A KASP molecular marker for detecting flowering and maturation trait genes in wheat and its application

Abstract

The present invention discloses a KASP molecular marker for detecting flowering and maturation trait genes of wheat and its application, which relates to the field of biotechnology. The primer set of the KASP molecular marker is primer set one and / or primer set two, and the specific sequences of primer set one and primer set two are given. At the same time, the KASP molecular marker is applied to wheat TaTPP‑ 7B The present invention develops a primer set based on wheat flowering and maturity traits, which can quickly and conveniently detect SNP sites and their superior haplotypes related to relevant traits, with the advantages of fast identification and high accuracy, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a KASP molecular marker for detecting flowering and maturation trait genes of wheat and an application thereof. Background Art

[0002] wheat( Triticum aestivum L Wheat is the world's largest cultivated crop, feeding approximately 3 billion people. Increasing and ensuring stable wheat yields are crucial measures for ensuring food security and improving people's living standards. The flowering and maturity stages of wheat are key factors influencing wheat yield formation and ecological adaptability. Flowering stage determines the number of wheat grains. Suitable environmental conditions during flowering (such as temperature, light, and moisture) are crucial for pollen viability, pollination efficiency, and fertilization success, ultimately affecting the number of grains per spike. Maturity, on the other hand, determines the degree of grain filling. During grain filling, the efficiency of photosynthetic product synthesis, transport, and accumulation in the grains directly influences thousand-kernel weight. Kernel number per spike and thousand-kernel weight are two key factors influencing the three key elements of wheat yield, influencing and determining wheat yield formation. Therefore, the different flowering and maturity stages of wheat varieties not only reflect wheat's adaptability to different light and temperature environments, but also jointly determine its yield potential and stability, making them an essential and core component of the yield formation process.

[0003] However, the current global climate change has led to temperature fluctuations and frequent extreme weather events, significantly increasing the risk of environmental stress (such as high temperatures and drought) during the flowering and maturity stages of wheat. This not only affects pollen viability, pollination efficiency, and grain number formation during flowering, but also induces the accumulation of reactive oxygen species (ROS), accelerates leaf senescence, shortens grain filling duration, and reduces grain plumpness, becoming a significant factor limiting yield potential. Under existing wheat cultivation techniques, management conditions, and climatic conditions, highly adaptable wheat varieties typically exhibit early flowering and early maturity (e.g., higher expression levels of heat shock proteins, stronger activity of antioxidant enzyme systems (such as SOD and CAT), and optimized carbohydrate distribution efficiency). Therefore, they can help wheat avoid adverse environmental stresses such as hot, dry winds and high temperatures during flowering and maturity, maintaining pollen viability and a high grain filling rate and duration under adverse conditions, thereby significantly ensuring food production and security.

[0004] In summary, the adaptive mechanisms of flowering and maturity in different wheat varieties are complex traits formed through long-term natural selection and artificial breeding. Optimizing these traits is crucial for improving wheat's yield potential and stability in diverse ecological environments. However, there are currently few reports on the detection and identification of flowering and maturity traits in wheat, and these detection and identification are often difficult and inaccurate. This has significantly limited the efficiency of assisted breeding for flowering and maturity traits and has hampered the development of the wheat industry. Therefore, in-depth research on the physiological mechanisms of these two stages and their interactions with the environment, as well as further exploring and utilizing these adaptive traits through genetic improvement and cultivation management, are important strategies for addressing future climate change challenges. This background provides an important theoretical basis and practical direction for related research and technological development. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a KASP molecular marker for detecting wheat flowering and maturity trait genes and its application. Based on the wheat flowering and maturity traits, a related primer set is developed, which can quickly and conveniently detect SNP sites and excellent haplotypes of related traits. It has the advantages of fast identification and high accuracy, and has good application prospects.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing a KASP molecular marker for detecting wheat flowering and maturation trait genes, and the primer set for detecting and identifying SNPs and haplotypes of the KASP molecular marker is:

[0007] Primer set 1, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively;

[0008] and / or,

[0009] Primer set 2 consists of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0010] Furthermore, the primer set of KASP molecular markers is used to identify early flowering / early maturing / short stem, or late flowering / late maturing / tall stem, of wheat.

