KASP molecular marker for detecting wheat flowering and maturing character gene and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for detecting wheat flowering and maturing character genes and application of the KASP molecular marker, and relates to the technical field of biology. A primer group of the KASP molecular marker is a primer group I and / or a primer group II, and specific sequences of the primer group I and the primer group II are given; meanwhile, the KASP molecular marker is applied to SNP of the wheat TaTPP-7B gene, excellent haplotype detection and identification, auxiliary identification of wheat flowering and maturation characters or molecular marker auxiliary breeding. The related primer group is developed on the basis of wheat flowering and ripening characters, SNP loci of related characters and excellent haplotypes of the SNP loci can be rapidly and conveniently detected, and the primer group has the advantages of being rapid in identification, high in accuracy and the like and has a good application prospect.

Description

Technical Field

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

[0002] wheat( Triticum aestivum L .) is the world's largest grain crop, feeding about 3 billion people worldwide. Increasing wheat yield and ensuring stable wheat yield are major strategic measures to ensure national food security and improve people's living standards. The flowering and maturity periods of wheat are key factors that determine wheat yield formation and ecological adaptability. Among them, the flowering period determines the number of wheat grains. The appropriate environmental conditions (such as temperature, light and water) during wheat flowering are crucial to pollen vitality, pollination efficiency and fertilization success rate, and ultimately affect the number of wheat grains per ear; while the maturity period determines the degree of grain filling. During the wheat filling period, the synthesis, transport and accumulation efficiency of photosynthetic products in the grains directly affect the thousand-grain weight of wheat. The number of grains per ear and the thousand-grain weight are two important factors that constitute the three elements of wheat yield, affecting and determining the formation of wheat yield. Therefore, the different flowering and maturity periods of wheat varieties are not only the specific manifestations of wheat's adaptation to different light and temperature environments, but also jointly determine the yield potential and stability of wheat, and are therefore an indispensable core link in the yield formation process.

[0003] However, the temperature fluctuations and frequent extreme weather events caused by the current global climate change have significantly increased the risk of environmental stress (such as high temperature and drought) during the flowering and maturity stages of wheat, which not only affects the vitality of wheat pollen, pollination efficiency and the formation of grain number during flowering; but also high temperature, drought or nutritional stress will induce the accumulation of reactive oxygen species (ROS), accelerate leaf senescence, shorten the duration of grain filling, and lead to a decrease in grain fullness, becoming an important factor limiting yield potential. Under the existing wheat cultivation technology management conditions and climatic environment, wheat varieties with strong adaptability usually have the characteristics of early flowering and early maturity (higher heat shock protein expression level, stronger antioxidant enzyme system (such as SOD, CAT) activity and optimized carbohydrate distribution efficiency). Therefore, they can help wheat avoid adverse environmental stresses such as hot dry wind and high temperature during the flowering and maturity stages, so that wheat can maintain pollen vitality and maintain a higher filling rate and duration in adversity, thereby greatly ensuring food production and safety.

[0004] In summary, the adaptive mechanisms of different wheat varieties to flowering and maturity are complex traits formed in the long-term natural selection and artificial breeding process of wheat, and their optimization is of great significance for improving the yield potential and stability of wheat in different ecological environments. However, there are currently few reports on the detection of wheat flowering and maturity traits, and there are problems such as difficulty and inaccuracy in its detection and identification, which greatly limits the efficiency of auxiliary breeding selection for wheat flowering and maturity traits and restricts the development of the wheat planting industry. Therefore, in-depth research on the physiological mechanisms of these two stages and the laws of their interaction with the environment, and further exploring and utilizing these adaptive characteristics through genetic improvement and cultivation management methods, are important strategies to cope with future climate change challenges. This background provides an important theoretical basis and practical direction for related research and technology 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. A related primer set is developed based on wheat flowering and maturity traits, which can quickly and conveniently detect SNP sites and excellent haplotypes of related traits, 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 of the KASP molecular marker for detecting and identifying SNPs and haplotypes 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 / or, 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.

[0007] 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.

[0008] 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.

[0009] Furthermore, the wheat flowering and maturation trait gene is TaTPP-7B The gene has a nucleotide sequence as shown in SEQ ID NO.7.

[0010] 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 SNP and excellent haplotypes of genes for flowering and maturity traits of wheat or production applications in molecular marker-assisted breeding.

[0011] The present invention also provides a method for detecting flowering and maturation traits of wheat, comprising the following steps: (1) Extracting genomic DNA from the wheat material to be tested; (2) using the genomic DNA as a template and using the above primer set 1 and / or primer set 2 to perform PCR amplification to obtain a PCR amplification product; (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 trait 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 maturing and tall stem wheat.

[0012] 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.

[0013] The present invention has the following beneficial effects: 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

[0014] Figure 1 It is a schematic diagram of the partial identification results of KASP genotyping of allelic SNP1 variant sites; Figure 2 Schematic diagram of partial identification results of KASP genotyping of allelic SNP2 variant site. DETAILED DESCRIPTION

[0015] 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.

