KASP molecular marker for regulating wheat grain number per ear and plant height gene and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for regulating wheat grain number and plant height genes and application of the KASP molecular marker, and relates to the technical field of biology. A primer group of the KASP molecular marker for identifying and regulating wheat grain number and plant height genes is composed of three primers with nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively. The invention also applies the KASP molecular marker to detection and identification of SNP allelic variation of the wheat TaTPP-7D gene, and assists in identification of grain number and plant height characters per ear of wheat or molecular marker assisted breeding. The related primer group is developed on the basis of the grain number per ear and the plant height character of the wheat, is used for detecting SNP allelic variation sites of the related character, has the advantages of rapidness, convenience, high identification speed, high accuracy and the like, improves the breeding efficiency of the wheat, promotes the development of the wheat industry, and has a wide application prospect.

Description

Technical Field

[0001] The 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 requirement to cope with the future global population explosion and climate warming as well as to ensure national food security. The number of grains per ear and plant height are two important agronomic traits that constitute and affect the formation of wheat yield. Among them, the number of grains per ear 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 ear 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 the crop, 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 use 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 spikelet number through genome-wide association analysis (GWAS), quantitative trait loci (QTL) mapping, and transcriptomics, such as TaGW2 , TaGNI and TaCKX etc. However, the molecular regulatory mechanisms and networks of biological processes such as spikelet meristem formation, inflorescence development and grain filling regulated by these genes are not yet fully understood. For a long time, the regulatory genes of spikelet number and plant height traits have been studied and reported separately, and there have been no reports on the research of genetic resources that combine the two. Therefore, it is crucial to explore and utilize new genetic resources that jointly regulate wheat spikelet number and plant height to increase wheat yield (increase the number of spikelets per unit area), optimize plant structure (moderate plant height), enhance environmental adaptability (resistance to lodging), and cultivate ideal plant type wheat varieties with 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 mining and utilization of the genes for the number of grains per ear and plant height in the present invention is no longer limited to the improvement of a single trait, but rather achieves the coordinated optimization of traits through "one factor with multiple effects" and achieves the purpose of multi-gene aggregation breeding. For example, high-yield genes are combined with dwarfing genes to cultivate high-yield and lodging-resistant varieties.

[0004] In summary, the mining and utilization of wheat grain number and plant height genes are key ways to increase wheat yield, optimize wheat plant type and enhance wheat environmental adaptability, which has far-reaching significance for ensuring global food security and promoting sustainable agricultural development. However, due to the large size of the wheat genome, the large number of repetitive sequences, the complex genetic background and regulatory network, there are still few reports on the relevant genes and their regulatory mechanisms that jointly regulate wheat grain number and plant height. Therefore, the mining and detection and identification of such gene resources are still very difficult, which greatly limits the efficiency of auxiliary breeding selection for wheat grain number and plant height and restricts the development of wheat planting industry. Therefore, the development of molecular markers that can identify wheat grain number and plant height traits is of great significance for high-yield breeding of wheat 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, which has the advantages of being quick and convenient, fast identification speed and high accuracy, 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 regulating wheat grain number per ear and plant height is provided. The KASP molecular marker is used for a primer set for identifying genes regulating wheat grain number per ear and plant height, and is composed 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, the wheat grain number and plant height genes are TaTPP-7D The gene has a nucleotide sequence as 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 that the genotype of the SNP site based on -1021 bp in the promoter region in the tested wheat genomic DNA is CC or TT genotype.

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

[0012] The present invention also provides a method for detecting wheat grain number and plant height traits, characterized in that it comprises the following steps: (1) Extracting genomic DNA from the wheat material to be tested; (2) using the genomic DNA as a template and the above primer set to perform PCR amplification to obtain a PCR amplification product; (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 ear, short stems and short ear 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.

[0013] The present invention also provides a kit for detecting wheat grain number per ear and plant height traits, comprising the KASP molecular marker for regulating wheat grain number per ear and plant height genes.

[0014] The present invention has the following beneficial effects: 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, and has the advantages of being quick and convenient, fast identification speed and high accuracy, which improves wheat breeding efficiency, promotes the development of the wheat industry, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the KASP genotyping partial identification results of allelic SNP variant sites. DETAILED DESCRIPTION

[0016] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0017] Example 1 1. Test wheat materials: Select multiple wheat materials distributed in different wheat regions in my country with large differences in the number of grains per ear and plant height (see Table 2 for specific material information) as materials for the discovery of polymorphic loci.

[0018] 2. Sequence alignment: Perform the following operations on each wheat tested: (1) Extracting genomic DNA from the wheat material to be tested; (2) using the genomic DNA as a template and using a primer set to perform PCR amplification, the amplification procedure is shown in Table 1, and a PCR amplification product is obtained; 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; (3) Clone and sequence the PCR amplification products; and perform sequence splicing and alignment.

[0019] Table 1 PCR amplification program

[0020] 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.

[0021] Based on the sequence alignment of the PCR amplification products of all tested wheats of 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.

[0022] The genotypes of the tested materials based on -1021 SNP are shown in Table 2.

[0023] Table 2 Genotypes of the tested materials

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] In Table 2, CC means 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 had the traits of large number of grains per ear, short stems and short ear length; TT means 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 had the traits of small number of grains per ear, tall stems and long ears.

