DNA of about 200 ng / μl, 10 μl of 2×Taq Plus Master Mix II (Dye Plus), 7 μl of ddH2O, 0.5 μM upstream primer, 0.5 μM downstream primer, a total of 20 μl;

[0094] Add the amplified product of Taq enzyme to 5 × TA / Blunt-Zero Cloning Mix and react at room temperature for 5 minutes. Take out the connected sample, add 50μL competent cells (Trans-T, stored at -80℃ before use) to the UV-sterilized workbench, mix gently, and place on ice for 20 minutes. After completion, move the sample to a water bath at 42℃ for 45 seconds, and then immediately place it on ice for 2 minutes. On the sterile operating table, add 700μL of antibiotic-free LB solid medium (formula per 100mL: 1g tryptone, 0.5g yeast extract, 0.5gNaCl, 1.5g agar powder, 100mL ultrapure water) to the sample and shake at 37℃ for 1-1.5h. Next, take 100-200 μL of bacterial solution and evenly spread it on LB solid medium with kanamycin (formula per 100 mL: 1g tryptone, 0.5g yeast extract, 0.5g NaCl, 1.5g agar powder, 100μL kanamycin solution, 100mL ultrapure water) in a sterile operating table, and incubate at 37°C for 15-16h. When the plaque grows to a suitable size, add 6μL of sterile water to the PCR sample well, use a pipette with a maximum range of 10μL to pick the bacteria into the well, gently pipette and mix, take out 4μL and add it to a centrifuge tube containing 1000μL LB liquid medium (formula per 100mL: 1g tryptone, 0.5g yeast extract, 0.5g NaCl, 100μL kanamycin solution, 100mL ultrapure water). Place the centrifuge tube in a 37°C environment and shake for 4-6 hours. At the same time, use the remaining 2 μL of bacterial solution in the sample well as a template and perform PCR detection using the universal primer M13 for bacterial solution sequencing. If the bacterial solution PCR reaction result is positive and contains the target fragment, the sample can be sent for sequencing.

[0095] The sequence of the M13 primer is:

[0096] SEQ ID NO.12: M13-F:5'-CAGGAAACAGCTATGACCATGAT-3';

[0097] SEQ ID NO.13:M13-R:5 '-GTAAAACGACGGCCAGTGC -3'

[0098] 2. Obtaining InDel Molecular Markers of Cotton Leaf Shape Control Gene LBL1

[0099] The full-length gene of LBL1 in duck-palm leaf, sub-chicken-claw leaf, chicken-claw leaf and super-chicken-claw leaf cotton materials was cloned, among which the full-length LBL1 gene of the duck-palm leaf material was 4306bp, and the nucleotide sequence was shown in sequence 4 in the sequence table, which included a promoter region formed by nucleotides 1-3000, a gene coding region formed by nucleotides 3001-3806, and a 3'-terminal UTR region formed by nucleotides 3807-4306. DNAMAN software was used to compare and analyze the full length of LBL1 gene of duck palm leaf, sub-chicken claw leaf, chicken claw leaf and super-chicken claw leaf cotton materials. It was found that: relative to the full length sequence of LBL1 gene of duck palm leaf and sub-chicken claw leaf cotton materials, the promoter region of the full length sequence of LBL1 gene of chicken claw leaf and super-chicken claw leaf cotton materials had a 133 bp nucleotide sequence inserted as shown in sequence 1, which was inserted between the 2331st and 2332nd positions of the full length sequence of LBL1 gene shown in sequence 4. The partial sequence comparison results of the promoter region of LBL1 gene of four leaf-shaped cotton materials can be found in Figure 2 A, wherein the blue highlighted portion is 133 bp shown in sequence 1, that is, there is no insertion of the 133 bp tandem repeat sequence at the corresponding position of the full-length sequence of the LBL1 gene of the duck-foot leaf and sub-chicken-claw leaf cotton materials, the molecular marker with the insertion of the 133 bp nucleotide sequence shown in sequence 1 is represented by LBL1-2332-In 133, and the molecular marker without the insertion of the 133 bp nucleotide sequence shown in sequence 1 is represented by LBL1-2332-del 133. Although some other differential sites, such as SNP sites, were found in the comparative analysis of the full-length gene sequences of the cotton materials of the four leaf types, the detection of these differential sites is not convenient for simply realizing the identification of the depth of cotton leaf fissures, and the InDel marker selected by the present invention can obtain the identification result by only one PCR and the result is reliable, and can be judged by the size of the electrophoresis band, rather than by the presence or absence of the band, avoiding the interference of false negatives caused by problems in the PCR system itself, and does not need to be judged by sequencing, which can save costs. Secondly, there is a 133 bp difference between the amplified products, which is of appropriate size and does not require 4% agarose gel electrophoresis, making it easy to configure. The InDel marker can be used as a molecular marker for tightly linked cotton leaf shape materials and applied to cotton leaf shape material breeding.

