A kasp marker and application thereof in identification of wheat crown layer green and cold type in late grain filling stage

Abstract

The application discloses a KASP marker and application thereof in identification of a crown layer green-keeping and cold-biased type in a late grain-filling stage of wheat. The application provides a substance for detecting the genotype of a SNP site AX-86174278 on a wheat chromosome 1B and / or a substance for detecting the genotype of a SNP site AX-109381183 on a wheat chromosome 4B, and application thereof in identification or auxiliary identification of a green-keeping and cold-biased type characteristic of a crown layer in a late grain-filling stage of a to-be-tested wheat. KASP marker special primers are designed, which can be used for identifying the green-keeping and cold-biased type characteristic in the late grain-filling stage of the crown layer, 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 auxiliary selection means.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a KASP marker and its application in the identification of wheat with green canopy and cold morphology during the late grain-filling stage. Background Technology

[0002] Leaves are the primary site of photosynthesis in plants, playing a crucial role in maintaining plant growth, development, yield, and quality. In wheat, grain yield is mainly contributed by photosynthesis during the flag leaf's senescence period (therefore, to achieve maximum yield, photosynthesis in the leaves must be maintained during the grain-filling stage). To achieve high yields, both a strong photosynthetic capacity of the photosynthetic source (especially the flag leaf) and a significant allocation of photosynthetic products from the leaves to the grains are required, thereby increasing grain weight and yield.

[0003] Green-holding wheat varieties have a longer leaf functional period and higher chlorophyll content than non-green-holding varieties, significantly impacting yield components such as spike number and thousand-grain weight, and mitigating the negative effects of drought stress during the grain-filling stage. During leaf senescence, photosynthesis is the most significantly altered and obvious physiological process. 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. Studies have shown that wheat plants with green-holding genes have lower canopy temperatures, easily achieving higher biomass and yields, and this trait can be inherited by offspring. Through hybridization with other wheat materials, it can positively influence the canopy temperature status of offspring. Therefore, green-holding not only delays senescence but may also possess undiscovered functions. Utilizing green-holding wheat materials to create high-yielding and superior varieties with better biological traits is feasible. Summary of the Invention

[0004] The purpose of this invention is to provide two SNP loci associated with canopy greenness and coolness during the late grain-filling stage (21 days after flowering) of wheat. Based on these loci, two KASP marker primers were developed. These KASP marker primers can be used to identify or assist in the identification of wheat senescence-related traits.

[0005] In a first aspect, the present invention provides the following B, A, and B, in any of the following applications:

[0006] 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;

[0007] 2) To identify or assist in identifying the greening retention of the wheat canopy during the later stages of grain filling;

[0008] 3) To identify or assist in identifying the temperature during the later stage of canopy grain filling in the wheat being tested;

[0009] 4) To identify or assist in identifying the degree of senescence in the late grain-filling stage of the wheat being tested;

[0010] 5) Select and breed wheat varieties that maintain greenness and are slightly cold during the later stages of canopy grain filling;

[0011] 6) Select and breed wheat varieties with low senescence levels;

[0012] 7) Select and breed high-yielding wheat varieties;

[0013] 8) Wheat genetics and breeding;

[0014] A is a substance used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B;

[0015] The substance referred to as B is a substance for detecting the genotype of the SNP locus AX-109381183 on wheat chromosome 4B;

[0016] The SNP site AX-86174278 is the 36th position of SEQ ID NO:7;

[0017] The SNP site AX-109381183 is the 36th position of SEQ ID NO:8.

[0018] In the applications described above,

[0019] The genotype of the SNP locus AX-86174278 is TT, CC, or CT;

[0020] The genotype of the SNP locus AX-109381183 is AA, GG, or AG.

[0021] In the applications described above,

[0022] The substance used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B is either A1 or A2.

[0023] A1) Primer set 1;

[0024] A2) PCR reagents or kits containing the complete set of primers 1;

[0025] The primer set 1 includes primer F1-1, primer F1-2, and primer 1-R;

[0026] The nucleotide sequence of primer F1-1 includes the sequence shown in positions 22-42 of SEQ ID NO:1;

[0027] The nucleotide sequence of primer F1-2 includes the sequence shown in positions 22-42 of SEQ ID NO:2;

[0028] The nucleotide sequence of primer 1-R is SEQ ID NO:3;

[0029] The substance used to detect the genotype of the SNP locus AX-109381183 on wheat chromosome 4B is either B1 or B2.

[0030] B1) Primer set 2;

[0031] B2) PCR reagents or kits containing the complete set of primers 2;

[0032] The primer set 3 includes primer F2-1, primer F2-2, and primer 2-R;

[0033] The nucleotide sequence of primer F2-1 includes the sequence shown in positions 22-39 of SEQ ID NO:4;

[0034] The nucleotide sequence of primer F2-2 includes the sequence shown in positions 22-39 of SEQ ID NO:5;

[0035] The nucleotide sequence of primer 2-R is SEQ ID NO:6.

