A KASP molecular marker for regulating the number of grains per spike and plant height genes in wheat and its application

Abstract

The present invention discloses a KASP molecular marker for regulating wheat grain number and plant height genes and its application, which relates to the field of biotechnology; the primer set of the KASP molecular marker for identifying wheat grain number and plant height genes comprises three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively. The present invention also applies the KASP molecular marker to wheat <h2 style=";text-align:left;direction:ltr">TaTPP-7D Detection, identification, and auxiliary identification of gene SNP allele variation in wheat grain number and plant height traits or molecular marker-assisted breeding. The present invention develops relevant primer sets based on wheat grain number and plant height traits for detecting SNP allele variation sites of related traits. This has the advantages of being quick and convenient, with fast identification speed and high accuracy, improving wheat breeding efficiency, promoting the development of the wheat industry, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a KASP molecular marker for regulating wheat grain number and plant height genes and an application thereof. Background Art

[0002] wheat( Triticum aestivum L. ) is one of the most important staple food crops in the world, feeding about 3 billion people worldwide. Continuously increasing wheat production is a rigid demand to cope with the future global population explosion and climate warming, as well as to ensure national food security. The number of grains per spike and plant height are two important agronomic traits that constitute and affect the formation of wheat yield. Among them, the number of grains per spike is one of the three elements that constitute wheat yield. Its genetic regulation mechanism is complex, controlled by multiple genes and easily affected by environmental factors. Studies have shown that the number of grains per spike is directly related to the grain yield per unit area, thus making a huge contribution to the increase in yield; while plant height affects the photosynthetic efficiency, nutrient distribution and lodging resistance of crops, and is an important target trait in wheat plant breeding. In the 1960s, the wheat dwarf gene Rht-B1 and Rht-D1 The discovery and application of has brought a new "green revolution" to wheat production and become a milestone in the history of wheat breeding. Its application in production has significantly improved the crop's resistance to lodging and achieved a significant breakthrough in yield. However, excessive dwarfing may lead to insufficient biomass and limited yield potential. In addition, scientists have identified several key genes related to grain number per spike through genome-wide association analysis (GWAS), quantitative trait loci (QTL) mapping, and transcriptomics. For example, TaGW2 、 TaGNI and TaCKX However, the molecular regulatory mechanisms and networks underlying these genes' involvement in regulating biological processes such as spikelet meristem formation, inflorescence development, and grain filling remain incompletely understood. For a long time, genes regulating grain number per spike and plant height have been studied and reported independently, but there have been no reports on genetic resources that jointly regulate both traits. Therefore, identifying and utilizing novel genetic resources that jointly regulate grain number per spike and plant height in wheat is crucial for increasing wheat yield (increasing grain number per unit area), optimizing plant architecture (achieving moderate plant height), enhancing environmental adaptability (lodging resistance), and cultivating wheat varieties with ideal plant types and high yield potential.

[0003] Historical practice has shown that varieties can contribute more than 40% to increasing crop yields, and the discovery and use of some key genes have played an important role in promoting the genetic improvement of wheat yields. Rht1 and Rht2Therefore, the present invention's exploration and utilization of genes for grain number per ear and plant height is no longer limited to improving a single trait. Instead, it achieves the coordinated optimization of traits through "one factor, multiple effects," thus achieving the goal of multi-gene aggregation breeding. For example, combining high-yield genes with dwarfing genes can cultivate high-yield varieties that are both high-yielding and resistant to lodging.

