KASP marker related to wheat canopy greenness and coldness in late grain-filling stage and application thereof

Abstract

The application discloses a KASP marker related to a wheat crown layer green-keeping and cold-biased type in a late grain-filling stage and application thereof. The application provides a substance for detecting the genotype of a SNP site AX-86174278 on a wheat chromosome 1B, a substance for detecting the genotype of a SNP site AX-861164768 on a wheat chromosome 3A and / or a substance for detecting the genotype of a SNP site AX-109381183 on a wheat chromosome 4B, and application in identifying or assisting in identifying the characteristics of a wheat crown layer green-keeping and cold-biased type in a late grain-filling stage. KASP marker special primers are designed, which can be used for identifying the green-keeping and cold-biased type in a late grain-filling stage, and then used for screening excellent wheat varieties with high yield, stress resistance and wide adaptability, thereby laying a theoretical foundation for breeding wheat varieties with high yield, stable quality and excellent quality and providing a molecular assisted selection method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a KASP marker associated with a green and cool canopy type in wheat during the late grain-filling stage (21 days after flowering) and its application. Background Technology

[0002] Wheat is the most widely cultivated crop globally and a major source of protein for the world's population, accounting for approximately 20% of daily intake in developing countries. Global climate change has led to frequent extreme heat events, making wheat susceptible to heat stress in its later growth stages. Heat stress accelerates the decline of wheat leaf function, shortens the grain-filling period, reduces grain weight, and ultimately leads to yield reduction. Therefore, preventing leaf senescence has become an important means of increasing wheat yield.

[0003] Green-holding and cold-climate-tolerant wheat varieties have longer leaf functional periods and higher chlorophyll content than non-green-holding varieties, significantly affecting yield components such as spike number and thousand-grain weight (Luo Yongli et al., 2016), and can alleviate the negative effects of drought stress during the grain-filling period. During leaf senescence, changes in chlorophyll content caused by chlorophyll degradation and synthesis are visible phenotypic traits of leaf senescence, and green-holding can repair or slow down this trait.

[0004] Molecular marker-assisted selection technology can track the transfer or aggregation of green genes, providing an effective technical means for breeding high-yielding and widely adaptable wheat varieties. Summary of the Invention

[0005] The purpose of this invention is to provide three SNP loci associated with canopy greenness and coolness during the late grain-filling stage (21 days after flowering) of wheat. Based on these loci, three KASP marker primers have been developed. These KASP marker primers can be used to identify or assist in the identification of wheat senescence-related traits.

[0006] In a first aspect, the present invention provides the following A, A and B, or A, B and C, in any of the following applications:

[0007] 1) To identify or assist in identifying the characteristics of the wheat canopy during the late grain-filling stage that are green and tend to be cold;

[0008] 2) To identify or assist in identifying the greening retention of the wheat canopy during the later stages of grain filling;

[0009] 3) To identify or assist in identifying the temperature during the later stage of canopy grain filling in the wheat being tested;

[0010] 4) To identify or assist in identifying the degree of senescence in the later stage of grain filling of the wheat to be tested;

[0011] 5) Select and breed wheat varieties that maintain greenness and are slightly cold during the later stages of canopy grain filling;

[0012] 6) Select and breed wheat varieties with low senescence levels;

[0013] 7) Select and breed high-yielding wheat varieties;

[0014] 8) Wheat genetics and breeding;

[0015] A is a substance used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B;

[0016] B is a substance used to detect the genotype of the SNP site AX-861164768 on wheat chromosome 3A;

[0017] C is a substance used to detect the genotype of the SNP site AX-109381183 on wheat chromosome 4B;

[0018] The SNP site AX-86174278 is the 36th position of SEQ ID NO:10;

[0019] The SNP site AX-861164768 is the 36th position of the reverse complementary sequence of SEQ ID NO:11;

[0020] The SNP site AX-109381183 is the 36th position of SEQ ID NO:12.

[0021] In the applications described above,

[0022] The genotype of the SNP locus AX-86174278 is TT, CC, or CT;

[0023] The genotype of the SNP locus AX-861164768 is AA, GG, or AG;

[0024] The genotype of the SNP locus AX-109381183 is AA, GG, or AG.

[0025] In the applications described above,

[0026] The substance used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B is either A1 or A2.

[0027] A1) Primer set 1;

[0028] A2) PCR reagents or kits containing the complete set of primers 1;

[0029] The primer set 1 includes primer F1-1, primer F1-2, and primer 1-R;

[0030] The nucleotide sequence of primer F1-1 includes the sequence shown in positions 22-42 of SEQ ID NO:1;

[0031] The nucleotide sequence of primer F1-2 includes the sequence shown in positions 22-42 of SEQ ID NO:2;

[0032] The nucleotide sequence of primer 1-R is SEQ ID NO:3;

[0033] The substance used to detect the genotype of the SNP locus AX-861164768 on wheat chromosome 3A is either B1 or B2.