[0036] In the applications described above,

[0037] The nucleotide sequence of primer F1-1 is SEQ ID NO:1;

[0038] The nucleotide sequence of primer F1-2 is SEQ ID NO:2;

[0039] Alternatively, the nucleotide sequence of primer F2-1 is SEQ ID NO:4;

[0040] The nucleotide sequence of primer F2-2 is SEQ ID NO:5.

[0041] Each of the aforementioned primer sets also includes fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0042] The nucleotide sequences of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B are SEQ ID NO:9-SEQ ID NO:12, respectively.

[0043] The ends of fluorescent probe A and fluorescent probe B are labeled with different fluorescent groups;

[0044] The quenching probe A and the quenching probe B are labeled with different quenching groups at their ends.

[0045] In a second aspect, the present invention provides any of the following substances:

[0046] The primer set 2 described in the first aspect;

[0047] Or the primer set 1 and primer set 2 described in the first aspect;

[0048] Or PCR reagents or kits containing the primer set described in the first aspect;

[0049] Or the DNA fragment shown in SEQ ID NO:7;

[0050] Or the DNA fragment shown in SEQ ID NO:8;

[0051] The PCR reagent consists of PCR reagent 1 and PCR reagent 2;

[0052] 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;

[0053] 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;

[0054] The nucleotide sequences of fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B are SEQ ID NO:9-SEQ ID NO:12, respectively.

[0055] The ends of fluorescent probe A and fluorescent probe B are labeled with different fluorescent groups;

[0056] The quenching probe A and the quenching probe B are labeled with different quenching groups at their ends.

[0057] 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.

[0058] Thirdly, the present invention provides the use of the primer set 2, the primer set 1, and the primer set 2 described in the first aspect, the PCR reagent or the kit described in the second aspect, in any of the following or in the preparation of products having any of the following characteristics:

[0059] 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;

[0060] 2) To identify or assist in identifying the greening retention of the wheat canopy during the later stages of grain filling;

[0061] 3) To identify or assist in identifying the temperature during the later stage of canopy grain filling in the wheat being tested;

[0062] 4) To identify or assist in identifying the degree of senescence in the late grain-filling stage of the wheat being tested;

[0063] 5) Select and breed wheat varieties that maintain greenness and are slightly cold during the later stages of canopy grain filling;

[0064] 6) Select and breed wheat varieties with low senescence levels;

[0065] 7) Select and breed high-yielding wheat varieties;

[0066] 8) Wheat genetics and breeding.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] The genetic breeding mentioned above, combined with a high-throughput phenotyping drone platform, has accelerated the application of drone spectroscopy in wheat genetic breeding.

[0074] Thirdly, the present invention provides any of the following methods:

[0075] 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, namely, method A2, or methods A1 and A2 as follows:

[0076] 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 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 CC or CT genotype.

[0077] The method shown in A2) 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:

[0078] The AA genotype wheat test wheat showed greater or greater green and cold-type characteristics in the late stage of canopy filling than the GG or AG genotype wheat test wheat.

[0079] B. A method for identifying or assisting in the identification of the greening retention of the wheat canopy during the later stage of grain filling, namely, method B2, or methods B1 and B2, or methods B1, B2 and B3:

[0080] 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.

[0081] The method shown in B2) 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:

[0082] 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.

[0083] C. A method for identifying or assisting in the identification of the temperature during the late stage of canopy filling in wheat, comprising: method C2, or methods C1 and C2, or methods C1, C2 and C3.

[0084] 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.

[0085] The method shown in C2) 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:

[0086] 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.

[0087] 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 D2, or methods D1 and D2, or methods D1, D2 and D3:

[0088] 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.

[0089] The method shown in D2) 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:

[0090] The AA genotype wheat samples showed a lower or lower degree of canopy senescence during the late canopy filling stage compared to the GG or AG genotype wheat samples.

[0091] In the method described above,

[0092] 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;

[0093] 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 2 described in the second aspect to obtain the genotype.

[0094] In the method of this invention, the genotype is obtained using KlusterCaller. TM The software determines the genotype based on fluorescence signals.

[0095] 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℃.

[0096] Fourthly, the present invention provides a method for selecting wheat with low senescence during the later stage of canopy filling, comprising the following 1) or / and 2):

[0097] 1) Select wheat with the TT genotype at the SNP locus AX-86174278 in the method described in the third aspect;

[0098] 2) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0099] 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) or / and 2):

[0100] 1) Select wheat with the TT genotype at the SNP site AX-86174278 in the method described in the third aspect;

[0101] 2) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0102] 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).