[0100] In order to identify the genotype of the InDel marker, amplification was performed using the following first PCR primer pair, and then the phenotype of the depth of cotton leaf cracking was determined based on the number and size of bands in the amplification results;

[0101] The first PCR primer pair includes:

[0102] Forward primer GH1-PF: 5'-GGTGATGGTTCGTCCTGTTG-3' (SEQ ID NO. 2)

[0103] Reverse primer GH1-PR: 5'-TTACATCAATCGTCAAATCATCACT-3' (SEQ ID NO. 3).

[0104] Using the cotton material genome as a template, the product sequence after amplification by this pair of PCR primers is shown in Sequence 6 or Sequence 7.

[0105] 3. Acquisition of dCAPS molecular markers related to cotton leaf shape control genes LBL1 and LBL2

[0106] Cleaved Amplified Polymorphism Sequences (CAPS) is also known as RFLP-PCR. The basic principle is to screen out DNA sequences containing known restriction sites and design corresponding specific PCR primers, then amplify the DNA fragments at the site through these specific PCR primers, and then use a restriction endonuclease with specificity that can recognize the site to cut the resulting amplified product, separate the restriction fragments by gel electrophoresis, stain and analyze the changes in the site. CAPS markers are co-dominant markers, which are relatively simple to operate and can be directly detected by agarose gel electrophoresis. However, CAPS markers must have suitable restriction endonucleases, so the workload during the development process is relatively large.

[0107] The full-length genes of LBL1 and LBL2 were cloned in duck-foot leaf, sub-chicken-claw leaf, chicken-claw leaf and super-chicken-claw leaf cotton materials.

[0108] The full length of LBL1 gene of duck-palm-shaped cotton material is 4306 bp, and its nucleotide sequence is shown in sequence 4 in the sequence table; DNAMAN software was used to compare and analyze the full length of LBL1 gene and the full length of LBL2 gene of duck-palm-shaped cotton materials, respectively, and multiple molecular markers were found. Among them, compared with the full length sequence of LBL1 gene of cotton materials with sub-paw, paw and super-paw, there is a 1 bp deletion in the 3'-UTR region of the full length sequence of LBL1 gene of duck-palm-shaped cotton material, that is, there is no 1 bp base insertion between the 3852nd and 3853rd positions of the nucleotide sequence shown in sequence 4, while there is a 1 bp base insertion in the corresponding position of the 3'-UTR region of the full length sequence of LBL1 gene of cotton materials with sub-paw, paw and super-paw, that is, there is a base G insertion. See Figure 2B, which is the partial sequence comparison result of the 3'-UTR region of the LBL1 gene of duck palm leaf, sub-chicken claw leaf, chicken claw leaf and super chicken claw leaf. Duck palm leaf lacks base G relative to sub-chicken claw leaf, chicken claw leaf and super chicken claw leaf. The molecular marker is named LBL1-3852. When LBL1-3852 is inserted with nucleotide G between positions 3852 and 3853 of the nucleotide sequence shown in Sequence 4, the marker indicates that LBL1-3852 is LBL1-3852-In G; when LBL1-3852 lacks nucleotide G between positions 3852 and 3853 of the nucleotide sequence shown in Sequence 4, the marker indicates that LBL1-3852 is LBL1-3852-del G.