[0034] B1) Primer set 2;

[0035] B2) PCR reagents or kits containing the complete set of primers 2;

[0036] The primer set 1 includes primer F2-1, primer F2-2, and primer 2-R;

[0037] The nucleotide sequence of primer F2-1 includes the sequence shown in positions 22-41 of SEQ ID NO:4;

[0038] The nucleotide sequence of primer F2-2 includes the sequence shown in positions 22-41 of SEQ ID NO:5;

[0039] The nucleotide sequence of primer 2-R is SEQ ID NO:6;

[0040] The substance used to detect the genotype of the SNP locus AX-109381183 on wheat chromosome 4B is either C1 or C2.

[0041] C1) Primer set 3;

[0042] C2) PCR reagents or kits containing the complete set of primers 3;

[0043] The primer set 3 includes primer F3-1, primer F3-2, and primer 3-R;

[0044] The nucleotide sequence of primer F3-1 includes the sequence shown in positions 22-39 of SEQ ID NO:7;

[0045] The nucleotide sequence of primer F3-2 includes the sequence shown in positions 22-39 of SEQ ID NO:8;

[0046] The nucleotide sequence of primer 3-R is SEQ ID NO:9.

[0047] In the applications described above,

[0048] The nucleotide sequence of primer F1-1 is SEQ ID NO:1;

[0049] The nucleotide sequence of primer F1-2 is SEQ ID NO:2;

[0050] Alternatively, the nucleotide sequence of primer F2-1 is SEQ ID NO:4;

[0051] The nucleotide sequence of primer F2-2 is SEQ ID NO:5;

[0052] Alternatively, the nucleotide sequence of primer F3-1 is SEQ ID NO:7;

[0053] The nucleotide sequence of primer F3-2 is SEQ ID NO:8.

[0054] Each of the aforementioned primer sets also includes fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0055] The nucleotide sequences of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B are SEQ ID NO:13-SEQ ID NO:16, respectively.

[0056] The ends of fluorescent probe A and fluorescent probe B are labeled with different fluorescent groups;

[0057] The quenching probe A and the quenching probe B are labeled with different quenching groups at their ends.

[0058] In a second aspect, the present invention provides any of the following substances:

[0059] The primer set 1 described in the first aspect;

[0060] Or the primer set 1 and primer set 2 described in the first aspect;

[0061] Or the primer set 1, primer set 2 and primer set 3 described in the first aspect;

[0062] Or PCR reagents or kits containing the primer set described in the first aspect;

[0063] Or the DNA fragment shown in SEQ ID NO:10;

[0064] Or the DNA fragment shown in SEQ ID NO:11 or its reverse complementary sequence;

[0065] Or the DNA fragment shown in SEQ ID NO:12;

[0066] The PCR reagent consists of PCR reagent 1, PCR reagent 2 and PCR reagent 3;

[0067] The PCR reagent 1 includes primer F1-1, primer F1-2, primer 1-R, fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0068] The PCR reagent 2 includes primer F2-1, primer F2-2, primer 2-R, fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0069] The PCR reagent 3 includes primer F3-1, primer F3-2, primer 3-R, fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0070] The nucleotide sequences of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B are SEQ ID NO:13-SEQ ID NO:16, respectively.

[0071] The ends of fluorescent probe A and fluorescent probe B are labeled with different fluorescent groups;

[0072] The quenching probe A and the quenching probe B are labeled with different quenching groups at their ends.

[0073] In each of the above PCR reagents, the molar ratio of primer F1, primer F2, and primer R is 0.1344:0.1344:0.336. In the embodiments of the present invention, the final concentration of primer F1 and primer F2 in the PCR amplification system is 0.1344 μM, and the final concentration of primer R in the PCR amplification system is 0.336 μM.

[0074] Thirdly, the present invention provides the use of the primer set 1, primer set 2, primer set 3, the PCR reagent or kit described in the first aspect, in any of the following or in the preparation of products having any of the following characteristics:

[0075] 1) To identify or assist in identifying the characteristics of the wheat canopy during the late grain-filling stage that are green and tend to be cold;

[0076] 2) To identify or assist in identifying the greening retention of the wheat canopy during the later stages of grain filling;

[0077] 3) To identify or assist in identifying the temperature during the later stage of canopy grain filling in the wheat being tested;

[0078] 4) To identify or assist in identifying the degree of senescence in the later stage of grain filling of the wheat to be tested;

[0079] 5) Select and breed wheat varieties that maintain greenness and are slightly cold during the later stages of canopy grain filling;

[0080] 6) Select and breed wheat varieties with low senescence levels;

[0081] 7) Select and breed high-yielding wheat varieties;

[0082] 8) Wheat genetics and breeding.

[0083] In the above text, the degree of senescence of the wheat in the late grain-filling stage was reflected by the greenness of the canopy in the late grain-filling stage, temperature and / or the greenness and cold-type characteristics.

[0084] Wheat with high green retention in the later stages of canopy filling showed a lower degree of senescence than wheat with low green retention in the later stages of canopy filling.

[0085] Alternatively, wheat grown at lower temperatures during the later stages of canopy filling exhibits a lower degree of senescence than wheat grown at higher temperatures during the later stages of canopy filling.

