SV molecular marker related to the vegetative branch number trait of gossypium hirsutum l. and use thereof

Abstract

The present invention relates to an SV molecular marker related to the vegetative branch number trait of Gossypium hirsutum L. and the use thereof. Provided are an SV molecular marker related to the vegetative branch number trait of Gossypium hirsutum L., primers shown as SEQ ID NO. 1-SEQ ID NO. 3 obtained on the basis of said SV molecular marker, and a detection kit. Therefore, the present invention can accurately predict and screen the vegetative branch number trait of Gossypium hirsutum L., and provides a scientific basis for cultivating Gossypium hirsutum L. having different vegetative branch types.

Description

SV molecular markers related to leaf and branch number traits in upland cotton and their applications

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on February 18, 2024, with application number CN 202410179509.0 and invention name “An SV molecular marker related to leaf branch number traits of upland cotton and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the technical field of biological agriculture, and specifically relates to SV molecular markers related to the leaf and branch number trait of upland cotton and their applications. Background Art

[0003] Cotton is a vital fiber crop worldwide. With the growth of productivity and rising labor costs, the demand for an ideal plant shape suitable for mechanization and dense planting is becoming increasingly urgent. The number of leaves and branches significantly influences cotton plant shape. Excessive leaves and branches can result in a loose plant shape, hindering dense planting and mechanization, increasing labor costs, and harboring pests and diseases.

[0004] In the existing technology, the plant type traits of upland cotton are mostly marked through field phenotyping to determine the number of leaves and branches of upland cotton. However, this method requires a lot of manpower and material resources, is not only time-consuming but also easily affected by the growth period of cotton.

[0005] Currently, there is no research or report on molecular markers related to the leaf and branch number trait in cotton. Therefore, how to use molecular markers to predict the leaf and branch number, improve the plant type and cultivate the ideal plant type in cotton is a technical problem that needs to be solved urgently in this field.

[0006] Summary of the Invention

[0007] The purpose of this application is to provide an SV molecular marker related to the leaf branch number trait of upland cotton and its application. This molecular marker can not only predict and screen the leaf branch number trait of upland cotton, but also realize the breeding of ideal cotton plant types.

[0008] In order to achieve the above objectives, this application provides the following technical solutions:

[0009] The present application provides an SV molecular marker related to the leaf branch number trait of upland cotton, wherein the SV molecular marker is a large fragment inserted at bases 620454 to 625557 on chromosome D11 of the upland cotton NDM8 genome; the nucleotide sequence of the large fragment is shown in SEQ ID NO.4;

[0010] The version of the upland cotton NDM8 genome is NDM8HEBAU.

[0011] The present application provides primers for amplifying the SV molecular marker described in the above technical solution, wherein the primers include forward primer MB-F, forward primer LB-F and reverse primer BR;

[0012] The nucleotide sequence of the forward primer MB-F is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the forward primer LB-F is shown in SEQ ID NO.2;

[0014] The nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.3.

[0015] The present application provides a detection kit containing the primers and PCR amplification reagents described in the above technical solution.

[0016] Preferably, the PCR amplification reagent includes DNA polymerase, dNTPs and Mg 2+ .

[0017] The present application provides the application of the SV molecular markers, primers or detection kits described in the above technical solutions, and the applications include one or more of the prediction and screening of the leaf and branch number traits of upland cotton and the cultivation of upland cotton with few leaf branches.

[0018] Preferably, the prediction of the leaf branch number trait of upland cotton includes identifying the leaf branch type of upland cotton;

[0019] When the leaf branch number of upland cotton is greater than 1, it is a multi-leaf branch type;

[0020] When the number of leaf branches of upland cotton is ≤1, the upland cotton is of the few-leaf-branch type.

[0021] Preferably, the upland cotton breeding includes cultivating upland cotton with few leaf branches.

