A cotton Indel marker and its application in cotton breeding

Abstract

The present invention belongs to the technical field of plant molecular breeding, and specifically relates to a cotton Indel marker and its application in cotton breeding. The Indel marker FP_D03_Indel described in the present invention is an Indel marker related to the flowering period and precocity of cotton, which can accurately identify the early or late flowering period and maturity (length of the growth period) of cotton, and improve the selection efficiency of early-maturing cotton varieties; at the same time, without affecting the yield and other agronomic traits, it is not affected by the environment, and the precocity of cotton varieties is improved quickly and efficiently, providing technical support for the selection and breeding of early-maturing cotton varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular breeding, and particularly relates to a cotton Indel marker and application thereof in cotton breeding. Background Art

[0002] Cotton (Gossypium spp.) is an important cash crop and the main raw material for the textile industry. Early-maturing cotton varieties have a short growth period, which can achieve double harvests of wheat-cotton, garlic-cotton or cotton-fodder crops in one year, improve land utilization, and greatly alleviate the contradiction between cotton and other crops in the competition for land resources. Therefore, the early maturity of varieties has always been an important target trait for cotton variety improvement.

[0003] The genetic mechanism of early maturity traits is complex, which seriously restricts the breeding of early-maturing cotton varieties. The rapid development of molecular marker technology and high-throughput sequencing technology has laid the foundation for the effective use of stable QTLs and related markers to carry out molecular design breeding of cotton early maturity. The early maturity of cotton is closely related to the flowering time, and the growth period of varieties that bloom early will also be advanced. QTLs related to the flowering period of cotton have been reported, but the number of varieties or genetic populations used in the QTL positioning population in early reports is limited, which limits the precise positioning of the main effect QTL of the flowering period, thereby affecting the accuracy of molecular marker-assisted selection. In addition, due to the large deviation of QTL positioning, it will also lead to the emergence of linkage drag in breeding, reducing the efficiency of breeding selection. Summary of the invention

[0004] The object of the present invention is to provide a cotton Indel marker and its application in cotton breeding. The Indel marker can accurately identify the early or late flowering period and maturity (length of growth period) of cotton, improve the selection efficiency of early-maturing cotton varieties, and can be used for the cultivation of early-maturing cotton varieties.

[0005] The present invention provides a cotton Indel marker FP_D03_Indel. The nucleotide sequence containing the cotton Indel marker FP_D03_Indel is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0006] The present invention also provides a primer pair for detecting the cotton Indel marker FP_D03_Indel described in the above technical solution, and the nucleotide sequences of the forward primer and the reverse primer of the primer pair are shown as SEQ ID NO.3 or SEQ ID NO.4 respectively.

[0007] The present invention also provides a kit, which comprises the primer pair and PCR amplification reagent described in the above technical solution.

[0008] The present invention also provides the use of the cotton Indel marker FP_D03_Indel described in the above technical solution, the primer pair described in the above technical solution or the kit described in the above technical solution in one or more of the following:

[0009] 1) Cotton breeding;

[0010] 2) Differentiate the maturity of cotton;

[0011] 3) Identify cotton maturity;

[0012] 4) Distinguish the flowering period of cotton;

[0013] 5) Identify the flowering period of cotton.

[0014] Preferably, the cotton breeding includes cultivating early-maturing cotton varieties or early-maturing cotton lines.

[0015] The present invention also provides a method for identifying the maturity and / or flowering period of cotton, comprising the following steps:

[0016] Using the primers described in the above technical solution to perform PCR amplification on the genomic DNA of the cotton sample to be tested to obtain an amplified product;

[0017] Sequencing the amplified product to obtain the amplified product sequence;

[0018] When the sequence of the amplified product is as shown in SEQ ID NO.1, the genotype of the cotton sample to be tested is homozygous FP_D03, and the cotton sample to be tested blooms late and opens bolls late;

[0019] When the sequence of the amplified product is as shown in SEQ ID NO. 2, the genotype of the cotton sample to be tested is homozygous fp_D03, and the cotton sample to be tested blooms early and opens bolls early;

[0020] When the sequence of the amplified product is as shown in SEQ ID NO.1 and SEQ ID NO.2, the genotype of the cotton sample to be tested is the heterozygous FP_D03 / fp_D03, and the flowering period and boll opening period of the sample to be tested are between the cotton varieties with the genotypes of FP_D03 and fp_D03.