[0086] Alternatively, wheat with high greenness and cold-type characteristics during the later stages of canopy filling may have a lower senescence rate than wheat with low greenness and cold-type characteristics.

[0087] The aforementioned green retention is reflected in the chlorophyll content in the later stages of canopy grouting. Specifically, high green retention in the later stages of canopy grouting means high chlorophyll content in the later stages of canopy grouting.

[0088] The aforementioned greening and cool-climate characteristics are largely reflected in the high greening rate and low temperature during the later stages of canopy grouting.

[0089] The genetic breeding mentioned above, combined with a high-throughput phenotyping drone platform, has accelerated the application of drone spectroscopy in wheat genetic breeding.

[0090] Thirdly, the present invention provides any of the following methods:

[0091] A. A method for identifying or assisting in the identification of the green and cool-type characteristics of the late grain-filling stage of the canopy of wheat, comprising the following method A1, or the following methods A1 and A2, or the following methods A1, A2 and A3:

[0092] The method shown in A1) includes the following steps: detecting whether the SNP site AX-86174278 in the genome of the wheat to be tested is TT, CC or CT, and the wheat to be tested with the TT genotype has a green and cold-type characteristic in the late stage of canopy filling that is greater than or has a candidate greater than the wheat to be tested with the CC or CT genotypes.

[0093] The method shown in A2) includes the following steps: Detecting the genotype of SNP site AX-861164768 in the wheat genome to be tested as AA, GG, or AG, and determining it as follows:

[0094] The AA genotype wheat test wheat showed greater or greater green and cooler characteristics in the late stage of canopy filling than the GG or AG genotype wheat test wheat.

[0095] The method shown in A3) includes the following steps: Detecting the genotype of SNP site AX-109381183 in the wheat genome to be tested to determine whether it is AA, GG, or AG, and then making the following judgment:

[0096] The AA genotype wheat test wheat showed greater or greater green and cooler characteristics in the late stage of canopy filling than the GG or AG genotype wheat test wheat.

[0097] B. A method for identifying or assisting in the identification of the greening retention of the wheat canopy during the late grain-filling stage, comprising the following method B1, or the following methods B1 and B2, or the following methods B1, B2 and B3:

[0098] The method shown in B1) includes the following steps: detecting whether the SNP site AX-86174278 in the genome of the wheat to be tested is TT, CC or CT, and the greening of the wheat to be tested with the TT genotype in the late stage of canopy filling is greater than or candidate greater than that of the wheat to be tested with the CC or CT genotypes.

[0099] The method shown in B2) includes the following steps: Detecting the genotype of the SNP site AX-861164768 in the wheat genome to be tested as AA, GG, or AG, and determining it as follows:

[0100] The green retention of the test wheat with the AA genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with the GG or AG genotypes.

[0101] The method shown in B3) includes the following steps: Detecting the genotype of SNP site AX-109381183 in the wheat genome to be tested to determine whether it is AA, GG, or AG, and then making the following judgment:

[0102] The green retention of the test wheat with the AA genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with the GG or AG genotypes.

[0103] C. A method for identifying or assisting in the identification of the temperature during the late stage of canopy filling in wheat, comprising the following method C1, or the following methods C1 and C2, or the following methods C1, C2 and C3:

[0104] The method shown in C1) includes the following steps: detecting whether the SNP site AX-86174278 in the genome of the wheat to be tested is TT, CC or CT, and the temperature of the wheat to be tested with the TT genotype in the later stage of canopy filling is lower than or lower than that of the wheat to be tested with the CC or CT genotypes.

[0105] The method shown in C2) includes the following steps: Detecting the genotype of SNP site AX-861164768 in the wheat genome to be tested as AA, GG, or AG, and determining it as follows:

[0106] The temperature during the late canopy filling stage of the AA genotype wheat was lower than or lower than that of the GG or AG genotype wheat.

[0107] The method shown in C3) includes the following steps: Detecting the genotype of SNP site AX-109381183 in the wheat genome to be tested to determine whether it is AA, GG, or AG, and then making the following judgment:

[0108] The temperature during the late canopy filling stage of the AA genotype wheat was lower than or lower than that of the GG or AG genotype wheat.

[0109] D. A method for identifying or assisting in the identification of the degree of senescence in the late stage of canopy filling in wheat, namely, method D1, or methods D1 and D2, or methods D1, D2 and D3:

[0110] The method shown in D1) includes the following steps: detecting whether the SNP site AX-86174278 in the genome of the wheat to be tested is TT, CC or CT, and the wheat to be tested with the TT genotype has a lower or lower degree of senescence in the late canopy filling stage than the wheat to be tested with the CC or CT genotype.

[0111] The method shown in D2) includes the following steps: Detecting the genotype of SNP site AX-861164768 in the wheat genome to be tested as AA, GG, or AG, and determining it as follows:

[0112] The degree of canopy senescence in the late stage of canopy filling of the test wheat with the AA genotype is less than or less than that of the test wheat with the GG or AG genotypes.

