Identification of Wheat with Green Canopy and Cold Type at Late Grain-filling Stage and Its Special KASP Markers

Abstract

The present application discloses a method for identifying a wheat crown layer green and cold type in the late filling stage and a KASP marker special for the method. The present application provides a substance for detecting the genotype of SNP site AX-861164768 on wheat chromosome 3A and / or a substance for detecting the genotype of SNP site AX-109381183 on wheat chromosome 4B, and an application of the substances in identifying or assisting in identifying a wheat crown layer green and cold type in the late filling stage. In addition, the present application designs KASP marker special primers, which can be used for identifying the wheat crown layer green and cold type in the late filling stage, and further 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 the identification of wheat canopy green and cool-toned type in the late grain-filling stage (21 days after flowering) and its specific KASP marker. Background Technology

[0002] Past research on wheat has mainly focused on high yield and disease resistance, lacking a deep understanding of the impact of high temperature stress.

[0003] Green retention, an important trait for drought resistance, refers to the ability of leaves to maintain their green color and prolong photosynthesis under high-temperature and drought stress. Green retention and stress resistance traits are closely linked, as evidenced in genetic research by the location of quantitative trait loci (QTLs) related to leaf senescence, drought, and temperature. These loci are found to be located in the same region of the genome, thus allowing for the selection of green retention traits while simultaneously improving drought stress resistance. Currently, the molecular mechanisms of green retention in wheat are not fully understood, and research on the processes of leaf development and senescence and yield formation is limited. This underscores the necessity and urgency of conducting in-depth research on wheat leaf green retention.

[0004] Greenness retention in wheat is a comprehensive manifestation of a series of intrinsic drought-resistance mechanisms, especially chlorophyll synthesis and degradation, and is also a quantitatively inherited trait controlled by multiple genes. Due to the complex influence of varietal genetic characteristics and environmental factors, greenness retention phenotypic values ​​cannot be studied using traditional cell biology and quantitative genetics methods. In recent years, the rapid development of molecular marker technology has provided an opportunity for research on the genetic mechanisms of greenness retention. Using molecular markers to perform genetic linkage analysis on greenness-related trait loci can provide a foundation for the breeding of drought-resistant and widely adaptable wheat varieties. Summary of the Invention

[0005] 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.

[0006] In a first aspect, the present invention provides the following A, A, and B, 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 late grain-filling stage of the wheat being 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 site AX-861164768 on wheat chromosome 3A;

[0016] The substance referred to as B is a substance for detecting the genotype of the SNP locus AX-109381183 on wheat chromosome 4B;

[0017] The SNP site AX-861164768 is the 36th position of the reverse complementary sequence of SEQ ID NO:7;

[0018] The SNP site AX-109381183 is the 36th position of SEQ ID NO:8.

[0019] In the applications described above,

[0020] The genotype of the SNP locus AX-861164768 is AA, GG, or AG;

[0021] The genotype of the SNP locus AX-109381183 is AA, GG, or AG.

[0022] In the applications described above,

[0023] The substance used to detect the genotype of the SNP locus AX-861164768 on wheat chromosome 3A is either A1 or A2.

[0024] A1) Primer set 1;

[0025] A2) PCR reagents or kits containing the complete set of primers 1;

[0026] The primer set 1 includes primer F1-1, primer F1-2, and primer 1-R;

[0027] The nucleotide sequence of primer F1-1 includes the sequence shown in positions 22-41 of SEQ ID NO:1;

[0028] The nucleotide sequence of primer F1-2 includes the sequence shown in positions 22-41 of SEQ ID NO:2;

[0029] The nucleotide sequence of primer 1-R is SEQ ID NO:3;

[0030] The substance used to detect the genotype of the SNP locus AX-109381183 on wheat chromosome 4B is either B1 or B2.

