A sv molecular marker related to leaf branch number trait of upland cotton and application thereof

Abstract

The present application belongs to the technical field of bio-agriculture, and particularly relates to a SV molecular marker related to leaf branch number trait of Gossypium hirsutum and application thereof. The present application provides a SV molecular marker related to leaf branch number trait of Gossypium hirsutum, and designs primers and detection kits as shown in SEQ ID NO. 1-SEQ ID NO. 3 according to the SV molecular marker, so that the prediction and screening of the leaf branch number trait of Gossypium hirsutum can be realized, and a scientific basis is provided for cultivating Gossypium hirsutum with different leaf branch types. In addition, the technical scheme provided by the present application does not need to consider the growth period and tissue type of cotton during the detection process, and does not need to perform field phenotype investigation, so that the genotype information of the sample can be accurately and quickly obtained, which is beneficial to accelerating the germplasm innovation of cotton ideal plant type and improving the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the technical field of bio-agriculture, specifically relating to an SV molecular marker related to the leaf branch number trait of upland cotton and its application. Background Technology

[0002] Cotton is a very important fiber crop worldwide. With the development of productivity and the continuous increase in labor costs, the demand for cotton with an ideal plant shape suitable for mechanization and dense planting is becoming increasingly urgent. The number of leaves and branches has a great impact on the cotton plant shape. Too many leaves and branches will lead to a loose cotton plant shape, which is not conducive to dense planting and mechanization, increases labor costs, and will also create hidden breeding grounds for pests and diseases.

[0003] Current techniques often rely on field phenotypic examinations to identify plant type traits in upland cotton and determine the number of leaves and branches. However, this method is resource-intensive, time-consuming, and susceptible to the influence of the cotton's growth stage. Currently, there are no studies or reports on molecular markers related to the number of leaves and branches in cotton. Therefore, how to utilize molecular markers to predict the number of leaves and branches in cotton, improve plant type, and cultivate an ideal plant type is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an SV molecular marker related to the leaf and branch number trait of upland cotton and its application. This molecular marker can not only predict and screen the leaf and branch number trait of upland cotton, but also realize the breeding of ideal cotton plant type.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides an SV molecular marker associated with the leaf-branch number trait of upland cotton. The SV molecular marker is a large fragment insertion occurring 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.

[0007] The version of the upland cotton NDM8 genome is NDM8HEBAU.

[0008] This invention provides primers for amplifying the SV molecular marker described in the above-mentioned technical solution, the primers including forward primer MB-F, forward primer LB-F and reverse primer BR;

[0009] The nucleotide sequence of the forward primer MB-F is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the forward primer LB-F is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.3.

[0012] This invention provides a detection kit containing the primers and PCR amplification reagents described in the above technical solution.

[0013] Preferably, the PCR amplification reagents include DNA polymerase, dNTPs, and Mg. 2+ .

[0014] This invention provides applications of the SV molecular markers, primers, or detection kits described in the above technical solutions, including one or more of the following: prediction and screening of the leaf-branch number trait in upland cotton and cultivation of upland cotton with few leaves and branches.

[0015] Preferably, the prediction of the leaf-branch number trait of upland cotton includes identifying the leaf-branch type of upland cotton;

[0016] When the number of leaf branches in upland cotton is greater than 1, then upland cotton is a multi-leaf branch type;

[0017] When the number of leaves and branches in upland cotton is ≤1, then upland cotton is a type with few leaves and branches.

[0018] Preferably, the upland cotton breeding includes breeding upland cotton with fewer leaves and branches.

[0019] This invention provides a method for identifying the leaf branch number trait of upland cotton, comprising the following steps:

[0020] 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 PCR amplification products; the PCR amplification products are then subjected to gel electrophoresis and the results are determined; when the PCR amplification product shows one band at a length of 510 bp, the sample to be tested is homozygous multi-leaved branch type; when the PCR amplification product shows one band at a length of 255 bp, the sample to be tested is homozygous few-leaved branch type; when the PCR amplification product shows one band at both the lengths of 255 bp and 510 bp, the sample to be tested is heterozygous leafy branch type.