[0021] Preferably, the PCR amplification system includes: 4.7 μL of ultrapure water, 1.0 μL of genomic DNA, 1.0 μL of 10×Buffer, 1.0 μL of 2.5 mM MgCl2, 0.2 μL of 10 mM dNTPs, 1.0 μL of 10 μM forward primer, 1.0 μL of reverse primer, and 0.1 μL of Taq DNA polymerase.

[0022] Preferably, the PCR amplification procedure includes: preheating at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0023] The present invention also provides a method for cultivating an early-maturing cotton strain, comprising the following steps:

[0024] SL EF_D03 as the donor parent, and hybridization with late-flowering cotton varieties as the recurrent parent to obtain the F1 generation;

[0025] The F1 generation plant containing homozygous fp_D03 was used as the donor parent and backcrossed 2-3 times with the recurrent parent to obtain BC n F1 generation, n is 2-3;

[0026] BC containing homozygous fp_D03 n The F1 generation plants are self-pollinated for 2-3 generations, and the homozygous plants with the genotype fp_D03 identified during the self-pollination are bred to obtain the BCnF2:m generation, which is the early-maturing cotton line, where m is 3-5;

[0027] The single segment replacement line SL EF _ D03 The breeding method includes: constructing an F2 population using Lumianyan No. 19 and Lumianyan No. 37 as parents;

[0028] The early-flowering plants in the F2 population were backcrossed with Lumianyan 37 for 6 times and then self-pollinated for 2 generations to obtain the BC6F2 generation;

[0029] The single plant containing the least genome component of Lumianyan No. 19 in the BC6F2 generation was self-pollinated to BC6F 2:4 The single segment replacement line SL EF_D03 .

[0030] Preferably, the late-flowering cotton varieties include one or more varieties of Lumian 361, Lumian 392, Lumian 3218, Lu6269, Lumian 3036, Lumian 378 and Lu7619.

[0031] Beneficial effects:

[0032] The present invention provides a cotton Indel marker FP_D03_Indel, and the nucleotide sequence containing the cotton Indel marker FP_D03_Indel is shown in SEQ ID NO.1 or SEQ ID NO.2. Based on the initial positioning of flowering traits using BSA-seq technology, the present invention further screens InDel markers for the major effect QTL interval of D03 chromosome, and uses BC6F2:3 and BC6F2:4 populations to finely locate the major effect QTL of flowering period, and identifies the major effect QTL site related to the flowering period of cotton through fine positioning, and develops a functional Indel marker FP_D03_Indel, which is an Indel marker related to the flowering period and precocity of cotton, can accurately identify the early or late flowering period and maturity (length of growth period) of cotton, and improve the selection efficiency of early-maturing cotton varieties; at the same time, without affecting the yield and other agronomic traits, it is not affected by the environment, and the precocity of cotton varieties is quickly and efficiently improved, providing technical support for the breeding of early-maturing cotton varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0034] Figure 1 This is a diagram showing the results of fine mapping of QTL for flowering period of cotton in Example 1;

[0035] Figure 2 This is a graph showing the comparison of flowering time between the improved offspring and the parents in Example 2. DETAILED DESCRIPTION

[0036] The present invention provides a cotton Indel marker FP_D03_Indel. The nucleotide sequence containing the cotton Indel marker FP_D03_Indel is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0037] The cotton Indel marker FP_D03_Indel of the present invention has two genotype sequences, namely, the FP_D03 genotype sequence and the fp_D03 genotype sequence. The nucleotide sequences of the FP_D03 genotype sequence and the fp_D03 genotype sequence are shown in SEQ ID NO.1 or SEQ ID NO.2, respectively, and are specifically:

[0038] SEQ ID NO.1:

[0039] 5'-GTGATAGTGTGGTTCCTGTTCAAACTTTGAGAACTAAGAATCAACC ACTACAAGAAGAGACACAATTGCAGAAACAGCAATTGCATAGT-3';

[0040] SEQ ID NO.2:

[0041] 5'-GTGATAGTGTGGTTCCTGTTCAAACTTTGAGAACTAAGAATCAACC ACTACAAGAAGAGACAATTGCAGAAACAGCAATTGCATAGT-3'.