[0031] B1) Primer set 2;

[0032] B2) PCR reagents or kits containing the complete set of primers 2;

[0033] The primer set 2 includes primer F2-1, primer F2-2, and primer 2-R;

[0034] The nucleotide sequence of primer F2-1 includes the sequence shown in positions 22-39 of SEQ ID NO:4;

[0035] The nucleotide sequence of primer F2-2 includes the sequence shown in positions 22-39 of SEQ ID NO:5;

[0036] The nucleotide sequence of primer 2-R is SEQ ID NO:6.

[0037] In the applications described above,

[0038] The nucleotide sequence of primer F1-1 is SEQ ID NO:1;

[0039] The nucleotide sequence of primer F1-2 is SEQ ID NO:2;

[0040] Alternatively, the nucleotide sequence of primer F2-1 is SEQ ID NO:4;

[0041] The nucleotide sequence of primer F2-2 is SEQ ID NO:5.

[0042] Each of the aforementioned primer sets also includes fluorescent probe A, fluorescent probe B, quencher probe A, and quencher probe B;

[0043] 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.

[0044] The ends of fluorescent probe A and fluorescent probe B are labeled with different fluorescent groups;

[0045] The quenching probe A and the quenching probe B are labeled with different quenching groups at their ends.

[0046] In a second aspect, the present invention provides any of the following substances:

[0047] The primer set 1 described in the first aspect;

[0048] Or the primer set 1 and primer set 2 described in the first aspect;

[0049] Or PCR reagents or kits containing the primer set described in the first aspect;

[0050] Or the DNA fragment shown in SEQ ID NO:7 or its reverse complementary sequence;

[0051] Or the DNA fragment shown in SEQ ID NO:8;

[0052] The PCR reagent consists of PCR reagent 1 and PCR reagent 2;

[0053] 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;

[0054] 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;

[0055] 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.

[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 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.

[0059] Thirdly, the present invention provides the use of the primer set 1, the primer set 2, the PCR reagent or the kit described in the first aspect, in any of the following or in the preparation of products having any of the following characteristics:

[0060] 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;

[0061] 2) To identify or assist in identifying the greening retention of the wheat canopy during the later stages of grain filling;

[0062] 3) To identify or assist in identifying the temperature during the later stage of canopy grain filling in the wheat being tested;

[0063] 4) To identify or assist in identifying the degree of senescence in the late grain-filling stage of the wheat being tested;

[0064] 5) Select and breed wheat varieties that maintain greenness and are slightly cold during the later stages of canopy grain filling;

[0065] 6) Select and breed wheat varieties with low senescence levels;

[0066] 7) Select and breed high-yielding wheat varieties;

[0067] 8) Wheat genetics and breeding.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] The genetic breeding mentioned above, combined with a high-throughput phenotyping drone platform, has accelerated the application of drone spectroscopy in wheat genetic breeding.

[0075] Thirdly, the present invention provides any of the following methods:

[0076] A. A method for identifying or assisting in the identification of the green and cool-type characteristics of the canopy during the late grain-filling stage of wheat, namely, method A1, or methods A1 and A2:

[0077] The method shown in A1) 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:

[0078] 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.

[0079] The method shown in A2) 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:

[0080] 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.

[0081] B. A method for identifying or assisting in the identification of the greening capacity of the wheat canopy during the late grain-filling stage, namely, method B1, or methods B1 and B2:

[0082] The method shown in B1) 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:

[0083] 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.

[0084] 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:

[0085] 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.

[0086] C. A method for identifying or assisting in the identification of the temperature during the late stage of canopy filling in wheat, comprising method C1, or methods C1 and C2:

[0087] The method shown in C1) 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:

[0088] 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.

[0089] 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:

[0090] 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.

[0091] 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 as follows:

[0092] The method shown in D1) 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:

[0093] The degree of senescence in the late canopy filling stage 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.

[0094] 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:

[0095] 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.

[0096] In the method described above,

[0097] 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 1 described in the second aspect to obtain the genotype;

[0098] 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.

