A method for screening different thousand kernel weight and ear phenotype of wheat and a special kit thereof

Abstract

The application discloses a method for screening wheat with different thousand-grain weights and ear phenotypes, and comprises the following steps: detecting whether the genotype of a wheat to be tested based on a TaNAC92-2A gene is genotype I or genotype II, the ear length, the number of grains per ear, the number of fertile spikelets and / or the number of spikelets per ear of the wheat with genotype I are greater than those of the wheat with genotype II; the thousand-grain weight of the wheat with genotype I is less than that of the wheat with genotype II; the genotype of the wheat with genotype I based on an A415G SNP site is AA homozygous type; the genotype of the wheat with genotype II based on the A415G SNP site is GG homozygous type; and the A415G SNP site is the 415th nucleotide from the 5' end in the wheat genome, as shown in SEQ ID NO: 1. By detecting the genotype of the wheat to be tested based on the TaNAC92-2A gene, the wheat thousand-grain weight and / or the ear phenotype can be screened. The application has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for screening different thousand-grain-weight and ear phenotype wheat and a special kit thereof. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in China. Increasing wheat yield per unit is an important way to meet the increasing demand for food. Ear number, grain number per ear and thousand-grain weight are the three elements of wheat yield. Coordinating the relationship among the three elements and improving the level of the three elements is the key to wheat high-yield breeding. Compared with ear number and grain number per ear, thousand-grain weight has the greatest impact on wheat yield and is less affected by the environment. Therefore, improving thousand-grain weight is an important way to increase wheat yield.

[0003] At present, researchers have located a large number of QTLs regulating wheat thousand-grain weight. However, due to the small phenotypic contribution rate of most QTLs and poor reproducibility in different years and environments, QTLs are difficult to apply to genetic improvement of wheat thousand-grain weight.

[0004] Single nucleotide polymorphism (SNP) refers to DNA sequence polymorphism caused by single nucleotide variation at the genome level, which is mainly caused by single base transition, transversion, insertion or deletion, among which the frequency of transition is significantly higher than that of other forms of variation. In addition, the distribution of SNPs on the whole genome level or in a certain gene is very uneven. Some SNPs may be distributed within the gene sequence, and some SNPs may be distributed in the non-coding sequence outside the gene. Among them, the number of SNPs in the non-coding sequence is more than that in the coding sequence. At the same time, according to the analysis of the influence of SNPs on biological genetic traits, it is found that the SNPs in the coding sequence are mainly divided into two types. One is synonymous cSNP, that is, the SNP does not change the amino acid sequence and does not affect the protein sequence translated by the gene; the other is non-synonymous cSNP, that is, the SNP causes the change of amino acid sequence, which changes the translated protein sequence and further changes the function of the protein. At present, it is found that SNP has the advantages of high density, strong representation, strong genetic stability and easy realization of automatic analysis, which is highly valued by breeders and used as a genetic marker in association analysis, construction of precise genetic map, genetic analysis and species evolution to assist breeders in selection breeding.

[0005] Association analysis is a new method to identify the relationship between phenotypic traits and genetic markers or candidate genes in a natural population based on linkage disequilibrium, and further to mine the functional sites controlling the target traits. According to the different scanning ranges of molecular markers, association analysis can be divided into genome-wide association studies (GWAS) and candidate gene association study (CGAS). GWAS mainly uses SNP markers at the whole genome level to screen variation sites that can cause differences in specific target traits, and further to mine candidate genes highly associated with target traits to reveal the relationship between genotype and phenotypic traits. CGAS mainly analyzes the candidate genes by sequencing, and mines the allelic variations with positive regulation on target traits by associating the variation sites screened from the reference sequence of the candidate genes with the phenotypic traits of the natural population. Both of them have the advantages of less time-consuming, wide variation range and high positioning accuracy, and are widely used in crop research. However, there are differences in specific operation. The molecular markers used in GWAS are the same in analyzing LD, kinship, population structure, genotype and phenotype association; while the molecular markers used in CGAS are molecular functional markers developed based on the polymorphism of the reference sequence of the candidate genes.