[0109] The full length of the LBL2 gene of the sub-chicken claw leaf shaped cotton material is 4767bp, and its nucleotide sequence is shown in sequence 5 in the sequence table, including a promoter region formed by nucleotides 1-3000, a gene coding region formed by nucleotides 3001-4267, and a 3'-terminal UTR region formed by nucleotide sequences 4268-4767. DNAMAN software was used to compare and analyze the full length of LBL2 gene of duck palm leaf, sub-chicken claw leaf, chicken claw leaf and super-chicken claw leaf cotton materials, and multiple molecular markers were found. One of the molecular markers was located in the 3' end UTR region of the full-length sequence of LBL2 gene, which contained or lacked the 14 bases shown in sequence 30, that is, the nucleotide sequence shown in sequence 30 was inserted or lacked between positions 4610-4625 in the nucleotide sequence shown in sequence 5. The corresponding positions of the full-length nucleotide sequence of LBL2 gene of sub-chicken claw leaf and chicken claw leaf cotton materials contained the 14 bases shown in sequence 30, while the corresponding positions of the full-length nucleotide sequence of LBL2 gene of duck palm leaf and super-chicken claw leaf cotton materials lacked the 14 bases shown in sequence 30, see Figure 2 C, which is the partial sequence comparison result of the 3'-UTR region of the LBL2 gene of duck palm leaf, sub-chicken claw leaf, chicken claw leaf and super-chicken claw leaf. Duck palm leaf and super-chicken claw leaf lack the 14 bases shown in sequence 30 relative to sub-chicken claw leaf and chicken claw leaf. The molecular marker is named LBL2-4610. When the nucleotide sequence shown in sequence 30 is inserted between positions 4610-4625 in the nucleotide sequence shown in sequence 5, the marker LBL2-4610 is expressed as LBL2-4610-In14; when the nucleotide sequence shown in sequence 30 is deleted between positions 4610-4625 in the nucleotide sequence shown in sequence 5, the marker LBL2-4610 is expressed as LBL2-4610-del14.

[0110] Specific primers (as shown in SEQ ID NOs. 14-17) were designed for the two deletions using PrimerPremier5.0 (http: / / www.premierbiosoft.com) software and developed into CAPS markers, namely LBL1-3852 and LBL2-4610, respectively. The dCAPS marker can be used as a molecular marker closely linked to cotton leaf shape materials and applied to cotton leaf shape material breeding.

[0111] The selection of these two markers from multiple markers is a result of extensive screening. Other markers are not easy to distinguish, and most of them have multiple restriction sites, which is not convenient for designing primers for PCR amplification and subsequent restriction experiments. In addition, the inventors have comprehensively considered the use of the three markers in combination with the Indel marker in the second part of Example 1 of the present invention. Whether the three molecular markers of the present invention are used alone or in combination, the identification procedure is relatively simple, and the products are easy to distinguish. In particular, when the Indel marker is used in combination with the dCAPS marker, it can achieve the distinction of the four leaf shapes of cotton in a simple way. For example, the Indel marker can be combined with the LBL1-3852 molecular marker to distinguish duck palm leaves and sub-chicken claw leaves, and the Indel marker can be combined with the LBL2-4610 molecular marker to distinguish chicken claw leaves and willow leaves (super chicken claw leaves).

[0112] The nucleotide sequences of the specific primers are as follows:

[0113] SEQ ID NO.14: LBL1-3852-F: 5'-CGAAAGTAGCTCGATTCGGA -3';

[0114] SEQ ID NO.15: LBL1-3852-R: 5'-ATACTCACTGATGCAGCCTT-3';

[0115] SEQ ID NO.16: LBL2-4610-F: 5'-TAAAAAGCCAAAAGGCTGCTT-3';

[0116] SEQ ID NO. 17: LBL2-4610-R: 5'-CGTATGGCAGGTTTGCTCTT-3'.

[0117] The cotton material genome is used as a template and PCR amplification is performed with the primer pair shown in SEQ ID NO: 14 and SEQ ID NO: 15. The sequence of the amplified product is shown in SEQ ID NO: 8 or SEQ ID NO: 9.