[0113] The method shown in D3) includes the following steps: Detecting the genotype of SNP site AX-109381183 in the wheat genome to be tested as AA, GG, or AG, and determining it as follows:

[0114] The AA genotype wheat samples showed a lower or lower degree of canopy senescence during the late grain-filling stage compared to the GG or AG genotype wheat samples.

[0115] In the method described above,

[0116] The method for detecting whether the SNP site AX-86174278 in the wheat genome to be tested is TT, CC or CT is as follows: using the wheat genome to be tested as a template, KASP detection is performed using PCR reagent 1 described in the second aspect to obtain the genotype;

[0117] Alternatively, the method for detecting the genotype of SNP site AX-861164768 in the wheat genome to be tested as AA, GG or AG is as follows: using the wheat genome to be tested as a template, KASP detection is performed using PCR reagent 2 described in the second aspect to obtain the genotype;

[0118] Alternatively, the method for detecting the genotype of SNP site AX-109381183 in the wheat genome to be tested as AA, GG or AG is as follows: using the wheat genome to be tested as a template, KASP detection is performed using PCR reagent 3 described in the second aspect to obtain the genotype.

[0119] In the method of this invention, the genotype is obtained using KlusterCaller. TM The software determines the genotype based on fluorescence signals.

[0120] The above KASP detection can be performed using a Touch-down PCR amplification program, as follows: 94℃ pre-denaturation for 15 min; (Touch-down program) 94℃ denaturation for 30 s, 61℃ annealing for 60 s, 72℃ extension for 30 s, 11 cycles, with the annealing temperature decreasing by 0.6℃ per cycle; (Amplification program) 94℃ denaturation for 30 s, 55℃ annealing for 60 s, 72℃ extension for 30 s, 26 cycles; 72℃ extension for 5 min; store at 10℃.

[0121] Fourthly, the present invention provides a method for selecting wheat with low senescence during the later stage of canopy filling, comprising the following 1), 2), or / and 3):

[0122] 1) Select wheat with the TT genotype at the SNP site AX-86174278 in the method described in the third aspect;

[0123] 2) Select wheat with the genotype AA for the SNP locus AX-861164768 in the method described in the third aspect;

[0124] 3) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0125] Fifthly, the present invention provides a method for selecting wheat varieties with high yields in the later stages of canopy filling, comprising the following 1), 2), or / and 3):

[0126] 1) Select wheat with the TT genotype at the SNP site AX-86174278 in the method described in the third aspect;

[0127] 2) Select wheat with the genotype AA for the SNP locus AX-861164768 in the method described in the third aspect;

[0128] 3) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0129] In this invention, wheat includes, but is not limited to, any one or more of the following varieties: Zhongmai 175 / Lunxuan 987RIL population (148 accessions), and natural populations widely planted in the Huang-Huai wheat region (160 accessions).

[0130] Beneficial effects of the present invention

[0131] This study integrated two traits—flag leaf chlorophyll content (CHL) and canopy temperature (CT)—during the late grain-filling stage. Using the Zhongmai 175 / Lunxuan 987RIL population, a novel CHL / CT index was defined to screen for "green-holding" and "cool-climate" traits during the late grain-filling stage and to identify QTL loci. Based on the conversion of tightly linked SNP markers to KASP markers, this study lays a theoretical foundation for breeding high-yielding, stable-yielding, and high-quality wheat varieties and provides a molecular-assisted selection method.

[0132] This invention relates to three SNP loci associated with the late-stage green canopy and cool-climate trait in wheat, located at positions 551.01, 557.96, and 443.45 Mb on wheat chromosomes 1B, 3A, and 4B, respectively. Based on these SNP loci, this invention also develops KASP marker-specific primers and kits containing these primers. The KASP marker-specific primers of this invention can be used to identify the genotype of wheat samples, and based on the genotype, can be used to screen wheat varieties with high potential for green canopy and high yield. The KASP marker-specific primers of this invention can be used to identify the late-stage green canopy and cool-climate trait, and further used to screen high-yielding, stress-resistant, and widely adaptable wheat varieties, laying a theoretical foundation and providing a molecular-assisted selection method for breeding high-yielding, stable-yielding, and high-quality wheat varieties. Attached Figure Description

[0133] Figure 1 Linkage map of SNP markers with QTL-caas-1B, QTL-caas-3A, and QTL-caas-4B2 genes.

[0134] Figure 2 The results of KASP marker detection for the canopy-green and cool-toned type of natural population varieties in the late grain-filling stage are shown. A, B and C are the detection results of AX-86174278 (denoted as AX-86174278-1B in the figure), AX-861164768 (denoted as AX-861164768-3A in the figure), and AX-109381183 (denoted as AX-109381183-4B2 in the figure), respectively. Detailed Implementation

[0135] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0136] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0137] The following examples are for illustrative purposes only and are not intended to limit the scope of protection of the invention. All quantitative experiments in the following examples were performed in duplicate, and the results were averaged. All primers in the following examples were synthesized by Beijing BGI Genomics Co., Ltd.; reagents used for KASP genotyping were provided by Beijing Jiacheng Biotechnology Co., Ltd.