[0022] The present application provides a method for identifying the leaf branch number trait of upland cotton, comprising the following steps:

[0023] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the primers described in the above technical solution to obtain a PCR amplification product; the PCR amplification product is subjected to gel electrophoresis and a judgment is made; when the PCR amplification product shows a band at a length of 510bp, the sample to be tested is a homozygous multi-leaf branch type; when the PCR amplification product shows a band at a length of 255bp, the sample to be tested is a homozygous few-leaf branch type; when the PCR amplification product shows a band at a length of 255bp and 510bp respectively, the sample to be tested is a heterozygous leaf branch type.

[0024] Preferably, the PCR amplification reaction system, based on 10 μL, includes: 2 μL PCRMasterMix, 1 μL 30-150 ng / μL genomic DNA template, 0.4 μL 10 μM forward primer MB-F, 0.4 μL 10 μM forward primer LB-F, 0.8 μL 10 μM reverse primer BR, and ddH2O to 10 μL.

[0025] Preferably, the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 67°C for 30 s, extension at 72°C for 20 s, for 34 cycles; and extension at 72°C for 1 min. Beneficial effects:

[0026] The present application provides an SV molecular marker related to the leaf branch number trait of upland cotton, wherein the SV molecular marker is a large fragment inserted at bases 620454 to 625557 on chromosome D11 of the upland cotton NDM8 genome, and the nucleotide sequence of the large fragment is shown in SEQ ID NO.4; the version of the upland cotton NDM8 genome is NDM8HEBAU, and the SV molecular marker is significantly correlated with the leaf branch number trait of upland cotton; in addition, the present application designs corresponding primers and detection kits based on the SV molecular marker, thereby realizing the prediction and screening of the leaf branch number trait of upland cotton, and providing a scientific basis for cultivating upland cotton with different leaf branch types. At the same time, during the detection process, there is no need to consider the growth period and tissue type of cotton, nor is there any need to conduct field phenotypic investigations. The genotype information of the sample can be accurately and quickly obtained, which is beneficial to accelerate the germplasm innovation of ideal cotton plant types and improve breeding efficiency.

[0027] Based on the above technical advantages, this application also provides a method for identifying the leaf branch number trait in upland cotton, comprising the following steps: using genomic DNA from a sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain a PCR amplification product; and subjecting the PCR amplification product to gel electrophoresis and making an assessment. Experiments have demonstrated that the SV marker provided in this application and the primers designed based on this marker can accurately detect the leaf branch number trait in upland cotton and effectively distinguish between pure heterozygous genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments.

[0029] FIG1 is a genome-wide association analysis of leaf and branch number data from 288 natural populations of upland cotton for two years in Example 1;

[0030] FIG2 is the sequencing comparison result of SV molecular marker intervals of different extreme upland cotton materials in Example 1;

[0031] FIG3 is a sequencing comparison result 2 of the SV molecular marker intervals of different extreme upland cotton materials in Example 1;

[0032] FIG4 is a sequencing comparison result 3 of the SV molecular marker intervals of different extreme upland cotton materials in Example 1;

[0033] FIG5 is a sequencing comparison result 4 of the SV molecular marker intervals of different extreme upland cotton materials in Example 1;

[0034] FIG6 shows the expression levels of the TCP18 gene in cotton materials with different numbers of leaves and branches in Example 2;

[0035] FIG7 shows the molecular detection results of the primer set in Example 3 in upland cotton materials with extreme leaf and branch numbers;

[0036] Figure 8 is a boxplot of the results of the investigation of leaf and branch numbers of different upland cotton materials based on molecular detection typing in Example 4;

[0037] Figure 9 shows the molecular detection results of different upland cotton materials in Example 5. DETAILED DESCRIPTION

[0038]

[0039] The present application provides primers for amplifying the SV molecular marker described in the above technical solution, comprising a forward primer MB-F, a forward primer LB-F, and a reverse primer BR; the nucleotide sequence of the forward primer MB-F is shown in SEQ ID NO.1, specifically 5'-ACAAGGATTTACGTGGTTCGG-3'; the nucleotide sequence of the forward primer LB-F is shown in SEQ ID NO.2, specifically 5'-AAACTGATCTGAAACTTGGGACT-3'; the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.3, specifically 5'-AGAGGGAGAATCGAATGGTCAAG-3'. The primers can specifically clone the SV molecular marker, thereby enabling prediction and screening of the leaf branch number trait of upland cotton.

