A molecular marker closely linked to a wheat total spikelet number qtl and application thereof

Abstract

This invention discloses a molecular marker closely linked to a QTL for the total number of spikelets in wheat and its applications. This invention provides the application of a specific fragment on the wheat 3D chromosome as a molecular marker in any of the following: identifying the total number of spikelets in wheat; selecting wheat plants, lines, strains, or varieties with a relatively high total number of spikelets; wherein the specific fragment is the DNA fragment shown in SEQ ID No. 5. This invention provides a new tool for marker-assisted selection breeding of wheat, which can accelerate the breeding process of high-yielding wheat varieties.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and molecular breeding, specifically to a molecular marker closely linked to the total number of spikelets in wheat (QTL, quantitative trait locus) and its application. Background Technology

[0002] Common wheat (Triticum aestivum L.) is one of my country's major food crops, and its yield directly affects the living standards of residents and national food security (He Zhonghu, Zhuang Qiaosheng, Cheng Shunhe et al. (2018) Development and scientific and technological progress of China's wheat industry. Acta Agronomica Sinica, 8:99-106.). High yield is the main goal of wheat breeding. The number of grains per spike is one of the three factors that constitute yield and is easily affected by the environment. The total number of spikelets is a direct component of the number of grains per spike and is significantly positively correlated with the number of grains per spike (Kuzay S. et al. (2019) Identification of a candidate gene for a QTL forspikelet number per spike on wheat chromosome arm 7AL by high-resolution genetic mapping. Theor Appl Genet, 132:2689-2705.). The differentiation of wheat spikelets begins at the two-ridge stage and ends with the appearance of the terminal spikelet during floret differentiation. This process is determined early in the reproductive development of wheat and is relatively unaffected by environmental conditions, exhibiting high heritability (Zhang J. Let et al. (2018) Identification and validation of QTL for grain yield and plant water status under contrasting water treatments in fall-sown spring wheats. Theor Appl Genet, 131:1741-1759.). Therefore, breeding varieties with a higher total number of spikelets to promote coordinated improvement of yield traits such as grain number per spike and continuously enhance yield potential is an effective approach to high-yield wheat breeding (He Zhonghu et al. (2011) Progress and Prospect of Wheat Breeding in China. Acta Agronomica Sinica, 37, 202-215.). Therefore, it is necessary to discover and clone major QTLs (quantitative trait loci) and their functional genes that control spikelet number in superior germplasm resources with multiple spikelet numbers, and to develop molecular markers to provide gene resources and available markers for the genetic improvement of total spikelet number and grain number per spike in wheat and for molecular marker-assisted breeding for high yield.

[0003] The correlation between total spikelet number and grain number per spike in wheat is a typical quantitative trait, controlled by multiple genes. QTL analysis can decompose complex quantitative traits into individual QTLs. Locating each QTL and analyzing its genetic effects helps to deeply understand the genetic basis of complex traits and lays the foundation for marker-assisted selection of related QTLs (Gupta PK (2008) Wheatgenetics in the post-genomics era. Curr Sci India, 95:1660-1662.). Marker-assisted selection breeding is an effective method that uses molecular markers associated with specific traits as an auxiliary means of breeding. It has the advantages of not being limited by environmental conditions, being detectable in all tissues and growth stages of plant development, and having high selection efficiency. At present, although some QTLs for the correlation between spikelet number and grain number per spike have been found in common wheat, there is still a lack of molecular markers for total spikelet number available for breeding. It is necessary to continue to explore new QTL loci for total spikelet number and develop molecular markers closely linked to them to improve the selection efficiency of total spikelet number in the breeding process and cultivate new high-yielding wheat varieties. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker closely linked to the QTL for the total number of spikelets in wheat and its application.

[0005] In a first aspect, the present invention claims protection for the use of a specific segment on the wheat 3D chromosome as a molecular marker in any of the following:

[0006] (A1) To identify or assist in the identification of the total number of spikelets in wheat;

[0007] (A2) Prepare products for identifying or assisting in the identification of the total number of spikelets in wheat;

[0008] (A3) Compare the total number of spikelets in the wheat samples;

[0009] (A4) Prepare a product for comparing the total number of spikelets in the wheat to be tested;

[0010] (A5) Select wheat plants, lines, strains or varieties with a relatively large total number of spikelets;

[0011] (A6) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively large total number of spikelets;

[0012] (A7) Select and breed wheat individual plants, lines, strains or varieties with a relatively small total number of spikelets;

[0013] (A8) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively small total number of spikelets;

[0014] The specific fragment is the DNA fragment shown in SEQ ID No. 5.