[0138] Example 1: Obtaining KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0139] I. Investigation and SNP marker analysis of canopy greening and cool-climate traits

[0140] 1. Investigation of chlorophyll content and canopy temperature characteristics

[0141] The RIL population of Zhongmai 175 / Lunxuan 987, including 148 families of parents, was selected as experimental materials for senescence trait testing. The chlorophyll content (CHL) of the flag leaf and the canopy temperature (CT) of wheat in the late grain-filling stage were investigated.

[0142] The experiment used plot planting, with each plot covering an area of ​​3.6m². 2 (1.2*3.0). Specific steps are as follows: To minimize marginal effects, use SPAD502 (Konica Minolta, Japan) to measure the chlorophyll content of the flag leaves of six wheat plants with consistent growth in the non-marginal area of ​​each plot during the late grain-filling stage. Take the average value as the chlorophyll content of that family. On a sunny, windless midday (11:00-13:00), use an infrared thermometer (Spectrum Tech., Inc. Aurora IL, USA) to measure the canopy temperature of each plot, ensuring consistent measurement angles. For specific trait surveys, refer to (Pask, Pietragalla, et al., 2012).

[0143] The higher the chlorophyll content (CHL) in the later stage of grain filling, the higher its yield potential; the lower the canopy temperature (CT) in the later stage of grain filling, the higher its yield potential.

[0144] Considering that high-yielding wheat families often exhibit "green-holding" and "cooler-temperature" canopy characteristics, we integrated wheat flag leaf chlorophyll content and canopy temperature to construct an index, CHL / CT, that comprehensively reflects the "green-holding" and "cooler-temperature" characteristics of wheat families in the late grain-filling stage. The formula is as follows:

[0145]

[0146] N represents the total number of plots, i represents the plot number, and j represents the late stage of grain filling; the higher the CHL / CT ratio, the more green the leaves are and the lower the canopy temperature, indicating that the family has a higher yield potential.

[0147] 2. SNP marker analysis

[0148] SNP (single nucleotide polymorphism) markers were used in the Zhongmai 175 / Lunxuan 987RIL population for 50k SNP microarray typing by CapitalBio Corporation (Beijing, China; http: / / bioservices.capitalbio.com) using Illumina SNP genotyping.

[0149] II. Gene localization and discovery of linkage markers AX-86174278, AX-861164768, and AX-109381183

[0150] Using Icimapping 4.1 software, combined with 50k SNP chip genotyping data and phenotypic results, associated loci were located. QTLs (QTL-caas-1B, QTL-caas-3A, QTL-caas-4B2) associated with the target trait were identified.

[0151] According to the International Wheat Genome Sequencing Consortium (IWGSC) Ref Seg 1.0 (IWGSC, 2018) http: / / plants.ensembl.org / index.html, the physical locations of QTL-caas-1B, QTL-caas-3A, and QTL-caas-4B2 on the chromosome were determined. Three SNP loci, AX-86174278, AX-861164768, and AX-109381183, were also identified. Figure 1 ).

[0152] SNP site AX-86174278 (also known as AX-86174278-1B) is the 36th position of SEQ ID NO:10. The base N at this site is C or T. It comes from the gene located at position 551.01Mb on the wheat chromosome 1B to be tested (Chinese Spring genome V1.0). The genotype of this site is TT, CC or CT.

[0153] SNP site AX-861164768 (also known as AX-861164768-3A) is position 36 of the reverse complementary sequence of SEQ ID NO:11. The base N at this site is A or G, and it comes from a gene located at position 557.96Mb on wheat chromosome 3A (Chinese Spring genome V1.0). The genotype of this site is AA, GG, or AG.

[0154] SNP site AX-109381183 (also denoted as AX-109381183-4B2) is position 36 of SEQ ID NO:12. The base N at this site is either A or G. It comes from a gene located at position 443.45 Mb on wheat chromosome 4B (Chinese Spring genome V1.0). The genotype of this site is AA, GG, or AG.

[0155] III. Development of KASP-specific primers

[0156] 1. Design of KASP marker-specific primers

[0157] This invention develops KASP marker-specific primers for the SNP sites obtained above. The KASP marker-specific primer sequences are shown in Table 1 below.

[0158] The KASP marker-specific primers developed for SNP sites consist of two upstream primers, namely primer F1 and primer F2, and one downstream primer, namely primer R.

[0159] Table 1 shows the marker primer sequence information.

[0160]

[0161] In the table above, positions 1-21 of each F1 primer are FAM fluorescent sequences, and the rest are specific primers; positions 1-21 of each F2 primer are HEX fluorescent sequences, and the rest are specific primers.

[0162] Specifically, for the AX-86174278 marker, primer F1 combined with primer R can amplify the fragment with the SNP genotype CC (the fragment with SEQ ID NO:10, and position 36 being C), and primer F2 combined with primer R can amplify the fragment with the SNP genotype TT (the fragment with SEQ ID NO:10, and position 36 being T). Primers F1 and F2, developed for the KASP marker at the AX-86174278 site, are used as forward primers, and primer R is the reverse primer.