[0040] The present application provides a detection kit, which contains the primers and PCR amplification reagents described in the above technical solution. The PCR amplification reagents described in the present application preferably include DNA polymerase, dNTPs and Mg 2+ ; The DNA polymerase, dNTPs and Mg 2+ There is no special limitation on the source and amount of the compound, and commercially available products in this field can be used.

[0041] The present application provides an application of the SV molecular marker, primer, or detection kit described in the above technical solution, wherein the application includes one or more of the prediction, screening, and breeding of leaf branch number traits of upland cotton, preferably including the prediction, screening, and breeding of leaf branch number traits of upland cotton. In the present application, the prediction of the leaf branch number trait of upland cotton preferably includes identifying the leaf branch type of upland cotton; when the leaf branch number of upland cotton is greater than 1, the upland cotton is preferably a multi-leaf branch type; when the leaf branch number of upland cotton is ≤1, the upland cotton is preferably a low-leaf branch type; and the breeding of upland cotton preferably includes cultivating upland cotton of the low-leaf branch type.

[0042] The present application provides a method for identifying the leaf branch number trait of upland cotton, comprising the following steps: using the genomic DNA of a sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain a PCR amplification product; performing gel electrophoresis on the PCR amplification product and making a judgment; when the PCR amplification product shows a band at a length of 510bp, the sample to be tested is a homozygous multi-leaf branch type; when the PCR amplification product shows a band at a length of 255bp, the sample to be tested is a homozygous few-leaf branch type; when the PCR amplification product shows a band at a length of 255bp and 510bp respectively, the sample to be tested is a heterozygous leaf branch type.

[0043] The present application preferably extracts genomic DNA from the sample to be tested to obtain the genomic DNA of the sample to be tested. In the present application, there are no special requirements for the extraction method of the genomic DNA of the sample to be tested, and techniques well known in the art can be used. In a specific embodiment of the present application, cotton genomic DNA is extracted using the CTAB method.

[0044] After obtaining the genomic DNA of the sample to be tested, the present application uses the genomic DNA of the sample to be tested as a template and performs PCR amplification using the primers described in the above technical solution to obtain a PCR amplification product. In the present application, the PCR amplification reaction system is preferably 10 μL, and includes: 2 μL PCRMasterMix, 1 μL of 30-150 ng / μL genomic DNA template, 0.4 μL of 10 μM forward primer MB-F, 0.4 μL of 10 μM forward primer LB-F, 0.8 μL of 10 μM reverse primer BR, and ddH2O to 10 μL. In a specific embodiment of the present application, the PCR Master Mix is ​​2×Hieff Canace Plus PCR Master Mix (With Dye).

[0045] The PCR amplification reaction procedure described herein is preferably: pre-denaturation at 95°C for 3 minutes; 34 cycles of denaturation at 95°C for 30 seconds, annealing at 67°C for 30 seconds, and extension at 72°C for 20 seconds; and extension at 72°C for 1 minute. After the extension described herein, the cells are preferably stored at 10°C for 5 minutes. This PCR amplification can be used to identify the leaf branch number trait of upland cotton.

[0046] After obtaining the PCR amplification product, the present application performs gel electrophoresis on the PCR amplification product and makes a judgment; there are no special requirements for the gel electrophoresis method, and techniques well known in the art can be used; when the PCR amplification product shows a band at a length of 510bp, the sample to be tested is a homozygous multi-leaf branch type; when the PCR amplification product shows a band at a length of 255bp, the sample to be tested is a homozygous low-leaf branch type; when the PCR amplification product shows a band at lengths of 255bp and 510bp respectively, the sample to be tested is a heterozygous leaf branch type. The above method is conducive to identifying the number of leaf branches in upland cotton, and then screening for the homozygous low-leaf branch type.