[0021] Preferably, the PCR amplification reaction system, in 10 μL, comprises: 2 μL PCRMasterMix, 1 μL genomic DNA template (30–150 ng / μL), 0.4 μL 10 μM MB-F, 0.4 μL 10 μM LB-F, 0.8 μL 10 μM BR, and ddH2O to bring the total to 10 μL.

[0022] Preferably, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 67℃ annealing for 30 s, 72℃ extension for 20 s, for 34 cycles; 72℃ extension for 1 min.

[0023] Beneficial effects:

[0024] This invention provides an SV molecular marker associated with the leaf-branch number trait in upland cotton. The SV molecular marker is a large fragment insertion at bases 620454–625557 on chromosome D11 of the upland cotton NDM8 genome, and the nucleotide sequence of this large fragment is shown in SEQ ID NO.4. The version of the upland cotton NDM8 genome is NDM8HEBAU. This SV molecular marker is significantly correlated with the leaf-branch number trait in upland cotton. Furthermore, this invention designs corresponding primers and detection kits based on the SV molecular marker, thereby enabling the prediction and screening of the leaf-branch number trait in upland cotton, providing a scientific basis for breeding upland cotton with different leaf-branch types. Simultaneously, the detection process does not require consideration of the cotton's growth stage or tissue type, nor does it require field phenotypic observation. It can accurately and quickly obtain the genotypic information of the samples, which is beneficial for accelerating the germplasm innovation of ideal cotton plant types and improving breeding efficiency.

[0025] Based on the above-mentioned technical advantages, this invention also provides a method for identifying the leaf-branch number trait of upland cotton, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain PCR amplification products; performing gel electrophoresis on the PCR amplification products and making a judgment. Experiments have shown that the SV marker provided by this invention and the primers designed based on the marker can accurately detect the leaf-branch number trait of upland cotton and effectively distinguish the homozygous type of genotype. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 The results of genome-wide association analysis of leaf and branch number data from 288 natural upland cotton populations in Example 1 over two years;

[0028] Figure 2 Example 1: Sequencing alignment results of SV molecular marker regions for different extreme upland cotton materials;

[0029] Figure 3 Example 1: Sequencing alignment results of SV molecular marker regions for different extreme upland cotton materials;

[0030] Figure 4 The sequencing alignment results of SV molecular marker regions for different extreme upland cotton materials in Example 1 are shown in Figure 3.

[0031] Figure 5 The sequencing alignment results of SV molecular marker regions for different extreme upland cotton materials in Example 1 are shown in Figure 4.

[0032] Figure 6 The expression level of the TCP18 gene in extreme cotton materials with different leaf-branch numbers in Example 2;

[0033] Figure 7 The results of molecular detection of the primer set in Example 3 in upland cotton material with extreme leaf and branch numbers;

[0034] Figure 8 Box plot of leaf branch number survey results for different upland cotton materials based on molecular detection typing in Example 4;

[0035] Figure 9 The results are molecular detection results for different upland cotton materials in Example 5. Detailed Implementation

[0036]

[0037] This invention provides primers for amplifying the SV molecular marker described in the above-mentioned technical solution. The primers include 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'; and the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.3, specifically 5'-AGAGGGAGAATCGAATGGTCAAG-3'. These primers can specifically clone the SV molecular marker, thereby enabling the prediction and screening of leaf and branch number traits in upland cotton.

[0038] This invention provides a detection kit containing the primers and PCR amplification reagents described in the preceding claims. The PCR amplification reagents of this invention preferably include DNA polymerase, dNTPs, and Mg. 2+ The source and amount of the DNA polymerase, dNTPs and buffer reagents are not particularly limited, and commercially available products in this field can be used.

[0039] This invention provides applications of the SV molecular markers, primers, or detection kits described in the above-mentioned technical solutions. These applications include one or more of the following: prediction and screening of the leaf-branch number trait in upland cotton, and upland cotton breeding. Preferably, these applications include prediction and screening of the leaf-branch number trait in upland cotton, and upland cotton breeding. In this invention, the prediction of the leaf-branch number trait in upland cotton preferably includes identifying the leaf-branch type of upland cotton; when the number of leaf-branch numbers in upland cotton is >1, the upland cotton is preferably a multi-leaf-branch type; when the number of leaf-branch numbers in upland cotton is ≤1, the upland cotton is preferably a few-leaf-branch type; the upland cotton breeding preferably includes cultivating upland cotton of the few-leaf-branch type.