[0006] CAPS marker is also called PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction), which is a kind of co-dominant molecular marker based on PCR, revealing the restriction length variation information of specific PCR fragments. Its basic principle is to amplify the target DNA by PCR, and then digest the amplification product into different size fragments by specific endonuclease, and distinguish directly on gel electrophoresis. Different alleles have different distribution of restriction enzyme cutting sites, producing different length of DNA fragment bands. The advantage is to avoid the cumbersome transfer and hybridization steps in RFLP analysis, and can maintain the accuracy of RFLP analysis. However, the SNP is less likely to be located at the restriction enzyme cutting site, therefore, dCAPS marker is proposed, that is, by introducing mismatched bases in the amplification primers, combining SNP sites to introduce new restriction endonuclease action sites, and producing polymorphism similar to CAPS marker. SUMMARY

[0007] The purpose of the present application is how to screen or assist in screening wheat with different thousand-grain weight, ear length, grain number per ear, fertile spikelet number and / or spikelet number per ear.

[0008] The present application first protects the screening or assisting in screening wheat with different thousand-grain weight, ear length, grain number per ear, fertile spikelet number and / or spikelet number per ear.

[0009] The application protects the screening or auxiliary screening of different thousand-grain weight, ear length, ear grain number, fertile spikelet number and / or spikelet number per ear of wheat, which can be method one, which can include the following steps: detecting whether the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype I or genotype II, the ear length, ear grain number, fertile spikelet number and / or spikelet number per ear of wheat of genotype I > wheat of genotype II; the thousand-grain weight of wheat of genotype I < wheat of genotype II;

[0010] The wheat of genotype I is wheat with AA homozygous genotype based on the A415G SNP site;

[0011] The wheat of genotype II is wheat with GG homozygous genotype based on the A415G SNP site;

[0012] The A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0013] The application protects the screening or auxiliary screening of different thousand-grain weight, ear length, ear grain number, fertile spikelet number and / or spikelet number per ear of wheat, which can be method two, which can include the following steps in turn:

[0014] (A1) Using the genomic DNA of the wheat to be tested as a template, using primer F1 and primer R1 to form a primer pair to perform PCR amplification, and obtaining PCR amplification product P1;

[0015] (A2) Using the PCR amplification product P1 as a template, using primer F2 and primer R2 to form a primer pair to perform PCR amplification, and obtaining PCR amplification product P2;

[0016] (A3) The PCR amplification product P2 is cut with restriction enzyme BglII to obtain a cut product; then the following judgment is made: if the cut product is two DNA fragments, the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype I; if the cut product is one DNA fragment, the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype II;

[0017] The ear length, ear grain number, fertile spikelet number and / or spikelet number per ear of wheat of genotype I > wheat of genotype II;

[0018] The thousand-grain weight of wheat of genotype I < wheat of genotype II;

[0019] The primer F1 is a single-stranded DNA molecule represented by SEQ ID NO: 4;

[0020] The primer R1 is a single-stranded DNA molecule represented by SEQ ID NO: 5;

[0021] The primer F2 is a single-stranded DNA molecule as shown in SEQ ID NO: 6;

[0022] The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO: 7.

[0023] The method for screening or assisting in screening wheat with different thousand-grain weight, ear length, grain number per ear, fertile spikelet number and / or spikelet number per ear can specifically be method three, which can sequentially comprise the following steps:

[0024] (B1) using the genomic DNA of the wheat to be tested as a template, performing PCR amplification with a primer pair composed of primer F1 and primer R1 to obtain a PCR amplification product P1;

[0025] (B2) using the PCR amplification product P1 as a template, performing PCR amplification with a primer pair composed of primer F2 and primer R2 to obtain a PCR amplification product P2;

[0026] (B3) performing enzyme digestion of the PCR amplification product P2 with restriction enzyme BglII to obtain an enzyme digestion product; and then performing the following evaluation: if the enzyme digestion product only has a 115bp DNA fragment and a 68bp DNA fragment, then the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype I; if the enzyme digestion product only has a 183bp DNA fragment, then the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype II;

[0027] The ear length, grain number per ear, fertile spikelet number and / or spikelet number per ear of the wheat of genotype I are greater than those of the wheat of genotype II;

[0028] The thousand-grain weight of the wheat of genotype I is less than that of the wheat of genotype II;

[0029] The primer F1 is a single-stranded DNA molecule as shown in SEQ ID NO: 4;

[0030] The primer R1 is a single-stranded DNA molecule as shown in SEQ ID NO: 5;

[0031] The primer F2 is a single-stranded DNA molecule as shown in SEQ ID NO: 6;

[0032] The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO: 7.