[0047] Experiments have shown that the SV marker provided in this application and the primers designed based on the marker can accurately detect the leaf and branch number traits of upland cotton and effectively distinguish the pure heterozygous types of genotypes. At the same time, there is no need to consider the growth period and tissue type of cotton during the detection process, nor is there any need to conduct field phenotypic investigations. The genotype information of the sample can be accurately and quickly obtained, which is beneficial to accelerate the germplasm innovation of ideal cotton plant types and improve breeding efficiency.

[0048] In order to further illustrate the present application, the SV molecular markers related to the leaf branch number trait of upland cotton provided in the present application and their applications are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0049] Example 1

[0050] Obtaining SV molecular markers related to leaf and branch number in upland cotton

[0051] (1) Planting of natural cotton population materials, investigation of leaf and branch number phenotypes and genome-wide association analysis:

[0052] From 2018 to 2019, 288 natural populations were planted in experimental fields in Korla, Xinjiang. Leaf and branch number traits were observed each July, collecting two years of leaf and branch number phenotypic data. The 288 upland cotton cultivars and the selected materials with extreme leaf and branch number phenotypes were all selected from upland cotton cultivars whose genomes had been resequenced by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (for details, see Wang et al. Asymmetric subgenome selection and cis-regular divergence during cotton domestication. Nat Genet, 2017, 49: 579-587).

[0053] Based on the 288 upland cotton cultivars whose genomes have been resequenced by the National Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University, the resequencing data were downloaded and combined with two years of phenotypic data for whole-genome association analysis. The results are shown in Figure 1 (in Figure 1, the left figure represents the whole-genome association analysis results of the leaf and branch number data of 288 upland cotton natural populations in 2018; the right figure represents the whole-genome association analysis results of the leaf and branch number data of 288 upland cotton natural populations in 2019).

[0054] As can be seen from Figure 1, the leaf and branch number data of the natural population of upland cotton are all located at extremely significant sites in the same region of chromosome D11.

[0055] (2) Sequencing comparison results of the SV molecular marker interval for materials with extreme leaf and branch numbers:

[0056] The leafy-branched upland cotton materials ZY161 and ZY207 (i.e., materials with leaf branches > 1) and the leafy-branched upland cotton materials J39 and ZY98 (i.e., materials with leaf branches ≤ 1) with the most extreme phenotypes and stable for two years in the above population were selected. The positioning interval of ZY161, ZY207, J39 and ZY98 (i.e., the natural population GWAS positioning interval is specifically the 171567-675567 base sequence) was sequenced and sequence alignment was verified. The results are shown in Figures 2 to 5 (in Figures 2 to 5, ZY161 and ZY207 are sequences of leafy-branched materials, J39 and ZY98 are sequences of leafy-branched materials, NDM8 is the reference genome sequence, and the reference genome sequence is of the leafy-branch type. The last row represents the reference results, where lowercase letters represent differences in the sequences involved in the alignment, and uppercase letters represent identical sequences involved in the alignment).

[0057] The sequencing results in Figures 2 to 5 show that the extreme accessions with many leafy branches (ZY161 and ZY207) have a ~5kb insertion compared to the extreme accessions with few leafy branches (J39 and ZY98). This ~5kb insertion is designated as an SV marker; this variation is a large insertion-deletion variation within the SV marker. This SV marker is directly associated with the leafy branch number trait in upland cotton; accessions with this insertion are likely to exhibit the leafy branch trait, while accessions without the 5kb insertion are likely to exhibit the leafy branch trait.