[0163] For the AX-86164768 marker, primers F1 and R can amplify the fragment with genotype CC at the SNP site (the fragment containing the reverse complementary sequence of SEQ ID NO:11, and position 36 of the reverse complementary sequence of SEQ ID NO:11 is G), and primers F2 and R can amplify the fragment with genotype TT at the SNP site (the fragment containing the reverse complementary sequence of SEQ ID NO:11, and position 36 of the reverse complementary sequence of SEQ ID NO:11 is A). KASP marker primers F1 and F2 developed for the AX-86164768 site are used as reverse primers, and primer R is the forward primer.

[0164] For the AX-109381183 marker, primer F1 combined with primer R can amplify the fragment with genotype AA at the SNP site (the fragment with SEQ ID NO:12, and position 36 being A), and primer F2 combined with primer R can amplify the fragment with genotype GG at the SNP site (the fragment with SEQ ID NO:12, and position 36 being G). Primers F1 and F2, developed for the KASP marker at the AX-109381183 site, are used as forward primers, and primer R is the reverse primer.

[0165] 2. Amplification using KASP-labeled primers

[0166] Genomic DNA was extracted from leaves of various wheat varieties. Using the genomic DNA as a template, PCR amplification was performed using KASP marker primers corresponding to different SNP sites, yielding PCR amplification products. PCR amplification products carrying the fluorescent sequence FAM showed red fluorescence upon irradiation, while those carrying the fluorescent sequence HEX showed blue fluorescence.

[0167] The PCR amplification system described above is as follows (total volume 5.0 μl): 2.0 μl 50 ng / μl template DNA, 1.5 μl 2×KASPreaction mix, 0.0336 μl primer mix (Assay mix), and 1.4664 μl ddH2O.

[0168] 2×KASP reaction mix reagent: The AQP genotyping universal kit (18241211 / 2218) manufactured by Beijing Jiacheng Biotechnology Co., Ltd. was used. The product contains fluorescent probe A, fluorescent probe B, quencher probe A, quencher probe B, HiGeno DNA Polymerase, PCR buffer, and dNTPs. For detailed principles and product information, please see: http: / / www.jasongen.com / newsdetail.aspx?channel_id=1017&id=1

[0169] The sequence of the fluorescent probe A is 5′-GAAGGTGACCAAGTTCATGCT-3′ (SEQ ID NO:13), with a fluorescent group FAM attached to the 5′ end;

[0170] The sequence of the fluorescent probe B is 5′-GAAGGTCGGAGTCAACGGATT-3′ (SEQ ID NO:14), with a fluorescent group HEX attached to the 5′ end;

[0171] The sequence of the above quenching probe A is 5′-AGCATGAACTTGGTCACCTTC-3′ (SEQ ID NO:15), with the quenching group BHQ attached to the 3′ end;

[0172] The sequence of the above-mentioned quenching probe B is 5′-AATCCGTTGACTCCGACCTTC-3′ (SEQ ID NO:16), with the quenching group BHQ attached to the 3′ end.

[0173] The above primer mix formula is as follows: 12.0 μM primer F1, 12.0 μM primer F2, 30.0 μM primer R, with the remainder being water; and the concentrations of primer F1 and primer F2 in the above PCR amplification system are both 0.1344 μM, and the final concentration of primer R in the PCR amplification system is 0.336 μM.

[0174] The above PCR amplification reactions were performed on a PTC-200 PCR instrument using a Touch-down PCR amplification program as follows: 94℃ pre-denaturation for 15 min; (Touch-down program) 94℃ denaturation for 30 s, 61℃ annealing for 60 s, 72℃ extension for 30 s, 11 cycles, with the annealing temperature decreasing by 0.6℃ per cycle; (Amplification program) 94℃ denaturation for 30 s, 55℃ annealing for 60 s, 72℃ extension for 30 s, 26 cycles; 72℃ extension for 5 min; stored at 10℃.

[0175] The PCR amplification products were processed in Pherastar. plus Genotyping was performed using a fluorescent microplate reader under fluorescent illumination, and then in KlusterCaller. TM The software reads the genotyped data and detects the genotypes of the AX-86174278, AX-86164768, and AX-109381183 loci.

[0176] IV. Identification of allele-specific markers at loci AX-86174278, AX-861164768, and AX-109381183: Whole genome DNA was extracted from 160 natural populations (Table 4) irrigated in Xinxiang in 2019.

[0177] Using genomic DNA as templates, genotyping was performed using the KASP marker primers corresponding to the SNP markers AX-86174278, AX-861164768, and AX-109381183, following the method described in section 2 above. This allowed for the detection of genotypes in different varieties.

[0178] The results are as follows Figure 2 As shown in Table 2, it can be seen that SNP markers can effectively identify the genotypes of wheat loci AX-86174278, AX-861164768, and AX-109381183.

[0179] Table 2 shows the segregation of allelic variations of markers AX-86174278, AX-861164768, and AX-109381183 in natural populations.