[0033] The method for screening or assisting in screening wheat with different thousand-grain weight, ear length, grain number per ear, fertile spikelet number and / or spikelet number per ear can specifically be method four, which can sequentially comprise the following steps:

[0034] (1) taking genomic DNA of the wheat to be tested as a template, performing PCR amplification on primer pair composed of primer F1 and primer R1 to obtain PCR amplification product P1;

[0035] The primer F1 is a single-stranded DNA molecule as shown in SEQ ID NO: 4;

[0036] The primer R1 is a single-stranded DNA molecule as shown in SEQ ID NO: 5;

[0037] (2) taking the PCR amplification product P1 as a template, performing PCR amplification on primer pair composed of primer F2 and primer R2 to obtain PCR amplification product P2;

[0038] The primer F2 is a single-stranded DNA molecule as shown in SEQ ID NO: 6;

[0039] The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO: 7;

[0040] (3) sequencing the PCR amplification product P2, and then judging as follows:

[0041] If the nucleotide sequence of the PCR amplification product P2 is as shown in SEQ ID NO: 2 from the 5' end 299-481, then the wheat to be tested is genotype I based on the TaNAC92-2A gene;

[0042] If the nucleotide sequence of the PCR amplification product P2 is as shown in SEQ ID NO: 3 from the 5' end 299-481, then the wheat to be tested is genotype II based on the TaNAC92-2A gene;

[0043] The ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike of the genotype I wheat > the genotype II wheat;

[0044] The thousand-grain weight of the genotype I wheat < the genotype II wheat.

[0045] The present application also protects a kit for identifying or assisting in identifying the thousand-grain weight, ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike of wheat. The kit can include substances for detecting whether the wheat to be tested is genotype I or genotype II based on the TaNAC92-2A gene;

[0046] The genotype I is the TaNAC92-2A gene with AA homozygous genotype based on the A415G SNP site;

[0047] The genotype II is the TaNAC92-2A gene with GG homozygous genotype based on the A415G SNP site;

[0048] The A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0049] The kit can specifically consist of a substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II.

[0050] The above-mentioned substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II can comprise primer pair A consisting of primer F1 and primer R1 and / or primer pair B consisting of primer F2 and primer R2.

[0051] The primer F1 is a single-stranded DNA molecule as shown in SEQ ID NO: 4.

[0052] The primer R1 is a single-stranded DNA molecule as shown in SEQ ID NO: 5.

[0053] The primer F2 is a single-stranded DNA molecule as shown in SEQ ID NO: 6.

[0054] The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO: 7.

[0055] The above-mentioned substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II can specifically consist of primer pair A and primer pair B.

[0056] The above-mentioned substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II can specifically consist of primer pair A.

[0057] The above-mentioned substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II can further comprise restriction enzyme BglII.

[0058] The above-mentioned substance for detecting whether the genotype of the TaNAC92-2A gene of the wheat to be tested is genotype I or genotype II can specifically consist of primer pair A, primer pair B and restriction enzyme BglII.

[0059] The present application also protects the molecular marker shown in SEQ ID NO: 1.

[0060] The present application also protects the use of the above-mentioned kit, which can be at least one of (z1)-(z4):

[0061] (z1) screening or assisting in screening wheat with different thousand kernel weight, ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike;

[0062] (z2) identifying or assisting in identifying wheat thousand kernel weight, ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike;

[0063] (z3) identifying or assisting in identifying genotype of wheat TaNAC92-2A gene;

[0064] (z4) wheat breeding.

[0065] The application also protects the use of the molecular marker shown in SEQ ID NO: 1 for at least one of (z1)-(z4):

[0066] (z1) screening or assisting in screening wheat with different thousand kernel weight, ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike;

[0067] (z2) identifying or assisting in identifying wheat thousand kernel weight, ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike;

[0068] (z3) identifying or assisting in identifying genotype of wheat TaNAC92-2A gene;

[0069] (z4) wheat breeding.