[0058] Example 2

[0059] In order to further determine the accuracy of the influence of the SV molecular marker on the leaf and branch number trait of upland cotton in Example 1, based on the upland cotton NDM8 genome Gossypium hirsutum (AD1), the NDM8HEBAU version determined that the physical location information of the SV molecular marker is: the nucleotide sequence shown by bases 620454 to 625557 on the upland cotton D11 chromosome. The downstream gene adjacent to this sequence is the TCP18 gene, which has been reported in many plants to be a key gene that negatively regulates plant lateral branch growth (References: Aguilar-Martinez JA. et al 2007. Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. Plant Cell 19:458-72; Martin-Trillo M. et al. 2011. Role of tomato BRANCHED1-like genes in the control of shoot branching. Plant J. 67:701-14; Minakuchi K. et al. 2010. FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice. Plant Cell Physiol. 51:1127-35.).

[0060] Extreme materials ZY161 and ZY207 with many leaves and branches, and extreme materials J39 and ZY98 with few leaves and branches were selected and planted. Under sufficient nutritional conditions, the axillary buds of the second leaf were taken when they reached the 4-5 leaf stage. Total RNA was extracted from different extreme materials using a kit method (RNA extraction kit purchased from Nanjing Novozymes Biotech Co., Ltd.), and the expression level of TCP18 was detected by qRT-PCR. The results are shown in Table 1 and Figure 6.

[0061] Table 1 Gene expression levels in different treatments

[0062] As shown in Table 1 and Figure 6, the TCP18 gene is highly expressed in the leafy-branch accessions J39 and ZY98, whereas it is essentially unexpressed in the leafy-branch accessions ZY161 and ZY207. This suggests that the 5 kb indel variant described in Example 1 regulates the leaf and branch number trait in cotton by modulating the expression of its downstream genes. Therefore, this large 5 kb indel variant is a key SV molecular marker affecting the leaf and branch number trait in upland cotton.

[0063] Example 3

[0064] (1) Based on the SV molecular marker (5 kb) in Example 1, a specific primer set for identifying the leaf branch number trait of upland cotton was designed. The specific primer set includes two forward primers (MB-F and LB-F) and a common reverse primer (BR);

[0065] MB-F: 5'-ACAAGGATTTACGTGGTTCGG-3' (SEQ ID NO. 1);

[0066] LB-F: 5'-AAACTGATCTGAAACTTGGGACT-3' (SEQ ID NO. 2);

[0067] BR: 5'-AGAGGGAGAATCGAATGGTCAAG-3' (SEQ ID NO. 3);

[0068] The forward primer MB-F and the reverse primer BR can specifically amplify the leafy branch genotype fragment with the SV molecular marker inserted, with a fragment size of 510bp; the forward primer LB-F and the shared reverse primer BR can specifically amplify the leafy branch genotype fragment without the SV molecular marker inserted, with a fragment size of 255bp.

[0069] (2) Verify the accuracy of the primer set in step (1):

[0070] The extreme materials ZY161 and ZY207 with many leaves and branches, and the extreme materials J39 and ZY98 with few leaves and branches were used as the test materials. The steps are as follows:

[0071] A. CTAB method was used to extract genomic DNA from cotton of different extreme materials;

[0072] B. PCR amplification: Using the cotton genomic DNA extracted in step A as a template, and using the primer set (SEQ ID NO.1 to SEQ ID NO.3) in step (1), PCR amplification was performed to obtain a PCR amplification product;

[0073] The PCR amplification system in 10 μL was as follows: 1 μL 30-150 ng / μL DNA template, 5 μL 2× HieffCanace Plus PCR Master Mix (With Dye), 0.4 μL forward primer MB-F (10 μM), 0.4 μL forward primer LB-F (10 μM), 0.8 μL reverse primer BR (10 μM), and 2.4 μL ddH2O.

[0074] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 67°C for 30 s, and extension at 72°C for 20 s; extension at 72°C for 1 min, and storage at 10°C for 5 min. HieffCanace Plus PCR MasterMix (With Dye) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0075] C. Agarose gel electrophoresis detection: The PCR amplification products in step B were detected using 1 wt.% agarose gel. The results are shown in Figure 7 (in Figure 7, the bands from left to right represent the results of J39 material, the results of J39 material, the results of J39 material, the results of J39 material, the results of ZY98 material, the results of ZY98 material, the results of ZY98 material, the results of ZY161 material, the results of ZY161 material, the results of ZY161 material, the results of ZY207 material, and the results of ZY207 material).