[0180] Tag Name genotype <![CDATA[Number a > AX-86174278 TT 105 CC 45 CT 0 ？ 10 AX-109381183 AA 53 GG 101 AG 0 ？ 6 AX-861164768 GG 133 AA 23 AG 3 ？ 1

[0181] a Number of corresponding genotypes

[0182] Table 3 shows the segregation of allelic variants of markers AX-86174278, AX-861164768, and AX-109381183 in four environments within a natural population.

[0183]

[0184]

[0185]

[0186] a Number of corresponding genotypes

[0187] b E1-E4 correspond to Xinxiang Irrigation in 2019, Xinxiang Water Saving in 2019, Luohe Irrigation in 2019, and Luohe Water Saving in 2019, respectively. c Mean ± Standard Deviation

[0188] d The t-test was performed on the two sets of phenotypic data. * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.

[0189] Table 4 shows information on natural populations.

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Therefore, the above results show that SNP markers AX-86174278, AX-861164768, and AX-109381183 can all be used to genotype individuals in natural populations. Furthermore, the favorable genotype TT of AX-86174278 is widely used in natural populations, while the favorable genotypes AA of AX-861164768 and AX-109381183 are less common, indicating significant potential for improvement. These markers can be used to quantify the leaf greenness and canopy coolness traits of wheat in the late grain-filling stage (21 days after flowering). Specific analysis follows:

[0197] 1) Determine whether the genotype of the wheat SNP locus AX-86174278 is TT, CC, or CT, as follows:

[0198] The green retention of the test wheat with the TT genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with the CC or CT genotypes.

[0199] Alternatively, the temperature during the late canopy filling stage of the test wheat with the TT genotype was lower than or lower than that of the test wheat with the CC or CT genotypes.

[0200] Alternatively, the TT genotype wheat test wheat has a green canopy and a colder phenotype during the later stages of canopy filling than or has a greater candidate phenotype than the CC or CT genotype wheat test wheat.

[0201] 2) The genotype of the wheat SNP locus AX-861164768 is determined to be AA, GG, or AG, as follows:

[0202] The green retention of the test wheat with the AA genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with the GG or AG genotypes.

[0203] Alternatively, the temperature during the late canopy filling stage of the AA genotype wheat was lower than or lower than that of the GG or AG genotype wheat.

[0204] Alternatively, the AA genotype wheat test wheat has a green canopy in the later stages of grain filling and a cold-type characteristic greater than or greater than the GG or AG genotype wheat test wheat.

[0205] 3) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0206] The green retention of the test wheat with AA genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with GG or AG genotype.

[0207] Alternatively, the temperature during the late canopy filling stage of the AA genotype wheat was lower than or lower than that of the GG or AG genotype wheat.

[0208] Alternatively, the AA genotype wheat test wheat has a green canopy in the later stages of grain filling and a cold-type characteristic greater than or greater than the GG or AG genotype wheat test wheat.

[0209] The aforementioned green retention is reflected in chlorophyll content; the higher the chlorophyll content, the greater the green retention.

[0210] The aforementioned green-holding and cool-loving characteristics are reflected by the ratio of chlorophyll content to canopy temperature (CHL / CT, formula described above). The higher the ratio, the better the green-holding and cool-loving characteristics, that is, the leaves are greener and the canopy temperature is lower, indicating that the family has higher yield potential.

[0211] Example 2: Application of KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0212] t-tests were performed on the markers AX-86174278, AX-861164768, and AX-109381183 in natural populations to verify the effectiveness of KASP markers and to assist in breeding decisions.

[0213] I. Testing canopy temperature and chlorophyll content of different wheat varieties during the later stages of irrigation

[0214] The method was the same as in Example 1. The results of canopy temperature (CT), chlorophyll content (CHL), and canopy greening and cool-type characteristics (CHL / CT) are shown in Table 3.

[0215] II. Genotyping of SNP loci in different wheat varieties

[0216] Genomic DNA was extracted from 160 natural varieties (as shown in Table 4 above) of Xinxiang Irrigation, Xinxiang Water Saving, Luohe Irrigation, and Luohe Water Saving Planting in 2019.

[0217] Using genomic DNA as a template, PCR amplification was performed using KASP-labeled primers corresponding to the SNP sites AX-86174278, AX-861164768, and AX-109381183, respectively, following the method described in section 2 of Example 1. The PCR amplification products were obtained. The PCR amplification product carrying the fluorescent sequence FAM showed a red color under fluorescence irradiation, while the PCR amplification product carrying the fluorescent sequence HEX showed a blue color under fluorescence irradiation.

[0218] The PCR amplification system described above is as follows (total volume 5.2 μl): 3.0 μl 20 ng / μl template DNA, 2.0 μl 2×KASP reaction mix, 0.1 μl primer mix (Assay mix), and 0.1 μl ddH2O. The 2×KASP reaction mix includes fluorescent probe A, fluorescent probe B, quencher probe A and quencher probe B, as well as high-fidelity Taq enzyme, dNTPs, etc.