[0070] In the above, if the genotype of the A415G SNP site is AA homozygous type, it is determined as wheat of genotype I; if the genotype of the A415G SNP site is GG homozygous type, it is determined as wheat of genotype II. The A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome. The thousand kernel weight of the "wheat of genotype I" is < the thousand kernel weight of the "wheat of genotype II". The ear length of the "wheat of genotype I" is > the ear length of the "wheat of genotype II". The grain number per spike of the "wheat of genotype I" is > the grain number per spike of the "wheat of genotype II". The fertile spikelet number of the "wheat of genotype I" is > the fertile spikelet number of the "wheat of genotype II". The spikelet number per spike of the "wheat of genotype I" is > the spikelet number per spike of the "wheat of genotype II". The "> " can be statistically > in particular. The "<" can be statistically < in particular.

[0071] Experiments prove that the method provided by the application can be used to screen or assist in screening wheat thousand kernel weight and / or ear phenotype (such as ear length, grain number per spike, fertile spikelet number and / or spikelet number per spike) based on the genotype of the TaNAC92-2A gene of the wheat to be detected. The application has important application value in the process of wheat molecular marker assisted breeding. DETAILED DESCRIPTION

[0072] The present application is further described in detail by the specific embodiments below, which are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0073] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0074] In the following examples, the natural population is composed of 389 wheat varieties from different wheat growing areas (including northern winter wheat area, Huanghuai winter wheat area, middle and lower reaches of Yangtze River winter wheat area, southwestern winter wheat area, southern winter wheat area, northeastern spring wheat area, northern spring wheat area, northwestern spring wheat area, Qinghai-Tibet winter and spring wheat area, and Xinjiang winter and spring wheat area). Since the wheat materials are all cultivated species, they are usually assumed to be highly homozygous plant materials, and the genotypes are all homozygous.

[0075] Example 1, discovery of four SNP sites in wheat TaNAC92-2A gene and establishment of wheat genotyping method based on TaNAC92-2A gene

[0076] I. Discovery of four SNP sites in wheat TaNAC92-2A gene

[0077] The inventors of the present application discovered four SNP sites in the wheat TaNAC92-2A gene (nucleotide sequence as shown in SEQ ID NO: 1) by analyzing the polymorphism of 32 wheat varieties with high polymorphism, which are named A415G SNP, C741T SNP, C1027G SNP and G1306T SNP in turn. The names of the 32 wheat varieties are shown in Table 1.

[0078] Table 1

[0079] No. Wheat variety name No. Name 1 Jin 2148-7 17 04-030 2 Lin Kang 5108 18 Beijing 14 3 Chang 6878 19 Beijing 10 4 Chang Le 5 20 An 85 Zhong 124-1 5 White Quaker 21 Beijing 8686 6 Chang Wu 131 22 04-044 7 Dali 1 23 Spring 229th-25 8 Hong Heshang 24 Jingpin 10 9 Bawangbian 25 Spring 049th-5-1 10 Jimai 41 26 Spring 454th-50-1 11 Jimai 6 27 Jing 411 12 Cangzhou Wheat 28 Dan R 8093 13 Yanduan 1 29 Feng Kang 13 14 Ziganbaimangxian 30 Jinghe 8922 15 Neixiang 188 31 Zhongguochun 16 Baishuomai 32 Pandas

[0080] The A415G SNP is located at position 415 from the 5' end of SEQ ID NO: 1, and the genotypes are AA homozygote and GG homozygote. The C741T SNP is located at position 741 from the 5' end of SEQ ID NO: 1, and the genotypes are TT homozygote and CC homozygote. The C1027G SNP is located at position 1027 from the 5' end of SEQ ID NO: 1, and the genotypes are CC homozygote and GG homozygote. The G1306T SNP is located at position 1306 from the 5' end of SEQ ID NO: 1, and the genotypes are GG homozygote and TT homozygote. Since the genomic DNA is a double-stranded DNA molecule composed of two single-stranded DNA molecules in reverse complement, the DNA molecule encoding the protein is generally named as the sense DNA molecule; the DNA molecule reverse complementary to the sense DNA molecule is named as the antisense DNA molecule. The genotypes of the A415G SNP, the C741T SNP, the C1027G SNP and the G1306T SNP are all the genotypes of the sense DNA.

[0081] According to the different wheat TaNAC92-2A genes, the wheat is divided into two genotypes: TaNAC92-2A-1 (hereinafter referred to as genotype I) and TaNAC92-2A-2 (hereinafter referred to as genotype II). The nucleotide sequence of the TaNAC92-2A gene of genotype I wheat is shown in SEQ ID NO: 2. The nucleotide sequence of the TaNAC92-2A gene of genotype II wheat is shown in SEQ ID NO: 3.