[0040] This invention provides a method for identifying the leaf-branch number trait of upland cotton, comprising the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the primers described in the above technical solution to obtain PCR amplification products; performing gel electrophoresis on the PCR amplification products and making a judgment; when the PCR amplification product shows one band at a length of 510 bp, the sample to be tested is a 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 few-leaf-branch type; when the PCR amplification product shows one band at lengths of 255 bp and 510 bp respectively, the sample to be tested is a heterozygous leaf-branch type.

[0041] This invention preferably extracts genomic DNA from the sample to be tested to obtain the genomic DNA of the sample. In this invention, there are no special requirements for the extraction method of the genomic DNA of the sample; techniques well known in the art can be used. In a specific embodiment of this invention, the CTAB method is used to extract genomic DNA from cotton.

[0042] After obtaining the genomic DNA of the sample to be tested, the present invention 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 PCR amplification products. In the present invention, the PCR amplification reaction system is preferably 10 μL, comprising: 2 μL PCR MasterMix, 1 μL of 30-150 ng / μL genomic DNA template, 0.4 μL of 10 μM MB-F, 0.4 μL of 10 μM LB-F, 0.8 μL of 10 μM BR, and ddH2O to bring the total to 10 μL. In a specific embodiment of the present invention, the PCR MasterMix is ​​2×HieffCanace Plus PCR MasterMix (With Dye).

[0043] The preferred PCR amplification reaction procedure of this invention is: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 67℃ for 30 s, extension at 72℃ for 20 s, for 34 cycles; and extension at 72℃ for 1 min. After extension, it is preferably stored at 10℃ for 5 min. The above PCR amplification can achieve the purpose of identifying the leaf and branch number trait of upland cotton.

[0044] After obtaining the PCR amplification product, the present invention performs gel electrophoresis on the PCR amplification product and makes a judgment. There are no special requirements for the gel electrophoresis method; techniques well known in the art can be used. When the PCR amplification product shows one band at a length of 510 bp, the sample is homozygous for a multi-leaved branch type; when the PCR amplification product shows one band at a length of 255 bp, the sample is homozygous for a few-leaved branch type; when the PCR amplification product shows one band at both 255 bp and 510 bp, the sample is heterozygous for a few-leaved branch type. This method is beneficial for identifying the number of leaves and branches in upland cotton, and thus screening out homozygous few-leaved branch types.

[0045] Experiments have shown that the SV marker provided by this invention and the primers designed based on the marker can accurately detect the leaf and branch number trait of upland cotton and effectively distinguish the homozygous type of genotype. At the same time, the detection process does not require consideration of the cotton's growth stage and tissue type, nor does it require field phenotypic investigation. It can accurately and quickly obtain the genotype information of the sample, which is beneficial to accelerating the germplasm innovation of ideal cotton plant type and improving breeding efficiency.

[0046] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an SV molecular marker related to the leaf branch number trait of upland cotton and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] Acquisition of SV molecular markers associated with leaf branch number trait in upland cotton

[0049] (1) Cultivation of natural cotton populations, investigation of leaf-branch number phenotype and genome-wide association analysis:

[0050] From 2018 to 2019, 288 natural populations were planted in an experimental field in Korla, Xinjiang. Leaf-branch number phenotypic data were collected in July each year, totaling two years' worth of data. The 288 upland cotton cultivars and the selected upland cotton materials with extreme leaf-branch number phenotypes were all selected from upland cotton cultivars whose genomes had been resequencing by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (see Wang et al. Asymmetric subgenome selection and cis-regulatory divergence during cotton domestication. Nat Genet, 2017, 49:579-587.).

[0051] Based on the 288 upland cotton cultivars whose genomes have been resequencing by the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University, resequencing data were downloaded and combined with two years of phenotypic data for genome-wide association analysis. The results are shown below. Figure 1 (exist Figure 1 In the middle, the left figure represents the genome-wide association study (GWAS) mapping results of 288 upland cotton natural population leaf-branch number data in 2018; the right figure represents the GWAS mapping results of 288 upland cotton natural population leaf-branch number data in 2019.