[0076] As shown in Figure 7, the PCR amplification product of the extreme material with few leafy branches showed only a single band at 255 bp after electrophoresis, while the PCR amplification product of the extreme material with many leafy branches showed a single band at 510 bp. This indicates that the primer set provided by this application, whose nucleotide sequences are represented by SEQ ID NO. 1 to SEQ ID NO. 3, can effectively use SV molecular markers to distinguish between leafy and leafless upland cotton plant types.

[0077] Example 4

[0078] (1) To further evaluate the accuracy of the SV markers in Example 1 and the primer set shown in SEQ ID NO. 1 to SEQ ID NO. 3 in Example 3, 17 upland cotton materials (ZY21, ZY81, J38, ZY85, ZY84, ZY309, J39, ZY98, J20, J23, ZY428, J70, ZY389, J68, ZY161, ZY207, and J668) were selected from a natural population for SV marker detection and typing. The specific steps were as follows:

[0079] A. The CTAB method was used to extract cotton genomic DNA from 17 upland cotton materials;

[0080] B. PCR amplification: the same method as step B in Example 3;

[0081] C. Agarose gel electrophoresis detection: The PCR amplification products in step B were detected using 1 wt.% agarose gel. The results are shown in Table 2 (in Table 2, M represents the leafy branch type, and L represents the leafless branch type).

[0082] Table 2 Typing results of 17 upland cotton materials

[0083] As can be seen from Table 2, 11 of the 17 upland cotton materials selected using SV molecular marker detection were of the multi-leaf branch type and 6 were of the few-leaf branch type.

[0084] (2) The 17 upland cotton materials of step (1) were planted in rows in Korla, Xinjiang and Wuhan, Hubei, and were managed routinely. Then, the leaf and branch number traits of the 17 materials were investigated in July (the results are shown in Tables 3 and 4). The phenotypic data obtained from the investigation and the typing results of the marker detection were used as the vertical axis and the horizontal axis to make a box plot, and the results are shown in Figure 8 (in Figure 8, the left figure shows the box plot of cotton in Wuhan, Hubei, and the right figure shows the box plot of cotton in Korla, Xinjiang. The vertical axis is the number of leaves and branches, and the horizontal axis represents the two types of leafy branches M and leafless branches L detected by molecular markers; the p value is calculated by variance analysis).

[0085] Table 3 Statistics of leaf and branch number of different materials 1 (unit: piece)

[0086] Table 4 Statistics of leaf and branch number traits of different materials 2 (unit: piece)

[0087] As can be seen from Tables 3 and 4, and Figure 8, the number of leaf branches of the low-leafed upland cotton materials detected using the SV marker primers in both Wuhan and Korla was much lower than that of the high-leafed upland cotton materials detected using the same molecular marker primers, with p-values ​​far below 0.001. This means that the SV molecular markers provided in this application can clearly distinguish between high-leafed and low-leafed upland cotton materials.

[0088] In summary, the SV molecular marker provided in this application and the primer set designed based on the marker (SEQ ID NO.1 to SEQ ID NO.3) can accurately detect and type the leaf and branch number trait of upland cotton.

[0089] Example 5

[0090] In order to determine whether the primer set shown in SEQ ID NO.1 to SEQ ID NO.3 in Example 3 can effectively distinguish the pure heterozygous types of leaf branch number genotypes of upland cotton, a verification experiment was carried out as follows:

[0091] (1) Using the extreme material J39 with few leafy branches as the male parent, and the extreme materials ZY161 and ZY207 with many leafy branches as the female parents, hybridize with J39 to obtain the F1 generation;

[0092] (2) The CTAB method was used to extract genomic DNA from F1, J39, ZY161, and ZY207 materials;

[0093] (3) performing PCR amplification using the genomic DNA extracted in step (2) as a template (the reaction system and reaction procedure of PCR amplification are the same as those of Example 3);

[0094] (4) The PCR amplification products obtained in step (3) were detected using 1 wt.% agarose gel. The results are shown in Figure 9.