[0118] The cotton material genome was used as a template and PCR amplification was performed with the primer pair shown in SEQ ID NO: 16 and SEQ ID NO: 17. The sequence of the amplified product was shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0119] Example 2 Identification of leaf fissure depth in cotton using InDel molecular markers

[0120] 1. Extraction of DNA from cotton

[0121] The CTAB method was used to extract the DNA of the cotton samples to be tested. The cotton sample resources were obtained from the Germplasm Resource Bank of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0122] 2. Identification of cotton leaf crack depth by PCR amplification results

[0123] The PCR reaction program was as follows: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds (denaturation), 55°C annealing for 15 seconds, 72°C extension for 30 seconds, 34 cycles; and then 72°C for 5 minutes to complete the product extension.

[0124] The reaction system (20 μL) of the PCR amplification reaction contains: 1.0 μl of cotton genomic DNA of about 200 ng / μl, 10 μl of 2×Taq Plus Master Mix II (Dye Plus), 7 μl of ddH2O, 0.5 μM upstream primer (sequence see SEQ ID NO. 2 in the sequence list), 0.5 μM downstream primer (sequence see SEQ ID NO. 3 in the sequence list), a total of 20 μl;

[0125] The polymorphism of the PCR amplification products was detected by agarose gel electrophoresis; the concentration of agarose gel used for electrophoresis was 4%, which was prepared according to the following method: weigh 4g agarose, add 100μl TBE buffer, heat it in a microwave oven until boiling, add 10μl EB solution after cooling slightly, and introduce it into a gel plate with a 25-hole comb inserted. After the gel solidifies, the PCR amplification product is spotted into the gel hole, electrophoresed at 135V for 13-18 minutes, and the polymorphism of the PCR amplification product is observed under ultraviolet light. The depth of the leaf fissure of the cotton to be tested is judged according to the PCR amplification results. If the PCR amplification result is a single band of 437bp in size, the leaf fissure of the cotton to be tested is shallow (duck palm leaf or sub-chicken claw leaf); if the PCR amplification result contains a single band of 570 bp in size, the leaf fissure of the cotton to be tested is deep (chicken claw leaf or super chicken claw leaf), and the electrophoresis result is as follows. Figure 3 The band size is obvious, and the identification result is consistent with the actual leaf fissure situation, indicating that the InDel marker typing is reliable. The results of the detection of the leaf fissure depth of the cotton materials to be tested in Table 1 (all homozygous materials) by the method of this embodiment are consistent with the actual leaf fissure depth of the corresponding cotton materials. For specific results, see Table 1 below.

[0126] Example 3 Identification of cotton leaf shape using a combination of InDel molecular markers and dCAPS molecular markers

[0127] (1) The depth of leaf fissure of the cotton material to be tested was preliminarily identified using InDel molecular markers. For the specific identification method, see Example 2. Based on the detection results of InDel molecular markers, the tested materials were classified into deep leaf fissure (chicken claw leaf or super chicken claw leaf) and shallow leaf fissure (duck palm leaf or sub-chicken claw leaf).

[0128] (2) The leaf type of the cotton material to be tested was identified using the two dCAPS markers (LBL1-3852 molecular marker and LBL2-4610) of the present invention.

[0129] The specific detection and identification methods are:

[0130] First, the cotton genome to be tested was used as a template and PCR amplification was performed using two dCAPS-labeled specific primer pairs, where:

[0131] The specific primer pair for detecting the LBL1-3852 molecular marker is the second primer pair, and the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively;

[0132] The specific primer pair for detecting the LBL2-4610 molecular marker is the third primer pair, and the nucleotide sequences of the forward primer and the reverse primer are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively;

[0133] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 minutes; 95°C for 15 seconds (denaturation), 55°C for 15 seconds, and extension at 72°C for 20 seconds, for 34 cycles; and then 72°C for 5 minutes to complete the extension of the product.

[0134] The reaction system (20 μL) for PCR amplification reaction contains: 1.0 μl of cotton genomic DNA of about 200 ng / μl, 10 μl of 2×Taq Plus Master Mix II (Dye Plus), 7 μl of ddH2O, 0.5 μM upstream primer, 0.5 μM downstream primer, a total of 20 μl.