[0052] Depend on Figure 1 It can be seen that the leaf and branch number data of the natural population of upland cotton were all located at highly significant sites in the same region of chromosome D11 throughout the whole genome.

[0053] (2) Sequencing alignment results of leaf branch number extreme materials in the SV molecular marker region:

[0054] The most phenotypically extreme and two-year-old stable upland cotton materials with multiple leaf branches (i.e., leaf branch number > 1) ZY161 and ZY207, and fewer leaf branches (i.e., leaf branch number ≤ 1) J39 and ZY98 were selected from the above populations. Sequencing and sequence alignment were performed on the mapping intervals (i.e., the natural population GWAS mapping intervals specifically from base 171567 to 675567) of ZY161, ZY207, J39, and ZY98. The results are shown in […]. Figures 2-5 (exist Figures 2-5 In the sequence, ZY161 and ZY207 are sequences of multi-leaved branches, J39 and ZY98 are sequences of few-leaved branches, and NDM8 is the reference genome sequence, which is of the multi-leaved branch type. The last line represents the reference results, where lowercase letters indicate differences in the sequences being compared, and uppercase letters indicate similarities.

[0055] Depend on Figures 2-5 Sequencing alignment results show that, compared to the extreme materials with fewer leaf branches (J39 and ZY98), the materials with more leaf branches (ZY161 and ZY207) have an insertion of approximately 5 kb. This 5 kb fragment is designated as the SV molecular marker. This variation type is a large fragment insertion / deletion variation within the SV molecular marker. In other words, this SV molecular marker is directly related to the leaf branch number trait in upland cotton; those with this insertion are likely to exhibit the more leaf branch trait, while those without the 5 kb insertion exhibit the less leaf branch trait.

[0056] Example 2

[0057] To further determine the accuracy of the effect of the SV molecular marker on the leaf and branch number trait of upland cotton in Example 1, the physical location information of the SV molecular marker was determined based on the Gossypium hirsutum (AD1) and NDM8HEBAU versions of the upland cotton NDM8 genome: the nucleotide sequence shown at bases 620454 to 625557 of chromosome D11 of upland cotton. The nearest downstream gene to this sequence is the TCP18 gene, which has been reported in various plants as a key gene negatively regulating lateral branching (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.).

[0058] Extremophiles with numerous leaves and branches (ZY161 and ZY207) and fewer leaves and branches (J39 and ZY98) were selected and planted. Under sufficient nutrient conditions, when the plants reached the 4-5 leaf stage, axillary buds of the second leaf from the bottom were harvested. Total RNA was extracted from each extremophile using a kit (RNA extraction kit purchased from Nanjing Novizan Biotechnology Co., Ltd.). The expression level of TCP18 was detected using qRT-PCR. The results are shown in Table 1 and [Table data missing]. Figure 6 .

[0059] Table 1 Gene expression levels under different treatments

[0060] deal with Repeat 1 Repeat 2 Repeat 3 Repeat 4 mean ZY161 0.000515102 0.000277071 0.000548453 0.000261901 0.000400632 ZY207 0.001178514 0.001162163 0.001012034 0.001287301 0.001160003 J39 0.054535987 0.081001588 0.104146257 0.09235864 0.083010618 ZY98 0.083762214 0.098147699 0.097027833 0.065510378 0.086112031

[0061] From Table 1 and Figure 6It can be seen that the TCP18 gene is highly expressed in the low-leaf-branch materials J39 and ZY98, while it is almost not expressed in the high-leaf-branch materials ZY161 and ZY207. This indicates that the 5kb insertion / deletion variant in Example 1 regulates the leaf-branch number trait in cotton by modulating the expression of its downstream genes. Therefore, this large 5kb insertion / deletion variant is a key SV molecular marker affecting the leaf-branch number trait in upland cotton.

[0062] Example 3

[0063] (1) Based on the SV molecular marker (5kb) in Example 1, a set of specific primers 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).

[0064] MB-F: 5'-ACAAGGATTTACGTGGTTCGG-3' (SEQ ID NO. 1);

[0065] LB-F: 5'-AAACTGATCTGAAACTTGGGACT-3' (SEQ ID NO. 2);

[0066] BR: 5'-AGAGGGAGAATCGAATGGTCAAG-3' (SEQ ID NO. 3);

[0067] Forward primer MB-F and reverse primer BR can specifically amplify multi-leaved branch genotype fragments with SV molecular marker insertion, with a fragment size of 510 bp; forward primer LB-F and the shared reverse primer BR can specifically amplify few-leaved branch genotype fragments without the SV molecular marker insertion, with a fragment size of 255 bp.