[0011] Furthermore, the polymorphism of the +806 SNP1 site corresponding to primer set 1 is T / C, and the polymorphism of the -701 SNP2 site corresponding to primer set 2 is A / G.

[0012] Furthermore, the wheat flowering and maturation trait gene is TaTPP-7B The gene, whose nucleotide sequence is shown in SEQ ID NO.7.

[0013] The present invention also provides the KASP molecular marker for detecting wheat flowering and maturation trait genes in wheat. TaTPP- 7B Detection, identification, and auxiliary identification of SNPs and excellent haplotypes of genes for wheat flowering and maturity traits or production applications in molecular marker-assisted breeding.

[0014] The present invention also provides a method for detecting flowering and maturation traits of wheat, comprising the following steps:

[0015] (1) Extracting genomic DNA from the wheat material to be tested;

[0016] (2) Using the genomic DNA as a template, PCR amplification is performed using the above-mentioned primer set 1 and / or primer set 2 to obtain a PCR amplification product;

[0017] (3) Compare the PCR amplification products; if the genotypes of the SNP1 site at +806 bp in the 3' UTR region and the SNP2 site at -701 bp in the promoter region in the genomic DNA are TT and AA respectively, TaTPP-7B The genes were identified as SNP1 TT, SNP2 AA and haplotype HapⅠ - T TCAC A AC, the wheat traits candidates to be tested are early flowering, early maturity and dwarf wheat; if the genotypes of the SNP1 site at +806 bp in the 3' UTR region and the SNP2 site at -701 bp in the promoter region in the genomic DNA are CC and GG respectively, TaTPP-7B The genes in the tested wheat were identified as SNP1 CC, SNP2 GG and haplotype HapⅡ - C ATTT G GT, the wheat trait candidates to be tested are late flowering, late maturity and tall stem wheat.

[0018] The present invention also provides a kit for detecting flowering and maturation traits of wheat, comprising the KASP molecular marker for detecting flowering and maturation trait genes of wheat.

[0019] The present invention has the following beneficial effects:

[0020] The present invention develops relevant primer sets based on the flowering and maturation traits of wheat, which can quickly and conveniently detect relevant traits, has the advantages of fast identification and high accuracy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the KASP genotyping results for the allelic SNP1 variant site;

[0022] Figure 2Schematic diagram of the KASP genotyping partial identification results of the allelic SNP2 variant site. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0024] Example 1

[0025] 1. Wheat materials for testing: Multiple wheat materials with large differences in flowering and maturation traits distributed in different wheat regions of my country were selected (see Table 2 for specific material information) as materials for discovering polymorphic loci.

[0026] 2. Sequence alignment: Perform the following operations on each wheat tested:

[0027] (1) Extracting genomic DNA from the wheat material to be tested;

[0028] (2) Using genomic DNA as a template, PCR amplification was performed using primer set 1 and / or primer set 2. The amplification procedure was as shown in Table 1 to obtain PCR amplification products. Primer set 1 consisted of three primers whose nucleotide sequences were shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; primer set 2 consisted of three primers whose nucleotide sequences were shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0029] (3) Clone and sequence the PCR amplified products; and perform sequence splicing and alignment.

[0030] Table 1 PCR amplification program

[0031]

[0032] The clone sequencing results of each wheat material were spliced and compared with the primer set 1 and primer set 2 respectively, and two SNP changes were found in the genomic PCR amplification products of different test wheats with primer set 1 and primer set 2.

[0033] Based on the sequence alignment of the PCR amplification products of all the tested wheats using primer set 1 and primer set 2, it was found that the polymorphism of SNP1 in primer set 1 was T / C, and the polymorphism of SNP2 in primer set 2 was A / G, which were named +806 SNP1 and -701 SNP2, respectively.

[0034] The genotypes of each test material based on +806 SNP1 and -701 SNP2 are shown in Table 2.