[0042] Compared with the TM-1 reference genome sequence (http: / / cotton.zju.edu.cn / index.htm), the base at +63-64bp of the FP_D03 genotype sequence of the present invention is CA; and the CA base is missing at +63-64bp of the fp_D03 genotype sequence.

[0043] The present invention also provides a primer pair for detecting the cotton Indel marker FP_D03_Indel described in the above technical solution, and the nucleotide sequences of the forward primer and the reverse primer of the primer pair are respectively shown in SEQ ID NO.3 or SEQ ID NO.4. The nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4 described in the present invention is as follows: SEQ ID NO.3: 5'-GTGATAGTGTGGTTCCTGTTCAAACTT-3' and SEQ ID NO.4: 5'-GTACTATGCAATTGCTGTTTCTGCAATT-3'.

[0044] The present invention also provides a kit, which includes the primer pair and PCR amplification reagent described in the above technical solution. The present invention has no special restrictions on the type of the PCR amplification reagent, and commercial finished product reagents or self-prepared reagents are all acceptable. For example, in one embodiment of the present invention, the PCR amplification reagent includes MgCl2, dNTPs and Taq DNA polymerase. The present invention has no special restrictions on the content of the PCR amplification reagent in the kit, and it can be selected conventionally according to needs.

[0045] The present invention also provides the use of the cotton Indel marker FP_D03_Indel described in the above technical solution or the primer pair described in the above technical solution or the kit described in the above technical solution in one or more of the following: 1) cotton breeding; 2) distinguishing cotton maturity; 3) identifying cotton maturity; 4) distinguishing cotton flowering period; 5) identifying cotton flowering period. The cotton breeding described in the present invention preferably includes cultivating early-maturing cotton varieties or early-maturing cotton lines; the cotton preferably includes upland cotton. The cotton maturity described in the present invention preferably includes early maturity, late maturity and maturity between early maturity and late maturity; the early maturity refers to the cotton growth period within 105 days, the late maturity refers to the cotton growth period more than 115 days, and the maturity between early maturity and late maturity refers to the cotton growth period between 105-115 days.

[0046] The present invention also provides a method for identifying the maturity and / or flowering period of cotton, comprising the following steps:

[0047] Using the primers described in the above technical solution to perform PCR amplification on the genomic DNA of the cotton sample to be tested to obtain an amplified product;

[0048] Sequencing the amplified product to obtain the amplified product sequence;

[0049] When the sequence of the amplified product is as shown in SEQ ID NO.1, the genotype of the cotton sample to be tested is homozygous FP_D03, and the cotton sample to be tested blooms late and opens bolls late;

[0050] When the sequence of the amplified product is as shown in SEQ ID NO. 2, the genotype of the cotton sample to be tested is homozygous fp_D03, and the cotton sample to be tested blooms early and opens bolls early;

[0051] When the sequence of the amplified product is as shown in SEQ ID NO.1 and SEQ ID NO.2, the genotype of the cotton sample to be tested is the heterozygous FP_D03 / fp_D03, and the flowering period and boll opening period of the sample to be tested are between the cotton varieties with the genotypes of FP_D03 and fp_D03.

[0052] The present invention uses the primers described in the above technical solution to perform PCR amplification on the genomic DNA of the cotton sample to be tested to obtain an amplified product. The present invention does not specifically limit the method for extracting the genomic DNA, and the conventional method for extracting genomic DNA in the art can be used. The system for PCR amplification of the present invention preferably includes: 4.7 μL of ultrapure water, 1.0 μL of genomic DNA, 1.0 μL of 10×Buffer, 1.0 μL of 2.5 mM MgCl2, 0.2 μL of 10 mM dNTPs, 1.0 μL of 10 μM forward primer, 1.0 μL of reverse primer, and 0.1 μL of Taq DNA polymerase. The procedure for PCR amplification of the present invention preferably includes: preheating at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and 30 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0053] After obtaining the PCR product, the present invention sequences the PCR product to obtain the amplified product sequence. The sequencing of the present invention is preferably high-throughput sequencing; the specific process of the high-throughput sequencing is not particularly limited, and the conventional high-throughput sequencing method in the art can be used.