[0068] (2) Verify the accuracy of the primer set in step (1):

[0069] The materials to be tested were ZY161 and ZY207, which have a high number of leaf branches, and J39 and ZY98, which have a low number of leaf branches. The steps were as follows:

[0070] A. Genomic DNA was extracted from cotton from different extreme materials using the CTAB method;

[0071] B. PCR amplification: Using the cotton genomic DNA extracted in step A as a template, PCR amplification was performed using the primer set (SEQ ID NO.1~SEQ ID NO.3) in step (1) to obtain the PCR amplification product;

[0072] The PCR amplification system, in 10 μL increments, consisted of: 1 μL of 30–150 ng / μL DNA template, 5 μL of 2×HieffCanace Plus PCRMasterMix (With Dye), 0.4 μL of MB-F (10 μM), 0.4 μL of ILB-F (10 μM), 0.8 μL of BR (10 μM), and 2.4 μL of ddH2O.

[0073] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 67℃ annealing for 30 s, 72℃ extension for 20 s, for 34 cycles; 72℃ extension for 1 min, and storage at 10℃ for 5 min. The HieffCanace Plus PCR MasterMix (With Dye) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0074] C. Agarose gel electrophoresis detection: The PCR amplification products from step B were detected using a 1 wt.% agarose gel. The results are shown in [Figure 1]. Figure 7 (exist Figure 7 In the image, the bars from left to right represent the results for materials J39, ZY98, ZY161, ZY161, ZY207, and ZY207, respectively.

[0075] Depend on Figure 7 As can be seen, the PCR amplification product of the sparse-leaved extreme material showed only one band at 255 bp after electrophoresis, while the PCR amplification product of the multi-leaved extreme material showed one band at 510 bp. Therefore, the primer sets provided by this invention, as shown in SEQ ID NO.1 to SEQ ID NO.3, can effectively utilize SV molecular markers to distinguish between multi-leaved and sparse-leaved upland cotton plant types.

[0076] Example 4

[0077] (1) To further evaluate the accuracy of the SV marker in Example 1 and the primer sets 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 the natural population for SV marker detection and genotyping. The specific steps are as follows:

[0078] A. Cotton genomic DNA was extracted from 17 upland cotton materials using the CTAB method;

[0079] B. PCR amplification: The method is the same as step B in Example 3;

[0080] 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 multi-leaved branch type and L represents the few-leaved branch type).

[0081] Table 217 Classification Results of Upland Cotton Materials

[0082] Material Classification results ZY21 M ZY81 L J38 L ZY85 L ZY84 M ZY309 L J39 L ZY98 L J20 M J23 M ZY428 M J70 M ZY389 M J68 M ZY161 M ZY207 M J668 M

[0083] As shown in Table 2, among the 17 selected upland cotton materials detected by SV molecular markers, 11 were multi-leaved and 6 were sparsely leafed.

[0084] (2) The 17 upland cotton materials from step (1) were planted in rows in Korla, Xinjiang and Wuhan, Hubei, and managed routinely. Then, in July, the leaf-branch number trait of the 17 materials was investigated (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 ordinate and abscissa, respectively, to create a box plot. The results are shown in Tables 3 and 4. Figure 8 (exist Figure 8 The left figure shows the box plot of cotton from Wuhan, Hubei Province, and the right figure shows the box plot of cotton from Korla, Xinjiang Province. The vertical axis represents the number of leaves and branches, and the horizontal axis represents the two types of branches (M with many leaves and L with few leaves) detected by molecular markers; the p-value is calculated by analysis of variance.

[0085] Table 3. Statistics on the number of leaves and branches of different materials (unit: number of branches)

[0086]

[0087]

[0088] Table 4. Statistics on the number of leaves and branches of different materials (2) (Unit: number of branches)

[0089]

[0090] From Tables 3 and 4, Figure 8 It can be seen that, regardless of whether in Wuhan or Korla, the leaf and branch count of upland cotton materials with few branches detected using the SV marker primers is much lower than that of upland cotton materials with many branches detected using the same molecular marker primers, with p values ​​much lower than 0.001. This means that the SV molecular marker provided by this invention can clearly distinguish between upland cotton materials with many branches and those with few branches.