[0004] In summary, the discovery and utilization of genes encoding wheat grain number and plant height are key approaches to increasing wheat yield, optimizing wheat plant architecture, and enhancing wheat's environmental adaptability. This has far-reaching implications for ensuring global food security and promoting sustainable agricultural development. However, due to the large size of the wheat genome, the abundance of repetitive sequences, and the complex genetic background and regulatory networks, the genes and their regulatory mechanisms that jointly regulate wheat grain number and plant height remain relatively scarce. Consequently, the discovery and detection of these genetic resources remain challenging, significantly limiting the efficiency of assisted breeding for traits such as grain number and plant height, and hindering the development of the wheat industry. Therefore, the development of molecular markers capable of identifying wheat grain number and plant height traits is of great significance for high-yield wheat breeding and genetic improvement of varieties. 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 regulating wheat grain number and plant height genes and its application. Based on the wheat grain number and plant height traits, a related primer set is developed to detect SNP allele variation sites of related traits. It has the advantages of being quick and convenient, fast identification speed and high accuracy, which improves the breeding efficiency of wheat, promotes the development of the wheat industry, and has broad application prospects.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a KASP molecular marker for identifying genes that regulate wheat grain number per spike and plant height is provided. The KASP molecular marker is used to identify a primer set for genes that regulate wheat grain number per spike and plant height, and consists of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0007] Furthermore, the primer set of the KASP molecular marker is used to identify or detect the wheat traits of high number of grains per ear / short stem / short ear length, or low number of grains per ear / tall stem / long ear length.

[0008] Furthermore, wheat grain number and plant height genes are TaTPP-7D The gene, whose nucleotide sequence is shown in SEQ ID NO.4.

[0009] Furthermore, the polymorphism of the SNP site corresponding to the primer set is T / C.

[0010] Furthermore, the primer set is used to detect whether the genotype of the SNP site based on the promoter region -1021 bp in the wheat genomic DNA to be tested is CC or TT genotype.

[0011] The present invention also provides the KASP molecular marker for regulating wheat grain number and plant height genes in wheat. TaTPP- 7D Detection, identification, and assisted identification of SNP allele variations in wheat grain number and plant height traits, or their application in molecular marker-assisted breeding.

[0012] The present invention also provides a method for detecting wheat grain number and plant height, which is characterized by comprising the following steps:

[0013] (1) Extracting genomic DNA from the wheat material to be tested;

[0014] (2) Using genomic DNA as a template, PCR amplification was performed using the above primer set to obtain a PCR amplification product;

[0015] (3) Compare the PCR amplification products; if the genotype of the SNP site based on the promoter region -1021 bp in the genomic DNA is CC genotype, TaTPP-7D The gene was identified as SNP CC in the wheat to be tested, and the candidate traits of the wheat to be tested are large number of grains per spike, short stem and short spike length; if the genotype of the SNP site based on the promoter region -1021 bp in the genomic DNA is TT genotype, TaTPP-7D The gene is identified as SNP TT in the wheat to be tested, and the candidate traits of the wheat to be tested are small number of grains per ear, tall stems and long ears.

[0016] The present invention also provides a kit for detecting wheat grain number per spike and plant height traits, comprising the KASP molecular markers for regulating wheat grain number per spike and plant height genes.

[0017] The present invention has the following beneficial effects:

[0018] The present invention develops relevant primer sets based on wheat grain number and plant height traits, which are used to detect SNP allele variation sites of relevant traits. It has the advantages of being quick and convenient, fast identification speed and high accuracy, improves wheat breeding efficiency, promotes the development of the wheat industry, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the KASP genotyping results for the allelic SNP variant sites. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1

[0022] 1. Wheat materials for testing: Multiple wheat materials with large differences in the number of grains per ear and plant height traits distributed in different wheat regions of my country were selected (see Table 2 for specific material information) as materials for the discovery of polymorphic loci.

[0023] 2. Sequence alignment: Perform the following operations on each wheat tested:

[0024] (1) Extracting genomic DNA from the wheat material to be tested;

[0025] (2) Using genomic DNA as a template, PCR amplification was performed using a primer set. The amplification procedure was as shown in Table 1 to obtain a PCR amplification product. The primer set consisted of three primers whose nucleotide sequences were shown as SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively.

[0026] (3) Clone and sequence the PCR amplified products; and perform sequence splicing and alignment.