[0054] After obtaining the sequence of the amplified product, the present invention uses the sequence of the amplified product to determine the flowering period and maturity of the cotton sample to be tested: when the sequence of the amplified product is as shown in SEQ ID NO.1, the genotype of the cotton sample to be tested is homozygous FP_D03, and the cotton sample to be tested blooms late and opens bolls late; when the sequence of the amplified product is as shown in SEQ ID NO.2, the genotype of the cotton sample to be tested is homozygous fp_D03, and the cotton sample to be tested blooms early and opens bolls early; when the sequence of the amplified product is as shown in SEQ ID NO.1 and SEQ ID NO.2, the genotype of the cotton sample to be tested is heterozygous FP_D03 / fp_D03, and the flowering period and boll opening period of the sample to be tested are between the cotton varieties with genotypes of FP_D03 and fp_D03. When the present invention makes the determination, it is preferred to use the sequence on chromosome D03 in the TM-1 reference genome (http: / / cotton.zju.edu.cn / index.htm) as a reference.

[0055] The present invention also provides a method for cultivating an early-maturing cotton strain, comprising the following steps:

[0056] SL EF_D03 as the donor parent, and hybridization with late-flowering varieties as the recurrent parent to obtain the F1 generation;

[0057] The F1 generation plant containing homozygous fp_D03 was used as the donor parent and backcrossed 2-3 times with the recurrent parent to obtain BCn F1 generation, n is 2-3;

[0058] BC containing homozygous fp_D03 n The F1 generation plants are self-pollinated for 2-3 generations, and the homozygous plants with the genotype fp_D03 identified during the self-pollination are bred to obtain the BCnF2:m generation, which is the early-maturing cotton line, where m is 3-5;

[0059] The single segment replacement line SL EF _ D03 The breeding method includes: constructing an F2 population using Lumianyan No. 19 and Lumianyan No. 37 as parents;

[0060] The early-flowering plants in the F2 population were backcrossed with Lumianyan 37 for 6 times and then self-pollinated for 2 generations to obtain the BC6F2 generation;

[0061] The single plant containing the least genome component of Lumianyan No. 19 in the BC6F2 generation was self-pollinated to BC6F 2:4 The single segment replacement line SL EF _ D03 .

[0062] The late-flowering varieties of the present invention preferably include one or more varieties of Lumian 361, Lumian 392, Lumian 3218, Lu 6269, Lumian 3036, Lumian 378 and Lu 7619, and more preferably Lumian 361, Lumian 392, Lumian 3218, Lu 6269, Lumian 3036, Lumian 378 or Lu 7619, that is, different early-flowering or early-maturing strains can be improved with different late-flowering varieties as recurrent parents. The present invention does not specifically limit the processes of hybridization, backcrossing and selfing in the above technical scheme, and the conventional hybridization, backcrossing and selfing processes in the art can be used. The method for identifying the F1 generation plants containing homozygous fp_D03 and the BC3F1 generation plants containing homozygous fp_D03 of the present invention is preferably the method for identifying the maturity and / or flowering period of cotton described in the above technical scheme, and the specific process will not be repeated. The breeding of the present invention is preferably generation-adding breeding, and the number of generations of the generation-adding breeding is preferably 3 generations.

[0063] In order to further illustrate the present invention, the technical effects provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0064] Terminology Note:

[0065] Flowering period: generally refers to the number of days from sowing to when 50% of the cotton plants bloom;

[0066] Boll opening period: generally refers to the number of days from the sowing period to the time when 50% of the cotton plants open the bolls.

[0067] Example 1

[0068] Fine mapping of cotton flowering period traits and identification of functional molecular markers:

[0069] In the early stage, our research group used the early-maturing upland cotton variety Lumianyan 19 (LMY19; flowering period is about 59 days) and the late-maturing variety Lumianyan 37 (LMY37; flowering period is about 68 days) to construct an F2 population. Through BSA-seq, a major QTL related to the flowering period trait was initially located on chromosome D03. The QTL interval was 9.6Mb and contained 334 genes. The fine positioning results are as follows Figure 1 shown.