[0035] Table 2 Genotypes of the tested materials

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] Among them, in Table 2, the haplotype composed of SNP1 TT and SNP2 AA HapⅠ The genotypes of the wheat materials tested are T TCAC A AC; haplotype consisting of SNP1 CC and SNP2 GG HapⅡ The genotypes of the wheat materials tested are C ATTT G GT; further, HapⅠ The haplotype represents the wheat material with early flowering, early maturity and short stem traits; HapⅡ The haplotype represents that the tested wheat material has the traits of late flowering, late maturity and tall stems.

[0044] As shown in Table 2, the allelic variation at the +806 SNP1 site and the -701 SNP2 site can form two haplotypes in both local varieties and selected varieties in the 262 Mini-Core Collection of my country. Among local varieties, those representing early flowering, early maturity, and dwarf traits have excellent haplotypes. HapⅠ Wheat varieties with late flowering, late maturity and tall stem traits account for only 76.09% of the 262 micro-core germplasms in my country, while HapⅡ The haplotype accounted for 23.91%; in the selected varieties, the proportion of excellent haplotypes in each major wheat region increased to 91.35%, and HapⅡ It dropped to 8.65%, indicating that in the long-term artificial breeding process in my country, TaTPP-7B The superior haplotypes of the gene have been strongly positively selected in all major wheat regions.

[0045] Example 2

[0046] Tissue sample testing:

[0047] 1. Extract genomic DNA from the tested wheat materials;

[0048] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primer set 1 and / or primer set 2. The amplification procedure is shown in Table 1. Primer set 1 consists of three primers whose nucleotide sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively; primer set 2 consists of three primers whose nucleotide sequences are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

[0049] PCR amplification was performed on the QuantStudio 7 instrument produced by ABI, and the genotyping results were automatically output. Figure 1 and Figure 2 shown.

[0050] The test materials were planted in Luoyang (2002, 2005, and 2006) and Shunyi (2010) under conventional water and fertilizer management. The results of association analysis for the local varieties of the test wheat genotypes are shown in Table 3, and the results of association analysis for the selected varieties are shown in Table 4. In Tables 3 and 4, 2002LY represents Luoyang (2002); 2005LY represents Luoyang (2005); 2006LY represents Luoyang (2006); and 2010SY represents Shunyi (2010). Data were statistically analyzed using the Tukey test, with significance levels set at 0.05 or 0.01 (* P <0.05,** P <0.01).

[0051] Table 3 TaTPP-7B Association analysis of important agronomic traits among different haplotypes in 262 local varieties in my country

[0052]

[0053] Table 3 shows that among the six agronomic traits of local varieties, namely, heading date (HD), maturity date (MD), plant height (PH), thousand kernel weight (TKW), grain number (GN), and spike length (SL), the two haplotypes showed statistically significant differences only in flowering date and maturity date. HapⅠ Compare HapⅡFlowering was earlier by 4.2 days (2006), 2.7 days (2005), 6 days (2002) and 2.7 days (2010); and maturity was earlier by 4.7 days (2006), 2.8 days (2005), 5.2 days (2002) and 2.8 days (2010). The differences were extremely significant or significant in all four years of testing (* P <0.05,** P <0.01); while plant height traits, although HapⅠ Plant height ratio HapⅡ The decreases were 2.1 cm (2006), 2.3 cm (2005), 2.1 cm (2002) and 2.8 cm (2010), respectively, but the differences were not statistically significant, indicating that TaTPP-7B The genes are mainly closely related to controlling the flowering and maturity traits of wheat.

[0054] Table 4 TaTPP-7B Association analysis of important agronomic traits among different haplotype types in 262 bred varieties in my country

[0055]