[0135] Then, the PCR amplification product is subjected to enzyme digestion treatment, and the specific enzyme digestion method is as follows:

[0136] The enzyme digestion system (30 μl) contains: 10 μl of PCR amplification product, 2 μl of 10× rCutSmart™ Buffer (purchased from NEW ENGLAND Biolabs), 1.0 μl of restriction endonuclease (purchased from NEW ENGLAND Biolabs), and sterile double distilled water to make up to 30 μl.

[0137] Different restriction endonucleases were used to digest the amplified products of different molecular markers. The amplified products of the LBL1-3852 molecular marker were digested with the restriction endonuclease Sau3A I, and the amplified products of the LBL2-4610 molecular marker were digested with the restriction endonuclease BcoDⅠ.

[0138] The reaction procedure of Sau3A I restriction enzyme digestion was as follows: stage 1: incubation at 37°C for 1 h; stage 2: inactivation at 65°C for 20 min; stage 3: storage at 4°C; the reaction procedure of BcoDⅠ restriction enzyme digestion was as follows: stage 1: incubation at 37°C for 15 min; stage 2: storage at 4°C.

[0139] After enzyme digestion, add 1 μL 10× DNA Loading Buffer to the sample, take 5 μL of the digestion product and perform electrophoresis on 4% agarose gel. Figure 4 When the size of the PCR product based on the LBL1-3852 molecular marker after Sau3A I restriction enzyme digestion is 298 bp, the cotton to be tested is duck claw leaf, and its genotype is Hap1, that is, LBL1-3852-delG: LBL1-3852-del G; when the size of the Sau3A I restriction enzyme digestion product is 55 bp and 243 bp, the genotype of the marker in the cotton to be tested is Hap2, that is, LBL1-3852-In G: LBL1-3852-InG, and the cotton is sub-chicken claw leaf, chicken claw leaf or super chicken claw leaf; the PCR product based on the LBL2-4610 molecular marker is digested by BcoD When the size of the product after digestion by BcoD I restriction endonuclease is 173bp and 194bp, the cotton to be tested is sub-chicken claw leaf shape or chicken claw leaf shape, and its genotype is Hap3, i.e., LBL2-4610-In14: LBL2-4610-In 14; when the size of the product after digestion by BcoD I restriction endonuclease is 368bp, the genotype of the marker in the cotton to be tested is Hap4, i.e., LBL2-4610-del 14: LBL2-4610-del 14, and the cotton to be tested is duck foot leaf shape or super chicken claw leaf shape.

[0140] In order to simplify the identification steps, the above steps (1) and (2) can be performed by the following method. After the identification in step (1), which dCAPS marker to be used for identification in step (2) is selected according to the identification result. For example, if the identification result in step (1) is shallow leaf fissure (duck palm leaf or sub-chicken claw leaf), then the LBL1-3852 molecular marker or the LBL2-4610 molecular marker is selected for identification in step (2). After identification by only one of the molecular markers, it can be distinguished whether the cotton leaf shape to be tested is duck palm leaf or sub-chicken claw leaf. When the identification result in step (1) is deep leaf fissure (chicken claw leaf or willow leaf), then the LBL2-4610 molecular marker is selected for identification in step (2). After identification by this molecular marker, it can be distinguished whether the cotton leaf shape to be tested is chicken claw leaf or willow leaf, and there is no need to perform genotyping on the LBL1-3852 molecular marker.

[0141] In this example, leaf shape identification was performed on cotton varieties in various germplasm resource banks, and the genotyping results of each material obtained were compared with the actual observed cotton leaf shape, and the consistency rate between the two was 100%, indicating that the Indel marker of the present invention was used in combination with the dCAPS molecular marker, and the detection accuracy of the four leaf shapes was 100%, which can be applied to cotton molecular marker-assisted selection breeding. The specific cotton planting and detection results to be tested are shown in Table 1.