[0103] 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.

[0104] Beneficial effects of the present invention

[0105] This invention relates to two SNP loci associated with the late-stage green canopy and cool-climate trait in wheat, located at positions 551.01 and 443.45 Mb on wheat chromosomes 1B 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

[0106] Figure 1 Linkage map of SNP markers with QTL-caas-1B and QTL-caas-4B2 genes.

[0107] Figure 2The results of KASP marker detection for the canopy-green and cool-type variety in the late grain-filling stage of the natural population are shown. A and BC are the detection results of AX-86174278 (denoted as AX-86174278-1B in the figure) and AX-109381183 (denoted as AX-109381183-4B2 in the figure), respectively. Detailed Implementation

[0108] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0109] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0110] 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.

[0111] Example 1: Obtaining KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0112] I. Investigation and SNP marker analysis of canopy greening and cool-climate traits

[0113] 1. Investigation of chlorophyll content and canopy temperature characteristics

[0114] 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.

[0115] 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).

[0116] 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.

[0117] To address the characteristic of high-yielding wheat families often exhibiting "green-holding" and "cooler-temperature" canopy temperatures, 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 during the late grain-filling stage. The formula is as follows:

[0118]

[0119] 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.

[0120] 2. SNP marker analysis

[0121] 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.

[0122] II. Gene localization and discovery of linkage markers AX-86174278 and AX-109381183

[0123] 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-4B2) associated with the target trait were identified.

[0124] 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 and QTL-caas-4B2 on the chromosome were determined. Two SNP loci, AX-86174278 and AX-109381183, were also identified. Figure 1 ).

[0125] SNP site AX-86174278 is position 36 of SEQ ID NO:7. The base N at this site is C or T, and it comes from the gene located at position 551.01Mb on the wheat chromosome 1B (Chinese Spring genome V1.0). The genotype of this site is TT, CC, or CT.

[0126] SNP site AX-109381183 is position 36 of SEQ ID NO:8. 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 at this site is AA, GG, or AG.

[0127] III. Development of KASP-specific primers

[0128] 1. Design of KASP marker-specific primers

[0129] 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.

[0130] 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.

[0131] Table 1 shows the marker primer sequence information.

[0132]

[0133] 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.

[0134] Specifically, for the AX-86174278 marker, primer F1 combined with primer R can amplify the fragment with genotype CC at the SNP site (the fragment with SEQ ID NO:7, and position 36 being C), and primer F2 combined with primer R can amplify the fragment with genotype TT at the SNP site (the fragment with SEQ ID NO:7, 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.

[0135] 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:8, 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:8, 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.

[0136] 2. Amplification using KASP-labeled primers

[0137] 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.

[0138] 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.

[0139] 2×KASP reaction mix reagent: The AQP genotyping universal kit manufactured by Beijing Jasongen Biotechnology Co., Ltd. was used. The product includes 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

[0140] The sequence of the fluorescent probe A is 5′-GAAGGTGACCAAGTTCATGCT-3′ (SEQ ID NO:9), with a fluorescent group FAM attached to the 5′ end;

[0141] The sequence of the fluorescent probe B is 5′-GAAGGTCGGAGTCAACGGATT-3′ (SEQ ID NO:10), with a fluorescent group HEX attached to the 5′ end;

[0142] The sequence of the above quenching probe A is 5′-AGCATGAACTTGGTCACCTTC-3′ (SEQ ID NO:11), with the quenching group BHQ attached to the 3′ end;

[0143] The sequence of the above-mentioned quenching probe B is 5′-AATCCGTTGACTCCGACCTTC-3′ (SEQ ID NO:12), with the quenching group BHQ attached to the 3′ end.

[0144] 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.

[0145] 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℃.

[0146] 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 at the AX-86174278 and AX-109381183 loci.

[0147] IV. Identification of allele-specific markers at loci AX-86174278 and AX-109381183

[0148] Whole genome DNA was extracted from 160 natural populations (Table 4) irrigated in Xinxiang in 2019.

[0149] Using genomic DNA as templates, genotyping was performed using the KASP marker primers corresponding to the SNP markers AX-86174278 and AX-109381183, respectively, using the method described in section 2 of the above three steps, to detect the genotypes of different varieties.

[0150] The results are as follows Figure 2As shown in Table 2, it can be seen that SNP markers can effectively identify the genotypes of wheat loci AX-86174278 and AX-109381183.

[0151] Table 2 shows the segregation of allelic variations of markers AX-86174278 and AX-109381183 in natural populations.

[0152]

[0153]

[0154] a Number of corresponding genotypes

[0155] Table 3 shows the segregation of allelic variants of AX-86174278 and AX-109381183 in four environments within a natural population.

[0156]

[0157]

[0158] a Number of corresponding genotypes

[0159] 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

[0160] 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.