[0070] On this basis, using the resequencing data of the two parents, InDel and SNP markers were developed for the target region to screen markers with polymorphism between the parents. Starting from the F2 generation in 2015, early flowering plants containing the QTL were selected and backcrossed with Lumianyan 37 in Linqing and Hainan, and then self-pollinated to the BC6F2 generation population, which contained 4220 plants. The newly developed InDel and SNP markers were used to scan the genotypes of the plants, identify the exchange plants, self-pollinate the plants, and collect seeds. The seeds harvested in 2020 were planted in two replicates in a single row to obtain the BC6F2 population. 2：3 The flowering time of the first flower was recorded every day during the flowering period. 15 plants were counted in each row, and the average value was used as the flowering period of the BC6F2 exchange plant. The flowering phenotypic traits of the exchange plant were combined with its genotype data to finely locate a major QTL locus for cotton flowering period. The QTL interval was narrowed to 345.56Kb, containing 10 genes. There was an Indel mutation in the interval between the two parents, that is, there was a 2-base deletion in the early-maturing parent Lumianyan 19 (Lumianyan 37: GACACA, Lumianyan 19: GACA). The Indel was named FP_D03_Indel, and Indel primers were designed in the flanking sequences on both sides of the deletion site. The forward primer FP_D03-F: 5'-GTGATAGTGTGGTTCCTGTTCAAACTT-3' (SEQ ID NO.3), and the reverse primer FP_D03-R: 5'-GTACTATGCAATTGCTGTTTCTGCAATT-3' (SEQ ID NO.4). Thirty varieties were used as samples to be tested (Table 1), DNA was extracted using the CTAB method, PCR amplification was performed using the primers, and high-throughput sequencing was performed on the amplified products.

[0071] PCR amplification system:

[0072] Ultrapure water (ddH2O) 4.7μL, template DNA 1.0μL, 10×Buffer 1.0μL, 2.5mM MgCl2 1.0μL, 10mM dNTPs 0.2μL, 10μM forward primer 1.0μL, reverse primer 1.0μL, Taq DNA polymerase 0.1μL.

[0073] Amplification reaction procedure:

[0074] Preheat at 95°C for 5 min, denature at 94°C for 45 s, anneal at 55°C for 45 s, extend at 72°C for 1 min, and cycle 30 times; extend at 72°C for 10 min; store at 4°C until removed, perform high-throughput sequencing on the PCR products, and record the genotype.

[0075] The flowering period and precocity of the cotton variety to be tested are determined according to the sequence of the amplified product: when the sequence +63-64bp of the amplified product is CA, the genotype of the cotton sample to be tested is FP_D03, and the cotton blooms late and the boll opening is also late; if there is a CA base deletion in the sequence +63-64bp of the amplified product, the genotype of the cotton sample to be tested is fp_D03, and the cotton blooms early and the boll opening is early; if the sequence of the amplified product is heterozygous, the genotype of the cotton sample to be tested is FP_D03 / fp_D03, and the flowering period and the boll opening period are between the two.

[0076] Table 130 cotton varieties genotype and flowering period survey results

[0077]

[0078]

[0079] Analysis of results: Thirty conventional varieties were screened with the molecular marker FP_D03_Indel. Based on genotype identification, it was found that the average flowering period of varieties containing the fp_D03 genotype was 59.89 days, while the average flowering period of varieties containing the FP_D03 genotype was 64.74 days (Table 1).

[0080] Example 2

[0081] Rapidly improve the early maturity of cotton varieties using FP_D03_Indel marker:

[0082] A single segment substitution line SL EF _ D03(SL: Substitution line; EF: Earlyflowering), which is the donor parent of the early flowering gene (♂), and the late flowering varieties (lines) Lumian 361, Lumian 392, Lumian 3218, Lu6269, Lumian 3036, Lumian 378 or Lu7619 are used as recurrent parents (♀), and the F1 generation is obtained by hybridization; 15 plants containing the fp_D03 marker genotype are selected from the F1 generation and backcrossed with the recurrent parents three times to obtain the BC3F1 generation; plants with the fp_D03 marker genotype are selected from the BC3F1 generation plants for 2 to 3 self-pollinations, and the FP_D03_Indel marker is used to identify the homozygous individual plants with the genotype of fp_D03 during the self-pollination process, and the homozygous individual plants are propagated for generations to obtain the early flowering line BC3F 2:3 .