[0095] As can be seen from Figure 9, the F1 generations of the two different hybrid combinations both have a 255bp band of the few-leaf branch genotype and a 510bp band of the many-leaf branch genotype, and the two bands can be clearly distinguished on the gel image; while the few-leaf branch material as the male parent can only detect the 255bp band of the few-leaf branch genotype, and the many-leaf branch materials ZY161 and ZY207 as the female parent can only detect the 510bp band of the many-leaf branch genotype.

[0096] It can be seen that the primer set shown in SEQ ID NO.1 to SEQ ID NO.3 provided in the present application can effectively distinguish the pure heterozygous types of genotypes and can be used as a co-dominant molecular marker in breeding.

[0097] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. An SV molecular marker related to the leaf branch number trait of upland cotton, characterized in that: The SV molecular marker is a large fragment inserted at bases 620454 to 625557 on chromosome D11 of the NDM8 genome of upland cotton; the nucleotide sequence of the large fragment is shown in SEQ ID NO.4; The version of the upland cotton NDM8 genome is NDM8HEBAU.

2. A primer for amplifying the SV molecular marker according to claim 1, characterized in that: The primers include forward primer MB-F, forward primer LB-F and reverse primer BR; The nucleotide sequence of the forward primer MB-F is shown in SEQ ID NO.1; The nucleotide sequence of the forward primer LB-F is shown in SEQ ID NO.2; The nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.

3.

3. A detection kit, characterized in that Contains the primers and PCR amplification reagents according to claim 2.

4. The detection kit according to claim 3, characterized in that The PCR amplification reagent includes DNA polymerase, dNTPs and Mg 2+ .

5. Use of the SV molecular marker according to claim 1, the primer according to claim 2, and the detection kit according to claim 3 or 4; The application includes one or more of prediction and screening of the leaf and branch number trait of upland cotton and breeding of upland cotton.

6. The use according to claim 5, characterized in that The prediction of the leaf branch number trait of upland cotton includes identifying the leaf branch type of upland cotton; When the leaf branch number of upland cotton is greater than 1, it is a multi-leaf branch type; When the number of leaf branches of upland cotton is ≤1, the upland cotton is of the few-leaf-branch type.

7. The use according to claim 5, characterized in that The upland cotton breeding includes cultivating upland cotton with few leaf branches.

8. A method for identifying the leaf branch number trait of upland cotton, characterized in that: The steps include: Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the primers described in claim 2 to obtain a PCR amplification product; performing gel electrophoresis on the PCR amplification product and making a judgment; When the PCR amplification product showed one band at a length of 510 bp, the sample to be tested was of the homozygous multi-leaf branch type; When the PCR amplification product shows one band at a length of 255 bp, the sample to be tested is a homozygous leafless branch type; When the PCR amplification product shows one band at the lengths of 255 bp and 510 bp respectively, the sample to be tested is of the heterozygous leaf branch type.

9. The method according to claim 8, characterized in that The PCR amplification reaction system (10 μL) includes: 2 μL PCR Master Mix, 1 μL 30-150 ng / μL genomic DNA template, 0.4 μL 10 μM forward primer MB-F, 0.4 μL 10 μM forward primer LB-F, 0.8 μL 10 μM reverse primer B-R, and ddH2O to 10 μL.

10. The method according to claim 9, characterized in that The PCR Master Mix includes 2×Hieff Canace Plus PCR Master Mix.

11. The method according to claim 8 or 9, characterized in that The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 67°C for 30 s, and extension at 72°C for 20 s, for 34 cycles; and extension at 72°C for 1 min.

12. The method according to claim 8 or 9, characterized in that The genomic DNA extraction method includes the CTAB method.

13. The method according to claim 8, characterized in that The gel electrophoresis was performed using 1 wt.% agarose gel.

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