[0056] As shown in Table 4, among the selected varieties, TaTPP-7B The change trend of the two haplotypes of the gene is the same as that of the local varieties. Among the six agronomic traits of heading date, maturity date, plant height, 1000-grain weight, number of grains per ear, and ear length, the two haplotypes showed statistically significant differences only in the two traits of flowering date and maturity date (supported by at least 3 years of average data). HapⅠ Compare HapⅡ Flowering was earlier by 7.8 days (2006), 4.5 days (2005), 4.8 days (2002) and 4.5 days (2010); and maturity was earlier by 6.1 days (2006), 4.0 days (2005), 5.2 days (2002) and 4.0 days (2010). The differences were extremely significant or significant in the three to four-year tests (* P <0.05,** P <0.01); plant height traits, HapⅠ Compare HapⅡ The plant heights of the 2006, 2005, 2002, and 2010 years decreased by 20.2 cm, 4.8 cm, 23.9 cm, and 11.6 cm, respectively, reaching a statistically significant level in 2006 and 2002 (** P<0.01), although there were two years without reaching the statistical difference level, but the change trend was exactly the same as the change trend of plant height of local varieties over the four years, and the plant height of selected varieties varied from 4.8 to 23.9 cm. In actual production, this plant height difference has reached an extremely significant level. These research results show that TaTPP-7B In addition to controlling the flowering and maturity periods of wheat, the gene is also closely related to the plant height trait of wheat.

[0057] In summary, +806 SNP1, -701 SNP2, and the two haplotypes formed by the two allele variant sites of +806 SNP1 and -701 SNP2 are all significantly associated with the flowering period, maturity period, and plant height of wheat; the KASP molecular marker developed from this can be used to detect the flowering period, maturity period, and plant height of different wheat varieties. TaTPP-7B The KASP molecular markers can be used to identify allelic variants (SNPs) and / or haplotypes associated with flowering, maturity, and plant height. Furthermore, superior allelic variants or haplotypes of these genes can be used in marker-assisted breeding. These KASP molecular markers enable breeders to quickly identify wheat germplasm carrying early flowering, early maturity, and dwarf traits and apply them to genetic improvement. Therefore, this invention can accelerate the genetic improvement of wheat varieties and improve breeding efficiency. It also provides strong technical support for the development of high-yield, high-quality wheat varieties adapted to diverse ecological environments and cultivation requirements.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of a KASP molecular marker identification primer set for detecting wheat flowering and maturation trait genes in identifying wheat flowering and maturation traits, characterized in that: The flowering and maturation trait of wheat is at least one of heading period and maturity period; the heading period trait is early flowering or late flowering, and the maturity period trait is early maturity or late maturity; The KASP molecular marker is located in the wheat flowering and maturation trait gene TaTPP-7B The 1570th and 5188th positions of the gene nucleotide sequence, TaTPP-7B The nucleotide sequence of the gene is shown in SEQ ID NO.7; based on TaTPP-7B The genotype of the SNP1 site at position 1570 of the gene nucleotide sequence is TT genotype or CC genotype, based on TaTPP-7B The genotype of the SNP2 site at position 5188 of the gene nucleotide sequence is an AA genotype or a GG genotype; when the genotypes of the SNP1 site and the SNP2 site are TT and AA respectively, the flowering and maturity traits of the wheat are at least one of early flowering and early maturity; when the genotypes of the SNP1 site and the SNP2 site are CC and GG respectively, the flowering and maturity traits of the wheat are at least one of late flowering and late maturity; The identification primer set for detecting the KASP molecular marker for wheat flowering and maturation trait genes is: Primer set 1, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; and primer set 2, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. A method for detecting flowering and maturation traits of wheat, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the wheat material to be tested; (2) Using genomic DNA as a template, PCR amplification was performed using primer set 1 and primer set 2 to obtain a PCR amplification product; The primer set 1 consists of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the primer set 2 consists of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively; The flowering and maturation trait of wheat is at least one of heading period and maturity period; the heading period trait is early flowering or late flowering, and the maturity period trait is early maturity or late maturity; (3) Compare the PCR amplification products; if the PCR amplification product in the genome to be tested is relative to that shown in SEQ ID NO.7 TaTPP-7B The genotypes of the SNP1 site at position 1570 and the SNP2 site at position 5188 of the gene nucleotide sequence are TT and AA respectively, and the wheat trait candidates to be tested are early flowering and early maturing wheat; if the PCR amplification product in the genome to be tested is relative to the genotype shown in SEQ ID NO.7 TaTPP-7B The genotypes of the SNP1 site at position 1570 and the SNP2 site at position 5188 of the gene nucleotide sequence are CC and GG, respectively. The candidate wheat traits to be tested are late flowering and late maturing wheat.

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