[0082] II. Synthesis of primer pair A and primer pair B for amplifying the target sequence comprising A415G SNP, C741T SNP, C1027G SNP and G1306T SNP

[0083] Primer pair A and primer pair B for amplifying the target sequence comprising A415G SNP, C741T SNP, C1027G SNP and G1306T SNP are designed and synthesized. Primer pair A is composed of primer F1 and primer R1. Primer pair B is composed of primer F2 and primer R2.

[0084] The nucleotide sequences of the respective primers are as follows:

[0085] Primer F1: 5'-AGCGTCCGATCAATCACAGA-3' (SEQ ID NO: 4)

[0086] Primer R1: 5'-GCATCAACAACAACACACGC-3' (SEQ ID NO: 5)

[0087] Primer F2: 5'-TGGGGGAGAAGGAGTGGTA-3' (SEQ ID NO: 6)

[0088] Primer R2: 5'-CGGTGTAGAAGACGAGCGT-3' (SEQ ID NO: 7)

[0089] The target sequence amplified by primer pair B is shown in SEQ ID NO: 1 from the 5' end of the 299th to the 481st position.

[0090] Three, establishment of a genotyping method for wheat based on TaNAC92-2A gene

[0091] 1. Extract the genomic DNA of the wheat to be tested.

[0092] 2. Use the genomic DNA of the wheat to be tested in step 1 as a template, and use primer pair A composed of primer F1 and primer R1 to perform PCR amplification to obtain PCR amplification product P1.

[0093] 3. After step 2 is completed, use PCR amplification product P1 as a template, and use primer pair B composed of primer F2 and primer R2 to perform PCR amplification to obtain PCR amplification product P2.

[0094] 4. Perform enzyme digestion on PCR amplification product P2 obtained in step 3 using restriction enzyme BglII to obtain an enzyme digestion product; perform 2.5% agarose gel electrophoresis detection on the enzyme digestion product, and make the following judgment: if the enzyme digestion product is band type A (showing two bands, 115 bp and 68 bp respectively), then the A415G SNP of the wheat to be tested is AA homozygous type, i.e., the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype I; if the enzyme digestion product is band type B (showing one band, 183 bp), then the A415G SNP of the wheat to be tested is GG homozygous type, i.e., the genotype of the wheat to be tested based on the TaNAC92-2A gene is genotype II.

[0095] Example 2, correlation analysis between the genotype of wheat based on the TaNAC92-2A gene and the thousand-grain weight and spike phenotype of wheat

[0096] Each wheat variety in the natural population was genotyped using the method in step three of Example 1. The natural population consisted of 389 wheat varieties (all hexaploid). The names of the wheat varieties are shown in Table 2.

[0097] The genotypes of 389 wheat varieties based on the TaNAC92-2A gene are shown in Table 2: the genotypes of 349 wheat varieties based on the TaNAC92-2A gene are genotype II, and the genotypes of 40 wheat varieties based on the TaNAC92-2A gene are genotype I.

[0098] Table 2. Genotypes based on the TaNAC92-2A gene in the natural population

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Note: I is genotype I, and II is genotype II.

[0111] II. Trait detection of thousand-grain weight, ear length, grain number per ear, fertile spikelet number, and spikelet number per ear

[0112] The 389 wheat varieties in the natural population were planted in K1-K5, a total of 5 environments. After harvesting, the average thousand-grain weight, average ear length, average grain number per ear, average fertile spikelet number, and average spikelet number per ear of the two genotypes of wheat were counted respectively.

[0113] 5 environments were 2018-SX-LN (K1), 2018-SX-N (K2), 2019-SX-LN (K3), 2019-SX-N (K4) and 2019-HB-N (K5), and the naming method was planting year-planting place-nitrogen treatment. Among them, SX was the experimental field of Shensheng of Shanxi Agricultural University, HB was the experimental field of Zhaoxian in Hebei, N was normal nitrogen treatment (that is, nitrogen application, the amount of nitrogen application was 18 kg / 667m2 (calculated according to pure nitrogen), 30%, 40% and 30% of the total amount of nitrogen fertilizer were applied before wintering, jointing stage and heading stage irrigation), and LN was low nitrogen treatment (that is, no nitrogen was applied during the whole growth period of wheat).

[0114] The statistical results are shown in Table 3.