[0083] Among them, the single segment replacement line SL EF _ D03 From the BC6F2 exchange plants containing the fp_D03 marker genotype obtained in Example 1, the exchange plant containing the least genomic component of the donor parent Lumianyan No. 19 was selected and self-crossed to BC6F 2:4 Obtained on behalf of.

[0084] In the method steps, the reaction system for amplifying the cotton sample to be tested using the FP_D03_Indel primer is 10 μL, specifically as follows:

[0085] Ultrapure water (ddH2O) 4.7μL, template DNA 1.0μL, 10×Buffer 1.0μL, 2.5mM MgCl2 1.0μL, 10mM dNTPs 0.2μL, 10μM forward primer 1.0μL, reverse primer 1.0μL, Taq DNA polymerase 0.1μL.

[0086] Amplification reaction procedure:

[0087] Preheat at 95°C for 5 min, denature at 94°C for 45 s, anneal at 55°C for 45 s, and extend at 72°C for 1 min; cycle 30 times, and extend at 72°C for 10 min; store at 4°C until removed, perform high-throughput sequencing on the PCR products, and record the genotype.

[0088] The flowering period of Lumian 361, Lumian 392, Lumian 3218, Lu6269, Lumian 3036, Lumian 378 and Lu7619 was investigated, and the results are shown in Table 2; at the same time, the flowering period of the cotton varieties improved by using Lumian 361, Lumian 392, Lumian 3218, Lu6269, Lumian 3036, Lumian 378 and Lu7619 as recurrent parents was investigated, and the results are shown in Table 2, and the flowering period comparison diagram of the recurrent parents and the improved cotton varieties is shown in Figure 2 As shown, Figure 2 ** indicates a difference of P less than 0.01.

[0089] Table 2 Flowering period of recurrent parents and their improved lines

[0090]

[0091] From Table 2 and Figure 2 It can be concluded that the method can stably replace the early-flowering genotype into the recurrent parent, and the method can advance the flowering period of cotton varieties by about 4 to 7 days.

[0092] It can be concluded from the above examples that the Indel markers described in the present invention can accurately identify the early or late flowering period and maturity (length of growth period) of cotton, improve the selection efficiency of early-maturing cotton varieties, and can be used for the breeding of early-maturing cotton varieties.

[0093] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cotton Indel marker FP_D03_Indel, characterized in that: The nucleotide sequence containing the cotton Indel marker FP_D03_Indel is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. Use of the cotton Indel marker FP_D03_Indel according to claim 1 in one or more of the following: 1) Early flowering cotton breeding; 2) Distinguish the flowering period of cotton; 3) Identify the flowering period of cotton.

3. A method for identifying the flowering period of cotton, characterized in that: The steps include: Using the primers shown in SEQ ID NO.3 and SEQ ID NO.4, PCR amplification is performed on the genomic DNA of the cotton sample to be tested to obtain an amplified product; Sequencing the amplified product to obtain the amplified product sequence; When the sequence of the amplified product is as shown in SEQ ID NO.1, the genotype of the cotton sample to be tested is homozygous FP_D03, and the cotton sample to be tested blooms late and opens bolls late; When the sequence of the amplified product is as shown in SEQ ID NO. 2, the genotype of the cotton sample to be tested is homozygous fp_D03, and the cotton sample to be tested blooms early and opens bolls early; When the sequence of the amplified product is as shown in SEQ ID NO.1 and SEQ ID NO.2, the genotype of the cotton sample to be tested is the heterozygous FP_D03 / fp_D03, and the flowering period and boll opening period of the sample to be tested are between the cotton varieties with the genotypes of FP_D03 and fp_D03.

4. The method according to claim 3, characterized in that The PCR amplification system includes: 4.7 μL of ultrapure water, 1.0 μL of genomic DNA, 1.0 μL of 10×Buffer, 1.0 μL of 2.5 mM MgCl2, 0.2 μL of 10 mM dNTPs, 1.0 μL of 10 μM forward primer, 1.0 μL of reverse primer, and 0.1 μL of TaqDNA polymerase.

5. The method according to claim 3 or 4, characterized in that: The PCR amplification procedure includes: preheating at 95°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 1 minute, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

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