[0161] Table 4 shows information on natural populations.

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168] Therefore, the above results show that the SNP markers AX-86174278 and AX-109381183 are...

[0169] Both markers can be used to genotype individuals in natural populations. The AX-86174278 marker with the favorable genotype TT is widely used in natural populations, while the AX-109381183 marker with the favorable genotype AA is less common, indicating significant potential for improvement. This marker 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 is as follows:

[0170] 1) Determine whether the genotype of the wheat SNP locus AX-86174278 is TT, CC, or CT, as follows:

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 2) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The aforementioned green retention is reflected in chlorophyll content; the higher the chlorophyll content, the greater the green retention.

[0179] 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.

[0180] Example 2: Application of KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0181] t-tests were performed on the markers AX-86174278 and AX-109381183 in natural populations to verify the effectiveness of KASP markers and to assist in breeding decisions.

[0182] I. Testing canopy temperature and chlorophyll content of different wheat varieties during the later stages of irrigation

[0183] 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.

[0184] II. Genotyping of SNP loci in different wheat varieties

[0185] 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.

[0186] Using genomic DNA as a template, PCR amplification was performed using KASP-labeled primers corresponding to the SNP markers AX-86174278 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 red fluorescence upon irradiation, while the PCR amplification product carrying the fluorescent sequence HEX showed blue fluorescence upon irradiation.

[0187] 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.

[0188] The sequence of the fluorescent probe A is 5′-GAAGGTGACCAAGTTCATGCT-3′ (SEQ ID NO:9), with a fluorescent group FAM attached to the 5′ end;

[0189] The sequence of the fluorescent probe B is 5′-GAAGGTCGGAGTCAACGGATT-3′ (SEQ ID NO:10), with a fluorescent group HEX attached to the 5′ end;

[0190] The sequence of the above quenching probe A is 5′-AGCATGAACTTGGTCACCTTC-3′ (SEQ ID NO:11), with the quenching group BHQ attached to the 3′ end;

[0191] The sequence of the above-mentioned quenching probe B is 5′-AATCCGTTGACTCCGACCTTC-3′ (SEQ ID NO:12), with the quenching group BHQ attached to the 3′ end.

[0192] 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.

[0193] 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℃.

[0194] The PCR amplification products were processed in Pherastar. plus Genotyping was performed using a fluorescent microplate reader with fluorescence illumination, and then in KlusterCaller. TM The software reads the data after fracturing.

[0195] The judgment method is as follows:

[0196] 1) Determine whether the genotype of the wheat SNP locus AX-86174278 is TT, CC, or CT, as follows:

[0197] The TT genotype wheat samples showed greater or greater green and cool-type characteristics in the late canopy filling stage compared to or candidate-greater than the CC or CT genotype wheat samples.

[0198] 2) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0199] 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.

[0200] 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 the four environments of the natural population and the verification of the green-green, cool-toned trait are shown in Table 3. Figure 2 As shown.

[0201] The above results indicate that wheat varieties with only TT genotype at the AX-86174278 locus and only AA genotype at the AX-109381183 locus 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.

[0202] 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 specifically used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B 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 maintain greenness and are slightly cold during the later stages of canopy grain filling; The SNP site AX-86174278 is the 36th position of SEQ ID NO:7; The genotype of the SNP locus AX-86174278 is TT, CC, or CT.

2. The application according to claim 1, characterized in that: The substance specifically used to detect the genotype of the SNP locus AX-86174278 on wheat chromosome 1B is either A1 or A2. A1) Primer set; A2) PCR reagents or kits containing the complete set of primers described above; The primer set includes primer F1-1, primer F1-2, and primer 1-R; The nucleotide sequence of primer F1-1 is SEQ ID NO:1; The nucleotide sequence of primer F1-2 is SEQ ID NO:2; The nucleotide sequence of primer 1-R is SEQ ID NO:

3.

3. A method for assisting in the identification of the green retention of the canopy in the late grain-filling stage of wheat, comprising the following steps: detecting whether the genotype of SNP site AX-86174278 in the genome of the wheat to be tested is TT, CC or CT, and the green retention of the wheat to be tested with the TT genotype in the late grain-filling stage is greater than or candidate greater than that of the wheat to be tested with the CC or CT genotypes; The SNP site AX-86174278 is the 36th position of SEQ ID NO:

7.

4. The method according to claim 3, characterized in that: The method for detecting whether the genotype of 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 to obtain the genotype; The PCR reagent contains a set of primers, including primer F1-1, primer F1-2, and primer 1-R; The nucleotide sequence of primer F1-1 is SEQ ID NO:1; The nucleotide sequence of primer F1-2 is SEQ ID NO:2; The nucleotide sequence of primer 1-R is SEQ ID NO:3.

Images