[0115] Table 3-1

[0116]

[0117] Note: P value is the significance level of correlation analysis, * P <0.05, ** P <0.01, *** P <0.001.

[0118] Table 3-2

[0119]

[0120] Note: P value is the significance level of correlation analysis, * P <0.05, ** P <0.01, *** P <0.001.

[0121] Table 3-3

[0122]

[0123]

[0124] Note: P value is the significance level of correlation analysis, * P <0.05, ** P <0.01, *** P <0.001.

[0125] Table 3-4

[0126]

[0127] Note: P value is the significance level of correlation analysis, ** P <0.01, ***This indicates that P < 0.001.

[0128] Table 3-5

[0129]

[0130] Note: P-value represents the significance level in the association analysis. * This indicates that P < 0.05. ** This indicates that P < 0.01. *** This indicates that P < 0.001.

[0131] III. Correlation Analysis

[0132] The general linear model (GLM) in Tassel 5.0 software was used to perform association analysis between the genotype of wheat TaNAC92-2A gene in natural population and thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets, and number of spikelets per spike. The results are shown in Table 3.

[0133] The results showed that in a natural population of 389 wheat varieties, the thousand-grain weight of genotype I wheat was less than that of genotype II wheat; the spike length of genotype I wheat was greater than that of genotype II wheat; the number of grains per spike of genotype I wheat was greater than that of genotype II wheat; the number of grain-filled spikelets of genotype I wheat was greater than that of genotype II wheat; and the number of spikelets per spike of genotype I wheat was greater than that of genotype II wheat. ">" indicates statistical significance, and "<" indicates statistical significance. The study of the natural population indicates that genotype I is the superior genotype for increasing spike length, number of grains per spike, number of grain-filled spikelets, and number of spikelets per spike, while genotype II is the superior genotype for increasing thousand-grain weight.

[0134] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for screening or assisting in screening wheat with different thousand-grain weights, spike lengths, number of grains per spike, number of grain-filled spikelets, and / or number of spikelets per spike, comprising the following steps: detecting the wheat to be tested based on... TaNAC92-2A The genotype of the gene is either genotype I or genotype II. Wheat with genotype I has a longer spike length, more grains per spike, more fertile spikelets, and / or more spikelets per spike than wheat with genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II; The wheat of genotype I is the AA homozygous wheat based on the A415G SNP site; The wheat of genotype II is the wheat with a genotype of GG homozygote based on the A415G SNP site; The A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO:1 in the wheat genome.

2. A method for screening or assisting in screening wheat with different thousand-grain weights, spike lengths, number of grains per spike, number of fertile spikelets, and / or number of spikelets per spike, comprising the following steps in sequence: (A1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer pair A composed of primers F1 and R1 to obtain PCR amplification product P1; (A2) Using the PCR amplification product P1 as a template, PCR amplification was performed on primer pair B composed of primer F2 and primer R2 to obtain PCR amplification product P2; (A3) The PCR amplification product P2 was digested with the restriction endonuclease BglII to obtain the digested product; then the following evaluation was performed: if the digested product consists of two DNA fragments, then the wheat sample tested is based on... TaNAC92-2A The gene's genotype is genotype I; if the enzyme digestion product is a DNA fragment, then the wheat being tested is based on... TaNAC92-2A The gene's genotype is genotype II; Wheat of genotype I has greater spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike than wheat of genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II; The primer F1 is the single-stranded DNA molecule shown in SEQ ID NO:4; The primer R1 is the single-stranded DNA molecule shown in SEQ ID NO:5; The primer F2 is the single-stranded DNA molecule shown in SEQ ID NO:6; The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO:

7.

3. A method for screening or assisting in screening wheat with different thousand-grain weights, spike lengths, number of grains per spike, number of fertile spikelets, and / or number of spikelets per spike, comprising the following steps in sequence: (B1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer pair A composed of primers F1 and R1 to obtain PCR amplification product P1; (B2) Using the PCR amplification product P1 as a template, PCR amplification was performed on primer pair B composed of primer F2 and primer R2 to obtain PCR amplification product P2; (B3) The PCR amplification product P2 was digested with the restriction endonuclease BglII to obtain the digested product; then the following evaluation was performed: if the digested product contained only a 115bp DNA fragment and a 68bp DNA fragment, then the wheat sample tested was based on... TaNAC92-2A The gene's genotype is genotype I; if the enzyme digestion product contains only a 183bp DNA fragment, then the wheat being tested is based on... TaNAC92-2A The gene's genotype is genotype II; Wheat of genotype I has greater spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike than wheat of genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II; The primer F1 is the single-stranded DNA molecule shown in SEQ ID NO:4; The primer R1 is the single-stranded DNA molecule shown in SEQ ID NO:5; The primer F2 is the single-stranded DNA molecule shown in SEQ ID NO:6; The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO:

7.