[0027] Table 1 PCR amplification program

[0028]

[0029] The clone sequencing results of each wheat material were subjected to genome sequence splicing and comparison analysis, and it was found that there were two types of genome PCR amplification products in different test wheats.

[0030] Based on the sequence alignment of the PCR amplification products of all tested wheat varieties using the primer set, it was found that the polymorphism of the SNP corresponding to the primer set was T / C, which was named -1021 SNP.

[0031] The genotypes of each test material based on the -1021 SNP are shown in Table 2.

[0032] Table 2 Genotypes of the tested materials

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] In Table 2, CC indicates that the genotype of the SNP site based on the promoter region -1021 bp in the genomic DNA is the CC genotype. TaTPP-7D The gene was identified as SNP CC in the wheat to be tested, and the wheat to be tested has the traits of large number of grains per spike, short stem and short spike length; TT indicates that the genotype of the SNP site based on the promoter region -1021 bp in the genomic DNA is the TT genotype, TaTPP-7D The gene was identified as SNP TT in the test wheat, and the test wheat has the traits of small number of grains per ear, tall stem and long ear length.

[0041] As shown in Table 2, for both local varieties and selected varieties, the allelic variation of the -1021 SNP locus has two main polymorphisms, T / C, in my country's 262 mini-core germplasm (Mini-Core Collection). Among local varieties, wheat varieties with the SNP CC genotype, which represents the traits of many grains per spike, short stems and short spike length, account for only 0.65% of my country's 262 mini-core germplasm, while wheat varieties with the SNP TT genotype, which represents the traits of few grains per spike, tall stems and long spike length, account for 99.35% of my country's 262 mini-core germplasm. Among selected varieties, the proportion of wheat varieties with the SNP CC genotype, which represents the traits of many grains per spike, short stems and short spike length, in China's 262 mini-core germplasm increased to 22.86%, while the proportion of wheat varieties with the SNP TT genotype, which represents the traits of few grains per spike, tall stems and long spike length, in my country's 262 mini-core germplasm decreased to 77.14%. This shows that different wheat-growing areas have different effects on the quality of wheat. TaTPP-7D The preferred breeding traits of genes are different. Generally speaking, in the 1960s and 1970s, major wheat regions tended to select CC genotypes, which have the traits of high number of grains per spike and short stem, thus greatly improving the yield of wheat in my country. However, this genotype also has the disadvantage of short spikes, which limits its further selection and utilization in breeding. So far, most wheat regions still have CC genotypes. TaTPP-7D In terms of gene breeding selection, the long-ear type is still preferred. In summary, in the long-term artificial breeding process in my country, TaTPP-7D Different genotypes of the gene have been subjected to strong artificial selection in major wheat-growing areas in my country, thus providing reliable genetic resources for the development of my country's wheat seed industry.

[0042] Example 2

[0043] Tissue sample testing:

[0044] 1. Extract genomic DNA from the tested wheat materials;

[0045] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using a primer set; the amplification procedure is shown in Table 1; the primer set consists of three primers whose nucleotide sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3, respectively;

[0046] PCR amplification was performed on the QuantStudio 7 instrument produced by ABI, and the genotyping results were automatically output. Figure 1 shown.

[0047] 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). The least significant difference method was used for statistical analysis, with a significance level of 0.05 or 0.01 (* P <0.05,** P <0.01).

[0048] Table 3 TaTPP-7D Association analysis of gene SNP allelic variation with important agronomic traits in 262 local varieties in my country

[0049]

[0050] Table 3 shows that among the six agronomic traits of local varieties, including grain number per spike (GN), plant height (PH), thousand kernel weight (TKW), spike length (SL), heading date (HD), and maturity date (MD), SNP alleles associated with these traits showed statistically significant differences only in grain number per spike, plant height, and thousand kernel weight. The SNP CC genotype had 9.2 more grains per spike (2006), 10.1 more (2002), and 5.7 more (2010) than the SNP TT genotype, respectively. Plant height was 27.0 cm (2006), 28.2 cm (2002), 26.5 cm (2010), and 27.5 cm (2005), respectively. The differences in grain number per spike across years were highly significant or significant for at least three years (except 2005) (* P <0.05,** P <0.01, while the differences in plant height were extremely significant or significant in all four years of data (* P <0.05,** P <0.01); the results showed that TaTPP-7D The genes are closely related to controlling the number of grains per ear and plant height of wheat.