[0142] Table 1 Molecular marker identification of cotton leaf shape in cotton germplasm test materials

[0143]

[0144]

[0145] In Table 1, the inferred leaf shape refers to the leaf shape inferred from the detection results of the markers of the present invention, and the actual leaf shape refers to the leaf shape obtained by visual observation after the cotton leaf shape is stabilized.

[0146] In Table 1, molecular marker LS001 refers to the result of Sau3AⅠ digestion, "-" refers to the genotype Hap2, i.e., LBL1-3852-InG: LBL1-3852-In G, the size of the digestion product is 55 bp and 243 bp, and the cotton is sub-chicken claw leaf, chicken claw leaf or super chicken claw leaf; "+" refers to the genotype Hap1, i.e., LBL1-3852- del G: LBL1-3852- del G, the size of the digestion product is 298 bp, and the cotton is duck claw leaf; molecular marker LS002 refers to the result of BcoDⅠ digestion, "+" refers to the genotype Hap3, i.e., LBL2-4610-In 14: LBL2-4610-In 14, the size of the digestion product by BcoDI restriction enzyme is 173 bp and 194 bp, and the cotton is sub-chicken claw leaf shape or chicken claw leaf shape; when BcoD When the size of the restriction endonuclease I digestion product is 368 bp, the genotype of the LBL2-4610 molecular marker in the cotton genome to be tested is Hap4, namely LBL2-4610-del 14: LBL2-4610-del14, represented by "-", and the cotton has a duck-foot leaf shape or a super-chicken-claw leaf shape. LS003 and LS004 represent InDel markers. LS003 is marked as “+”, which means that the PCR product is a single band with a size of 437 bp, that is, the leaf fissure of the cotton to be tested is shallow (duck claw leaves and sub-chicken claw leaves), and the genotype is LBL1-2331-del 133:LBL1-2331-del 133, otherwise it is indicated by “-”; LS004 is marked as “+”, which means that the PCR amplification product is a single band with a size of 570 bp, that is, the leaf fissure of the cotton to be tested is deep (chicken claw leaves and super-chicken claw leaves), and the genotype is LBL1-2332-In 133:LBL1-2331-In 133, otherwise it is indicated by “-”.

[0147] The combination of the above markers can make a relatively accurate prediction of the four leaf shapes of cotton, indicating that both InDel markers and dCAPS molecular markers can assist cotton breeding.