[0091] In summary, the SV molecular markers provided by this invention and the primer sets (SEQ ID NO.1 to SEQ ID NO.3) designed based on these markers can accurately detect and classify the leaf and branch number traits of upland cotton.

[0092] Example 5

[0093] To determine whether the primer sets shown in SEQ ID NO.1 to SEQ ID NO.3 in Example 3 can effectively distinguish the homozygous type of the leaf branch number genotype in upland cotton, the following verification experiment was conducted:

[0094] (1) Using the few-leaf-branch extreme material J39 as the male parent, and using the multi-leaf-branch extreme materials ZY161 and ZY207 as the female parents respectively, J39 was crossed to obtain the F1 generation;

[0095] (2) Genomic DNA was extracted from F1, J39, ZY161 and ZY207 materials using the CTAB method;

[0096] (3) PCR amplification was performed using the genomic DNA extracted in step (2) as a template (the PCR amplification reaction system and reaction procedure were the same as those in Example 3).

[0097] (4) The PCR amplification products obtained in step (3) were detected using a 1wt.% agarose gel electrophoresis. The results are shown in the figure. Figure 9 .

[0098] Depend on Figure 9 It can be seen that the F1 generation of both different hybrid combinations simultaneously possesses 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; however, the few-leaf-branch material used as the male parent can only detect the 255bp band of the few-leaf-branch genotype, while the many-leaf-branch materials ZY161 and ZY207 used as the female parent can only detect the 510bp band of the many-leaf-branch genotype.

[0099] Therefore, the primer sets provided by this invention, as shown in SEQ ID NO.1 to SEQ ID NO.3, can effectively distinguish between homozygous genotypes and can be used as molecular markers for codominance in breeding.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of SV molecular markers or primers for detecting said SV molecular markers or detection kits containing said primers; said application is one or more of the following: prediction, screening, and breeding of leaf branch number trait in upland cotton; The SV molecular marker is a large insertion at bases 620454-625557 on chromosome D11 of the upland cotton NDM8 genome, and the nucleotide sequence of the large insertion is shown in SEQ ID NO.4; the version of the upland cotton NDM8 genome is NDM8HEBAU; 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; and the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The test kit also contains PCR amplification reagents.

3. The application according to claim 2, characterized in that, The PCR amplification reagents include DNA polymerase, dNTPs, and Mg. 2+ .

4. The application according to claim 1, 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 number of leaf branches in upland cotton is greater than 1, then upland cotton is a multi-leaf branch type; When the number of leaves and branches in upland cotton is ≤1, then upland cotton is a type with few leaves and branches.

5. The application according to claim 4, characterized in that, The upland cotton breeding includes the cultivation of upland cotton with fewer leaves and branches.

6. A method for identifying the leaf-branch number trait of upland cotton, characterized in that, Includes the following steps: Using the genomic DNA of the sample to be tested as a template, PCR amplification was performed using primers to obtain PCR amplification products; the PCR amplification products were then subjected to gel electrophoresis and an assessment was made. 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; and the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO.

3. When the PCR amplification product shows a single band at a length of 510 bp, the sample to be tested is a homozygous multi-leaved branch type. When the PCR amplification product shows a single band at a length of 255 bp, the sample to be tested is a homozygous sparse-leaved type. When the PCR amplification product shows one band at lengths of 255bp and 510bp, the sample to be tested is of the heterozygous leaf-branch type.

7. The method according to claim 6, characterized in that, The PCR amplification reaction system, in 10 μL increments, includes: 2 μL PCR MasterMix, 1 μL genomic DNA template (30-150 ng / μL), 0.4 μL 10 μM MB-F, 0.4 μL 10 μM LB-F, 0.8 μL 10 μM BR, and ddH2O to bring the total to 10 μL.

8. The method according to claim 6 or 7, characterized in that, The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 67℃ annealing for 30 s, 72℃ extension for 20 s, for 34 cycles; 72℃ extension for 1 min.

Images