[0051] Table 4 TaTPP-7D Association analysis of gene SNP allelic variation with important agronomic traits in 262 bred varieties in my country

[0052]

[0053] As shown in Table 4, among the selected varieties, TaTPP-7D The changing trends of the SNP CC and SNP TT genotypes corresponding to the gene are also the same as those of local varieties. Among the six agronomic traits, including number of grains per ear, plant height, 1000-grain weight, ear length, heading period, and maturity period, only the number of grains per ear and plant height showed statistically significant differences. Among them, the SNP CC genotype had 7.8 more grains per ear (2006), 6.5 more grains (2002), 6.1 more grains (2010), and 5.95 more grains (2005) than the SNP TT genotype; the plant height was 7.4 cm (2006), 12.7 cm (2002), 7.7 cm (2010), and 12.8 cm (2005) higher, respectively. The above differences in number of grains per ear and plant height were extremely significant or significant in all four years (* P <0.05,** P <0.01), and its changing trend is the same as that of local varieties. The above research results show that among the selected varieties,TaTPP-7D The gene is also closely related to controlling the number of grains per ear and plant height of wheat.

[0054] In summary, the genotypes formed by the -1021 SNP allele variation site are significantly associated with the number of grains per spike and plant height in wheat; and the KASP molecular marker developed from this can be used to detect the genotypes in different wheat varieties. TaTPP-7D The SNP polymorphism associated with the gene for kernel number per spike and plant height can also be further applied to molecular marker-assisted breeding. Using this KASP molecular marker, breeders can quickly identify wheat germplasm with traits such as high kernel number per spike, short stems, or long spike length, and apply this to genetic improvement. Therefore, this invention can accelerate the genetic improvement of wheat varieties, improve breeding efficiency, and provide strong technical support for cultivating high-yield, high-quality wheat varieties that are adaptable to different ecological environments and cultivation requirements.

[0055] 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. Used to detect wheat TaTPP-7D The application of KASP molecular markers for SNP allele variation of genes in identifying wheat grain number and plant height traits is characterized by: The wheat TaTPP-7D The nucleotide sequence of the gene is shown in SEQ ID NO.

4. The KASP molecular marker is a wheat TaTPP-7D The SNP site at position 5384 of the gene nucleotide sequence is a CC genotype or a TT genotype.

2. The use according to claim 1, characterized in that The primer set for detecting the KASP molecular marker consists of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

3. The use according to claim 2, characterized in that The primer set is used to detect whether the genotype of the SNP site at position 5384 based on the nucleotide sequence shown in SEQ ID NO. 4 in the genomic DNA of the wheat to be tested is the CC genotype or the TT genotype.

4. A method for detecting wheat grain number and plant height, 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 and employing the primer set described in claim 2 to perform PCR amplification to obtain a PCR amplification product; (3) Compare the PCR amplification products; if the genotype of the SNP site at position 5384 based on the nucleotide sequence shown in SEQ ID NO.4 in the genomic DNA is CC genotype, TaTPP-7D The gene is identified as SNP CC in the wheat to be tested, and the candidate traits of the wheat to be tested are large number of grains per spike, short stem, and short spike length; if the genotype of the SNP site at position 5384 based on the nucleotide sequence shown in SEQ ID NO.4 in the genomic DNA is TT genotype, TaTPP-7D The gene is identified as SNP TT in the wheat to be tested, and the candidate traits of the wheat to be tested are small number of grains per ear, tall stems and long ears.

5. Use of the primer set for detecting the KASP molecular marker according to claim 2 in preparing a kit for detecting wheat grain number and plant height traits.

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