[0099] In the method of this invention, the genotype is obtained using KlusterCaller. TM The software determines the genotype based on fluorescence signals.

[0100] 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℃.

[0101] 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):

[0102] 1) Select wheat with the genotype AA for the SNP locus AX-861164768 in the method described in the third aspect;

[0103] 2) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0104] 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):

[0105] 1) Select wheat with the genotype AA for the SNP locus AX-861164768 in the method described in the third aspect;

[0106] 2) Select wheat with the genotype AA for the SNP locus AX-109381183 in the method described in the third aspect.

[0107] 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).

[0108] 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.

[0109] Beneficial effects of the present invention

[0110] This invention relates to two SNP loci associated with the late-stage green canopy and cool-climate trait in wheat, located at positions 557.96 and 443.45 Mb on wheat chromosomes 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 for 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

[0111] Figure 1 Linkage map of SNP markers with QTL-caas-3A and QTL-caas-4B2 genes.

[0112] 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 B are the detection results of AX-861164768 (denoted as AX-861164768-3A in the figure) and AX-109381183 (denoted as AX-109381183-4B2 in the figure), respectively. Detailed Implementation

[0113] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0114] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0115] 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.

[0116] Example 1: Obtaining KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0117] I. Investigation and SNP marker analysis of canopy greening and cool-climate traits

[0118] 1. Investigation of chlorophyll content and canopy temperature characteristics

[0119] 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.

[0120] 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).

[0121] 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.

[0122] 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:

[0123]

[0124] 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.

[0125] 2. SNP marker analysis

[0126] 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.

[0127] II. Gene localization and discovery of linkage markers AX-861164768 and AX-109381183

[0128] Using Icimapping 4.1 software, combined with 50k SNP chip genotyping data and phenotypic results, associated loci were located. Three QTLs (QTL-caas-3A, QTL-caas-4B2) associated with the target trait were identified.

[0129] 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-3A and QTL-caas-4B2 on the chromosome were determined. Two SNP loci, AX-861164768 and AX-109381183, were also identified. Figure 1 ).

[0130] SNP site AX-861164768 (also known as AX-861164768-3A) is the 36th position of the reverse complementary sequence of SEQ ID NO:7. 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.

[0131] SNP site AX-109381183 (also known as AX-109381183-4B2) is the 36th position of SEQ ID NO:8. The base N at this site is A or G, and it comes from the 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.

[0132] III. Development of KASP-specific primers

[0133] 1. Design of KASP marker-specific primers

[0134] 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.

[0135] Each SNP site has a dedicated KASP marker primer consisting of two upstream primers, namely primer F1 and primer F2, and one downstream primer, namely primer R.

[0136] Table 1 shows the marker primer sequence information.

[0137]

[0138] 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.

[0139] Specifically, for the AX-86164768 marker, primer F1 combined with primer R can amplify the fragment with genotype GG at the SNP site (the fragment containing the reverse complementary sequence of SEQ ID NO:7, and position 36 of the reverse complementary sequence of SEQ ID NO:7 is G), and primer F2 combined with primer R can amplify the fragment with genotype AA at the SNP site (the fragment containing the reverse complementary sequence of SEQ ID NO:7, and position 36 of the reverse complementary sequence of SEQ ID NO:7 is A). Primers F1 and F2, developed for the KASP marker at the AX-86164768 site, are used as reverse primers, and primer R is the forward primer.

[0140] 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.

[0141] 2. Amplification using KASP-labeled primers

[0142] 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.

[0143] 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.

[0144] 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

[0145] 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;

[0146] 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;

[0147] 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;

[0148] 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.

[0149] 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.

[0150] 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℃.

[0151] 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-861164768 and AX-109381183 loci.

[0152] IV. Identification of allele-specific markers at loci AX-861164768 and AX-109381183

[0153] Whole genome DNA was extracted from 160 natural populations (Table 4) irrigated in Xinxiang in 2019.