Claims

1. Application of a reagent for detecting polymorphism or genotype of molecular markers related to cotton leaf shape control genes in identifying or breeding cotton leaf shape varieties; characterized in that: The molecular markers include: An InDel molecular marker located in the promoter region of the cotton leaf shape control gene LBL1, wherein the polymorphism of the InDel molecular marker is an insertion or deletion of the nucleotide sequence shown in sequence 1 between positions 2331 and 2332 of the nucleotide sequence shown in sequence 4 in the sequence table; when the InDel molecular marker is an insertion of the nucleotide sequence shown in sequence 1, the InDel molecular marker is expressed as LBL1-2331-In 133; when the InDel molecular marker is a deletion of the nucleotide sequence shown in sequence 1, the InDel molecular marker is expressed as LBL1-2331-del 133; A dCAPS marker LBL1-3852 located in the 3'-UTR region of the cotton leaf shape control gene LBL1, wherein the polymorphism of the marker LBL1-3852 is the insertion or deletion of nucleotide G between positions 3852 and 3853 of the nucleotide sequence shown in SEQ ID NO: 4; when the marker LBL1-3852 is the insertion of nucleotide G, the marker LBL1-3852 is expressed as LBL1-3852-In G; when the marker LBL1-3852 is the deletion of nucleotide G, the marker LBL1-3852 is expressed as LBL1-3852-del G; and A dCAPS marker LBL2-4610 located in the 3'-UTR region of the cotton leaf shape control gene LBL2, wherein the polymorphism of the marker LBL2-4610 is an insertion or deletion of the nucleotide sequence shown in sequence 30 between the 4610th and 4625th positions of the nucleotide sequence shown in sequence 5; when the marker LBL2-4610 is an insertion of the nucleotide sequence shown in sequence 30, the marker LBL2-4610 is expressed as LBL2-4610-In14; when the marker LBL2-4610 is a deletion of the nucleotide sequence shown in sequence 30, the marker LBL2-4610 is expressed as LBL2-4610-del 14; The reagents include: a first primer pair for amplifying the InDel molecular marker, a second primer pair for amplifying the marker LBL1-3852, and a third primer pair for amplifying the marker LBL2-4610; a first restriction endonuclease for enzymatically cleaving the amplified product of the second primer pair; and a second restriction endonuclease for enzymatically cleaving the amplified product of the third primer pair; The cotton leaf shapes include duck palm leaf, sub-chicken claw leaf, chicken claw leaf, and willow leaf; Cotton varieties with different leaf shapes are jointly identified or bred using a reagent for detecting the polymorphism or genotype of the InDel molecular marker and a reagent for detecting the polymorphism or genotype of the markers LBL1-3852 and LBL2-4610, wherein: When the genotype of the InDel molecular marker is LBL1-2331-In 133: LBL1-2331-In 133, the leaf fissure of the cotton is deep, and the leaf shape corresponding to the deep leaf fissure is chicken claw leaf or willow leaf; when the genotype of the InDel molecular marker is LBL1-2331-del 133: LBL1-2331- del 133, the leaf fissure of the cotton is shallow, and the leaf shape corresponding to the shallow leaf fissure is duck palm leaf or sub-chicken claw leaf; When the genotype of the marker LBL1-3852 is LBL1-3852-In G: LBL1-3852-In G, the leaf shape of the cotton is sub-chicken claw leaf, chicken claw leaf or willow leaf; when the genotype of the marker LBL1-3852 is LBL1-3852-del G: LBL1-3852-del G, the leaf shape of the cotton is duck foot leaf; When the genotype of the marker LBL2-4610 is LBL2-4610-In14: LBL2-4610-In14, the leaf shape of the cotton is sub-chicken claw leaf or chicken claw leaf; when the genotype of the marker LBL2-4610 is LBL2-4610-del 14: LBL2-4610-del 14, the leaf shape of the cotton is duck foot leaf or willow leaf.

2. The use according to claim 1, characterized in that: The base sequence of the forward primer of the first primer pair is shown as SEQ ID NO. 2 in the sequence listing, and the base sequence of the reverse primer is shown as SEQ ID NO. 3 in the sequence listing.

3. The use according to claim 1 or 2, characterized in that: The nucleotide sequence of the forward primer of the second primer pair is shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.15; The first restriction endonuclease is Sau3A I; The nucleotide sequence of the forward primer of the third primer pair is shown in SEQ ID NO.16, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.17; The second restriction endonuclease is BcoD I.