4. A method for screening or assisting in screening wheat with different thousand-grain weights, spike lengths, number of grains per spike, number of fertile spikelets, and / or number of spikelets per spike, comprising the following steps in sequence: (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer pair A composed of primers F1 and R1 to obtain PCR amplification product P1; The primer F1 is the single-stranded DNA molecule shown in SEQ ID NO:4; The primer R1 is the single-stranded DNA molecule shown in SEQ ID NO:5; (2) Using the PCR amplification product P1 as a template, primer pair B composed of primer F2 and primer R2 was used for PCR amplification to obtain PCR amplification product P2; The primer F2 is the single-stranded DNA molecule shown in SEQ ID NO:6; The primer R2 is the single-stranded DNA molecule shown in SEQ ID NO:7; (3) Sequencing the PCR amplification product P2 and then evaluating it as follows: If the nucleotide sequence of the PCR amplification product P2 is as shown in SEQ ID NO:2 from position 299 to 481 starting from the 5' end, then the wheat to be tested is based on TaNAC92-2A The gene's genotype is genotype I; If the nucleotide sequence of the PCR amplification product P2 is as shown in SEQ ID NO:3 from position 299 to 481 starting from the 5' end, then the wheat to be tested is based on TaNAC92-2A The gene's genotype is genotype II; Wheat of genotype I has greater spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike than wheat of genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II.

5. The application of the reagent kit is at least one of (z1)-(z4): (z1) Screening or assisted screening of wheat with different thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; (z2) To identify or assist in the identification of wheat thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; (z3) Identification or auxiliary identification of wheat TaNAC92-2A The genotype of a gene; (z4) Breeding of wheat based on thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; The kit includes a detection method for wheat based on... TaNAC92-2A The substance whose genotype is genotype I or genotype II; Genotype I is the AA homozygous genotype based on the A415G SNP locus. TaNAC92-2A Gene; Genotype II is the GG homozygous type based on the A415G SNP locus. TaNAC92-2A Gene; The A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO:1 in the wheat genome; Wheat of genotype I has greater spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike than wheat of genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II.

6. The application according to claim 5, characterized in that: The detection of wheat is based on TaNAC92-2A The substances that determine whether the gene is genotype I or genotype II include primer pair A consisting of primer F1 and primer R1 and / or primer pair B consisting of primer F2 and primer R2; The primer F1 is the single-stranded DNA molecule shown in SEQ ID NO:4; The primer R1 is the single-stranded DNA molecule shown in SEQ ID NO:5; The primer F2 is the single-stranded DNA molecule shown in SEQ ID NO:6; The primer R2 is a single-stranded DNA molecule as shown in SEQ ID NO:

7.

7. The application according to claim 6, characterized in that: The detection of wheat is based on TaNAC92-2A The genotype of a gene, whether it is genotype I or genotype II, also includes the restriction endonuclease BglII.

8. The application of the molecular marker shown in SEQ ID NO:1, for at least one of (z1)-(z4): (z1) Screening or assisted screening of wheat with different thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; (z2) To identify or assist in the identification of wheat thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; (z3) Identification or auxiliary identification of wheat TaNAC92-2A The genotype of a gene; (z4) Breeding of wheat based on thousand-grain weight, spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike; The application is achieved through the A415G SNP site; the A415G SNP site is the 415th nucleotide from the 5' end of SEQ ID NO:1 in the wheat genome; If the genotype based on the A415G SNP locus is homozygous for AA, then based on TaNAC92-2A The gene's genotype is genotype I; If the genotype based on the A415G SNP locus is GG homozygous, then based on TaNAC92-2A The gene's genotype is genotype II; Wheat of genotype I has greater spike length, number of grains per spike, number of fertile spikelets and / or number of spikelets per spike than wheat of genotype II. The thousand-grain weight of wheat of genotype I is less than that of wheat of genotype II.

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