[0154] Using genomic DNA as templates, genotyping was performed using the KASP marker primers corresponding to the SNP markers AX-861164768 and AX-109381183, respectively, using the method described in section 2 of the above three steps, to detect the genotypes of different varieties.

[0155] The results are as follows Figure 2As shown in Table 2, it can be seen that these SNP markers can effectively identify the genotypes of wheat loci AX-861164768 and AX-109381183.

[0156] Table 2 shows the segregation of allelic variations of markers AX-861164768 and AX-109381183 in natural populations.

[0157] Tag Name genotype <![CDATA[Number a > AX-109381183 AA 53 GG 101 AG 0 ？ 6 AX-861164768 GG 133 AA 23 AG 3 ？ 1

[0158] a Number of corresponding genotypes

[0159] Table 3 shows the segregation of allelic variants of AX-861164768 and AX-109381183 in four environments within a natural population.

[0160]

[0161]

[0162] a Number of corresponding genotypes

[0163] 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

[0164] 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.

[0165] Table 4 shows information on natural populations.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] Therefore, the above results show that SNP markers AX-861164768 and AX-109381183 can both be used to genotype individuals in natural populations. The favorable genotype AA of these two markers is relatively rare in natural populations, indicating significant potential for improvement. This genotype can be used to quantify the leaf greenness and cool canopy characteristics of wheat in the late grain-filling stage (21 days after flowering). Specific analysis follows:

[0173] 1) The genotype of the wheat SNP locus AX-861164768 is determined to be AA, GG, or AG, as follows:

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 2) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0178] 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.

[0179] 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.

[0180] 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.

[0181] The aforementioned green retention is reflected in chlorophyll content; the higher the chlorophyll content, the greater the green retention.

[0182] 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.

[0183] Example 2: Application of KASP marker-specific primers associated with wheat canopy greenness and coolness traits

[0184] t-tests were performed on the markers AX-861164768 and AX-109381183 in natural populations to verify the effectiveness of KASP markers and to assist in breeding decisions.

[0185] I. Testing canopy temperature and chlorophyll content of different wheat varieties during the later stages of irrigation

[0186] 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.

[0187] II. Genotyping of SNP loci in different wheat varieties

[0188] 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.

[0189] Using genomic DNA as a template, PCR amplification was performed using KASP-labeled primers corresponding to the SNP markers AX-861164768 and AX-109381183, 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.

[0190] 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.

[0191] 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;

[0192] 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;

[0193] 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;

[0194] 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.

[0195] 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.

[0196] 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℃.

[0197] 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.

[0198] The judgment method is as follows:

[0199] 1) The genotype of the wheat SNP locus AX-861164768 is determined to be AA, GG, or AG, as follows:

[0200] The AA genotype wheat tested had a green canopy and a cool-type characteristic greater than or greater than the GG or AG genotype wheat tested.

[0201] 2) The genotype of the wheat SNP locus AX-109381183 is determined to be AA, GG, or AG, as follows:

[0202] The AA genotype wheat tested had a green canopy and a cool-type characteristic greater than or greater than that of the GG or AG genotype wheat tested.

[0203] 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.

[0204] The above results indicate that wheat varieties with the genotype AA at loci AX-861164768 and AX-109381183 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.

[0205] 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-861164768 on wheat chromosome 3A 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-861164768 is the 36th position of the reverse complementary sequence of SEQ ID NO:7; The genotype of the SNP locus AX-861164768 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-861164768 on wheat chromosome 3A is either A1 or A2. A1) Primer set; A2) PCR reagents or kits containing the complete set of primers described above; The primer set 1 consists of 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 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-861164768 If the genotype is AA, GG, or AG, determine the following: 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-861164768 is the 36th position of the reverse complementary sequence of SEQ ID NO:

7.

4. The method according to claim 3, characterized in that: The specific detection of SNP sites in the wheat genome to be tested AX-861164768 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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