4. A method for identifying cotton leaf shape using a reagent for detecting the polymorphism or genotype of a molecular marker related to a cotton leaf shape controlling gene, characterized in that: The molecular markers include: An InDel molecular marker located in the promoter region of the cotton leaf shape control gene LBL1, wherein the polymorphism of the InDel molecular marker is an insertion or deletion of the nucleotide sequence shown in sequence 1 between positions 2331 and 2332 of the nucleotide sequence shown in sequence 4 in the sequence table; when the InDel molecular marker is an insertion of the nucleotide sequence shown in sequence 1, the InDel molecular marker is expressed as LBL1-2331-In 133; when the InDel molecular marker is a deletion of the nucleotide sequence shown in sequence 1, the InDel molecular marker is expressed as LBL1-2331-del 133; A dCAPS marker LBL1-3852 located in the 3'-UTR region of the cotton leaf shape control gene LBL1, wherein the polymorphism of the marker LBL1-3852 is the insertion or deletion of nucleotide G between positions 3852 and 3853 of the nucleotide sequence shown in SEQ ID NO: 4; when the marker LBL1-3852 is the insertion of nucleotide G, the marker LBL1-3852 is expressed as LBL1-3852-In G; when the marker LBL1-3852 is the deletion of nucleotide G, the marker LBL1-3852 is expressed as LBL1-3852-del G; and A dCAPS marker LBL2-4610 located in the 3'-UTR region of the cotton leaf shape control gene LBL2, wherein the polymorphism of the marker LBL2-4610 is an insertion or deletion of the nucleotide sequence shown in sequence 30 between the 4610th and 4625th positions of the nucleotide sequence shown in sequence 5; when the marker LBL2-4610 is an insertion of the nucleotide sequence shown in sequence 30, the marker LBL2-4610 is expressed as LBL2-4610-In14; when the marker LBL2-4610 is a deletion of the nucleotide sequence shown in sequence 30, the marker LBL2-4610 is expressed as LBL2-4610-del 14; The reagents include: a first primer pair for amplifying the InDel molecular marker, a second primer pair for amplifying the marker LBL1-3852, and a third primer pair for amplifying the marker LBL2-4610; a first restriction endonuclease for enzymatically cleaving the amplified product of the second primer pair; and a second restriction endonuclease for enzymatically cleaving the amplified product of the third primer pair; The base sequence of the forward primer of the first primer pair is shown in SEQ ID NO. 2 in the sequence listing, and the base sequence of the reverse primer is shown in SEQ ID NO. 3 in the sequence listing; The nucleotide sequence of the forward primer of the second primer pair is shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.15; The first restriction endonuclease is Sau3A I; The nucleotide sequence of the forward primer of the third primer pair is shown in SEQ ID NO.16, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.17; The second restriction endonuclease is BcoD I; The method comprises the following steps: S1. Extracting genomic DNA of cotton to be tested; S2, using the genomic DNA as a template, using the first primer pair to perform PCR amplification, and determining the leaf fissure depth of the cotton to be tested by identification; The identification is to perform electrophoresis or sequencing on the PCR product. When the PCR product is 437 bp, the cotton to be tested has shallow leaf lobes, and the shallow leaf lobes are duck palm leaves or sub-chicken claw leaves; when the PCR product is 570 bp, the cotton to be tested has deep leaf lobes, and the deep leaf lobes are chicken claw leaves or willow leaves; S3, using the genomic DNA of the cotton to be tested as a template, and using the second primer pair to perform PCR amplification to obtain an amplification product containing the marker LBL1-3852; and / or PCR amplification is performed using the third primer pair to obtain an amplification product containing the marker LBL2-4610; S4, treating the amplification product containing the marker LBL1-3852 with the first restriction endonuclease, and verifying the genotype of the marker LBL1-3852 by digesting the product with the enzyme; When the enzyme digestion product is 55 bp and 243 bp, the leaf shape of the cotton to be tested is sub-chicken claw leaf, chicken claw leaf or willow leaf, and the genotype of marker LBL1-3852 is LBL1-3852-In G: LBL1-3852-In G; when the enzyme digestion product is 298 bp, the leaf shape of the cotton to be tested is duck foot leaf, and the genotype of marker LBL1-3852 is LBL1-3852-del G: LBL1-3852-del G; and / or The amplification product containing the marker LBL2-4610 is treated with a second restriction endonuclease, and the genotype of the marker LBL2-4610 is verified by the enzyme digestion product. When the size of the enzyme digestion product is 173bp and 194bp, the leaf shape of the cotton to be tested is sub-chicken claw leaf shape or chicken claw leaf shape, and the genotype of the marker LBL2-4610 is LBL2-4610-In14: LBL2-4610-In14; when the size of the enzyme digestion product is 368bp, the leaf shape of the cotton to be tested is duck palm leaf shape or willow leaf shape, and the genotype of the marker LBL2-4610 is LBL2-4610- del 14: LBL2-4610- del 14.

5. The method according to claim 4, characterized in that The method comprises: When the identification result of step S2 is shallow leaf lobation, the genotype of marker LBL1-3852 is further detected by using the second primer pair and the first restriction endonuclease, or the genotype of marker LBL2-4610 is further detected by using the third primer pair and the second restriction endonuclease, thereby distinguishing the cotton of duck palm leaf variety from the cotton of sub-chicken claw leaf variety; When the identification result of step S2 is deep leaf fissure, the third primer pair and the second restriction endonuclease are used to further detect the genotype of marker LBL2-4610, thereby distinguishing the cotton of the chicken-claw leaf variety from the cotton of the willow leaf variety.

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