[0016] Example 1 1. Test wheat materials: Select multiple wheat materials with large differences in flowering and maturation traits distributed in different wheat regions of my country (see Table 2 for specific material information) as materials for discovering polymorphic loci.

[0017] 2. Sequence alignment: Perform the following operations on each wheat tested: (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 / or primer set 2, and the amplification program was as shown in Table 1 to obtain a PCR amplification product; 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; (3) Clone and sequence the PCR amplification products; and perform sequence splicing and alignment.

[0018] Table 1 PCR amplification program

[0019] The clone sequencing results of each wheat material were spliced ​​and compared with the primer set 1 and primer set 2 respectively, and it was found that there were two kinds of SNP changes in the genomic PCR amplification products of different test wheats with primer set 1 and primer set 2.

[0020] Based on the sequence alignment of the PCR amplification products of all tested wheats with 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.

[0021] The genotypes of each test material based on +806 SNP1 and -701 SNP2 are shown in Table 2.

[0022] Table 2 Genotypes of the tested materials

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] Among them, in Table 2, the haplotype composed of SNP1 TT and SNP2 AA HapⅠ The genotypes of wheat materials tested are T TCAC A AC; haplotype consisting of SNP1 CC and SNP2 GG HapⅡ The genotypes of 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.

[0030] As shown in Table 2, for both local varieties and selected varieties, the allele variation of the +806 SNP1 locus and the -701 SNP2 locus can form two haplotypes in the 262 Mini-Core Collection of my country. Among local varieties, those representing early flowering, early maturity and short stem 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%; among the selected varieties, the proportion of excellent haplotypes in major wheat regions 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.

[0031] 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 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; 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.

[0032] 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); and 2010SY means Shunyi (2010). The data statistical software used Tukey's test, and the significant difference level was 0.05 or 0.01 (* P <0.05,** P <0.01).

[0033] Table 3 TaTPP-7B Association analysis of important agronomic traits among different haplotypes in 262 local varieties in my country

[0034] As shown in Table 3, among the six agronomic traits of 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 the two traits of flowering date and maturity date. HapⅠ Compare HapⅡ The flowering time was 4.2 days earlier (2006), 2.7 days earlier (2005), 6 days earlier (2002) and 2.7 days earlier (2010); the maturity time was 4.7 days earlier (2006), 2.8 days earlier (2005), 5.2 days earlier (2002) and 2.8 days earlier (2010). The differences were extremely significant or significant in the four-year tests (*P <0.05,** P <0.01); while plant height trait, 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.

[0035] Table 4 TaTPP-7B Association analysis of important agronomic traits among different haplotypes in 262 selected varieties in my country

[0036] From Table 4, we can see that among the selected varieties, TaTPP-7B The change trend of the two haplotypes of the gene is also the same as that of local varieties. Among the six agronomic traits of heading period, maturity period, 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 period and maturity period (supported by at least 3 years of average data). HapⅠ Compare HapⅡ The flowering time was 7.8 days earlier (2006), 4.5 days (2005), 4.8 days (2002) and 4.5 days (2010) respectively; the maturity time was 6.1 (2006), 4.0 (2005), 5.2 (2002) and 4.0 days (2010) earlier. The difference was extremely significant or significant in the three to four years of testing (* 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 statistical differences, but the change trend was exactly the same as that of the local varieties in the four years, and the plant height of the selected varieties varied from 4.8 to 23.9 cm. In actual production, this plant height difference has reached a very 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.

[0037] In summary, +806 SNP1, -701 SNP2 and the two haplotypes formed by the two alleles of +806 SNP1 and -701 SNP2 are 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 allele variation (SNP) and / or haplotype associated with the flowering period, maturity period, and plant height traits of the gene, and further, the excellent allele variation or haplotype of the gene can be applied to molecular marker-assisted breeding. Through these KASP molecular markers, breeders can quickly screen out wheat germplasm carrying early flowering, early maturity, and short stem traits 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.

[0038] 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 detecting flowering and maturation trait genes in wheat, characterized in that: The primer set used by the KASP molecular marker to detect and identify SNP and haplotype 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 / or, 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.

2. The KASP molecular marker for detecting wheat flowering and maturation trait genes according to claim 1, characterized in that: The wheat flowering and maturation trait gene is TaTPP-7B The gene has a nucleotide sequence as shown in SEQ ID NO.

7.

3. The KASP molecular marker for detecting the flowering and maturation trait gene of wheat according to any one of claims 1 to 2 is used in wheat TaTPP-7B Detection, identification and auxiliary identification of SNP and excellent haplotypes of genes for flowering and maturity traits of wheat or production applications in molecular marker-assisted breeding.

4. 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 and using primer set 1 and / or primer set 2 in claim 1 to perform PCR amplification to obtain a PCR amplification product; (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 trait 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 maturing and tall stem wheat.

5. A kit for detecting flowering and maturation traits of wheat, characterized in that: The invention comprises the KASP molecular marker for detecting the wheat flowering and maturation trait gene as described in any one of claims 1 to 2.

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