[0219] The sequence of the fluorescent probe A is 5′-GAAGGTGACCAAGTTCATGCT-3′ (SEQ ID NO:13), with a fluorescent group FAM attached to the 5′ end;

[0220] The sequence of the fluorescent probe B is 5′-GAAGGTCGGAGTCAACGGATT-3′ (SEQ ID NO:14), with a fluorescent group HEX attached to the 5′ end;

[0221] The sequence of the above quenching probe A is 5′-AGCATGAACTTGGTCACCTTC-3′ (SEQ ID NO:15), with the quenching group BHQ attached to the 3′ end;

[0222] The sequence of the above-mentioned quenching probe B is 5′-AATCCGTTGACTCCGACCTTC-3′ (SEQ ID NO:16), with the quenching group BHQ attached to the 3′ end.

[0223] The primer mixture mentioned above includes primers F1, F2, and R. The final concentrations of primers F1 and F2 in the PCR amplification system are both 0.1344 μM, and the final concentration of primer R in the PCR amplification system is 0.336 μM.

[0224] PCR amplification reactions were performed on a PTC-200 PCR instrument using a Touch-down PCR program: 94℃ pre-denaturation for 15 min; (Touch-down program) 94℃ denaturation for 30 s, 61℃ annealing for 60 s, 72℃ extension for 30 s, 11 cycles, with the annealing temperature decreasing by 0.6℃ per cycle; (Amplification program) 94℃ denaturation for 30 s, 55℃ annealing for 60 s, 72℃ extension for 30 s, 26 cycles; 72℃ extension for 5 min; stored at 10℃.

[0225] The PCR amplification products were processed in Pherastar. plus Genotyping was performed using a fluorescent microplate reader under fluorescent illumination, and then in KlusterCaller. TM The software reads the data after fracturing.

[0226] The judgment method is as follows:

[0227] 1) Determine whether the genotype of the wheat SNP locus AX-86174278 is TT, CC, or CT, as follows:

[0228] The TT genotype wheat samples showed greater or greater candidate characteristics of green canopy retention and cold-type characteristics during the late grain-filling stage compared to the CC or CT genotype wheat samples.

[0229] 2) The genotype of the wheat SNP locus AX-861164768 is determined to be AA, GG, or AG, as follows:

[0230] The AA genotype wheat test wheat showed greater or greater green and cooler characteristics in the late stage of canopy filling than the GG or AG genotype wheat test wheat.

[0231] 3) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0232] The AA genotype wheat samples exhibited greater or greater green canopy retention and cold-type characteristics during the later stages of canopy filling compared to or candidate wheat samples with the GG or AG genotypes.

[0233] Pairwise comparisons were performed on the PCR amplification products of each wheat variety, and t-tests were conducted based on color. The genotyping results of the three markers in four environments of the natural population and the verification of the green-green, cool-biased trait are shown in Table 3. Figure 2 As shown.

[0234] The above results indicate that wheat varieties with only TT genotype at the AX-86174278 locus and only AA genotype at the AX-861164768 and AX-109381183 loci have higher chlorophyll content and lower canopy temperature. This suggests that this locus can rapidly and accurately identify wheat senescence-related traits (specifically manifested in high chlorophyll content and / or low canopy temperature) or predict their yield, thus assisting in breeding decisions.

[0235] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of a substance that specifically detects the genotype of the SNP locus AX-109381183 on wheat chromosome 4B in any of the following: 1) To assist in identifying the greening retention of the wheat canopy during the later stages of grain filling; 2) Select and breed wheat varieties that retain greenness during the later stages of canopy grain filling; The SNP site AX-109381183 is the 36th position of SEQ ID NO:12; The genotype of the SNP locus AX-109381183 is AA, GG, or AG.

2. The application according to claim 1, characterized in that: The substance specifically used to detect the genotype of the SNP locus AX-109381183 on wheat chromosome 4B is either A1 or A2. A1) Primer set; A2) PCR reagents or kits containing the complete set of primers described above; The primer set consists of primer F3-1, primer F3-2, and primer 3-R; The nucleotide sequence of primer F3-1 is SEQ ID NO:7; The nucleotide sequence of primer F3-2 is SEQ ID NO:8; The nucleotide sequence of primer 3-R is SEQ ID NO:

9.

3. A method for assisting in the identification of green retention in the late grain-filling stage of wheat canopy, comprising the following steps: specifically detecting SNP sites in the genome of the wheat to be tested. AX-109381183 The genotype is AA, GG, or AG. Determine as follows: The green retention of the test wheat with the AA genotype in the later stage of canopy filling was greater than or greater than that of the test wheat with the GG or AG genotypes. The SNP site AX-109381183 is the 36th position of SEQ ID NO:

12.

4. The method according to claim 3, characterized in that: The specific detection of SNP sites in the wheat genome to be tested AX-109381183 The method for determining the genotype as AA, GG, or AG is as follows: using the wheat genome to be tested as a template, KASP detection is performed using the PCR reagent described in claim 2 to obtain the genotype.

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