[0031] As shown in Table 2, no matter it is a local variety or a selected variety, 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 the local varieties, wheat varieties representing the traits of more grains per ear, short stems and short ear length, namely the SNP CC genotype, account for only 0.65% of my country's 262 mini-core germplasm, while wheat varieties representing the traits of fewer grains per ear, tall stems and long ear length, namely the SNP TT genotype, account for 99.35% of my country's 262 mini-core germplasm. Among the selected varieties, wheat varieties representing the traits of more grains per ear, short stems and short ear length, namely the SNP CC genotype, account for 22.86% of my country's 262 mini-core germplasm, while wheat varieties representing the traits of fewer grains per ear, tall stems and long ear length, namely the SNP TT genotype, account for 77.14% of my country's 262 mini-core germplasm. This shows that different wheat-growing areas have different effects on wheat production. 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, i.e., more spikelets and shorter stems, which greatly increased the yield of wheat in my country. However, this genotype also has the disadvantage of short spikelets, which limits its further selection and utilization in breeding. So far, most wheat regions still prefer 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.

[0032] Example 2 Tissue sample testing: 1. Extract genomic DNA from the tested wheat materials; 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; PCR amplification was performed on the QuantStudio 7 instrument produced by ABI, and the genotyping results were automatically output. Figure 1 shown.

[0033] The test materials were planted in Luoyang (2002, 2005, 2006) and Shunyi (2010) under conventional water and fertilizer management. The results of association analysis of local varieties in the test wheat materials of each genotype are shown in Table 3, and the results of association analysis of selected varieties are shown in Table 4. In Tables 3 and 4, 2002LY means Luoyang (2002); 2005LY means Luoyang (2005); 2006LY means Luoyang (2006); 2010SY means Shunyi (2010). The least significant difference method was used in the data statistical software, and the significant difference level was 0.05 or 0.01 (* P <0.05,** P <0.01).

[0034] Table 3 TaTPP-7D Association analysis of gene SNP allele variation with important agronomic traits in 262 local varieties in my country

[0035] As shown in Table 3, among the six agronomic traits of local varieties, namely, grain number (GN), plant height (PH), thousand kernel weight (TKW), spike length (SL), heading date (HD) and maturity date (MD), the SNP allele variation-associated traits only showed statistically significant differences in grain number, plant height and thousand kernel weight. Among them, the number of grains per ear of SNP CC genotype was 9.2 more (2006), 10.1 more (2002) and 5.7 more (2010) than that of SNP TT genotype; the plant height was 27.0 cm (2006), 28.2 cm (2002), 26.5 cm (2010) and 27.5 cm (2005) higher respectively; the difference in grain number per ear in each year reached extremely significant or significant levels 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 show that TaTPP-7D The genes are mainly closely related to controlling the number of grains per ear and plant height of wheat.

[0036] Table 4 TaTPP-7D Association analysis of gene SNP allelic variation with important agronomic traits in 262 bred varieties in my country

[0037] It can be seen from Table 4 that 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 the 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 number of grains per ear of the SNP CC genotype was 7.8 grains more (2006), 6.5 grains (2002), 6.1 grains (2010) and 5.95 grains (2005) than that of 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 the 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 genes are also closely related to controlling the number of grains per ear and plant height of wheat.

[0038] In summary, the genotypes formed by the -1021 SNP allele variation site are significantly associated with wheat grain number and plant height; 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 number of grains per ear and plant height traits of the gene can also be further applied to molecular marker-assisted breeding. Through the KASP molecular marker, breeders can quickly screen out wheat germplasm with the traits of more grains per ear, shorter stems or longer ear length and apply them to genetic improvement of germplasm. Therefore, the present invention can accelerate the genetic improvement process of wheat varieties and improve breeding efficiency; and provide strong technical support for the cultivation of high-yield and high-quality wheat varieties that adapt to different ecological environments and cultivation requirements.

[0039] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A KASP molecular marker for regulating wheat grain number and plant height genes, characterized in that: The KASP molecular marker is used for identifying a primer set for genes regulating wheat grain number per ear and plant height, and is composed of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

2. The KASP molecular marker for regulating wheat grain number and plant height genes according to claim 1, characterized in that: The wheat grain number and plant height gene is TaTPP-7D The gene has a nucleotide sequence as shown in SEQ ID NO.

4.

3. The KASP molecular marker for regulating wheat grain number and plant height genes according to claim 1, characterized in that: The primer set is used to detect whether the genotype of the SNP site based on -1021 bp in the promoter region in the genomic DNA of the wheat to be tested is a CC genotype or a TT genotype.

4. The KASP molecular marker for regulating wheat grain number and plant height according to any one of claims 1 to 3 is used in wheat TaTPP-7D Detection and identification of SNP allele variation of genes, and application in auxiliary identification of wheat grain number per ear and plant height traits or molecular marker-assisted breeding.

5. 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 the primer set described in claim 1 to perform PCR amplification to obtain a PCR amplification product; (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 ear, short stems and short ear 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.

6. A kit for detecting wheat grain number and plant height, characterized in that: The invention comprises the KASP molecular marker for regulating wheat grain number per ear and plant height gene as described in any one of claims 1 to 3.

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