[0015] Secondly, the present invention claims protection for the use of a substance for detecting whether a specific segment is present on the 3D chromosome of wheat in any of the following:

[0016] (A1) To identify or assist in the identification of the total number of spikelets in wheat;

[0017] (A2) Prepare products for identifying or assisting in the identification of the total number of spikelets in wheat;

[0018] (A3) Compare the total number of spikelets in the wheat samples;

[0019] (A4) Prepare a product for comparing the total number of spikelets in the wheat to be tested;

[0020] (A5) Select wheat plants, lines, strains or varieties with a relatively large total number of spikelets;

[0021] (A6) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively large total number of spikelets;

[0022] (A7) Select and breed wheat individual plants, lines, strains or varieties with a relatively small total number of spikelets;

[0023] (A8) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively small total number of spikelets;

[0024] The specific fragment is the DNA fragment shown in SEQ ID No. 5.

[0025] The specific fragment is located at molecular marker 3D-Ind7308, and the closely linked QTL is named QTss-3D. QTss-3D is located in the region between molecular markers AX-108807742 and AX-111064903, corresponding to a physical region of 3.46 Mb (121.98-125.44 Mb) on chromosome 3DS of the Chinese Spring reference genome (Refseq v1.0). The specific fragment corresponds to position 125387308 on chromosome 3D of the Chinese Spring reference genome (Refseq v1.0).

[0026] The substance used to detect whether a specific segment is present on the wheat 3D chromosome can be a primer pair used to amplify the specific segment.

[0027] Furthermore, the upstream primer in the primer pair can be designed based on a sequence in the wheat 3D chromosome located upstream of the specific fragment, and the downstream primer can be designed based on a sequence in the wheat 3D chromosome located downstream of the specific fragment.

[0028] In a specific embodiment of the present invention, the primer pair consists of a single-stranded DNA molecule shown in SEQ ID No. 3 and a single-stranded DNA molecule shown in SEQ ID No. 4.

[0029] Thirdly, the present invention claims a method for identifying or assisting in the identification of the total number of spikelets in wheat.

[0030] The method for identifying or assisting in the identification of the total number of spikelets in wheat, as claimed in this invention, may include the following steps:

[0031] (B1) Detect whether the wheat 3D chromosome contains a specific segment;

[0032] (B2) Based on the results of (B1), the total number of spikelets of the wheat to be tested is determined as follows: the total number of spikelets of the wheat to be tested (homozygous) that does not contain the specific segment in the 3D chromosome (i.e., the "specific segment" is missing in the 3D chromosome) is greater than or more candidates than the total number of spikelets of the wheat to be tested (homozygous) that contains the specific segment in the 3D chromosome (i.e., the "specific segment" is not missing in the 3D chromosome);

[0033] The specific fragment is the DNA fragment shown in SEQ ID No. 5.

[0034] Fourthly, the present invention claims protection for a method for breeding wheat single plants, lines, strains, or varieties with a relatively large total number of spikelets, which may include the following steps (C1) and (C2):

[0035] (C1) Detect whether the wheat 3D chromosome contains a specific segment;

[0036] (C2) Based on the results of (C1), select the test wheat (homozygous) whose 3D chromosome does not contain the specific segment (i.e., the 3D chromosome is missing the "specific segment") as the parent for breeding, and select wheat (homozygous) whose 3D chromosome does not contain the specific segment (i.e., the 3D chromosome is missing the "specific segment") in each generation of breeding, and finally obtain wheat single plants or lines or varieties with a relatively large total number of spikelets.

[0037] The specific fragment is the DNA fragment shown in SEQ ID No. 5.

[0038] Fifthly, the present invention claims a method for breeding wheat plants, lines, strains, or varieties with a relatively small total number of spikelets, comprising the following steps (D1) and (D2):

[0039] (D1) Detect whether the wheat 3D chromosome contains a specific segment;

[0040] (D2) Based on the results of (D1), select the test wheat (homozygous) containing the specific segment in the 3D chromosome (i.e., the 3D chromosome does not have the missing "specific segment") as the parent for breeding, and select wheat (homozygous) containing the specific segment in the 3D chromosome (i.e., the 3D chromosome does not have the missing "specific segment") in each generation of breeding, and finally obtain wheat single plants or lines or varieties with a relatively small total number of spikelets.

[0041] The specific fragment is the DNA fragment shown in SEQ ID No. 5.

[0042] In the fourth and fifth aspects above, the method for detecting whether the wheat 3D chromosome to be tested contains a specific segment may be as follows (E1) or (E2):

[0043] (E1) direct sequencing;

[0044] (E2) Using the genome of the wheat to be tested as a template, PCR amplification is performed using the primer pair described in the second aspect above, and the specific fragment is determined based on the amplification results.

[0045] Furthermore, using the genome of the wheat to be tested as a template, PCR amplification is performed using the primer pair (SEQ ID No. 3 and SEQ ID No. 4). If the amplification product is a 132bp target fragment, then the wheat 3D chromosome to be tested contains the specific fragment; if the amplification product is a 90bp target fragment, then the wheat 3D chromosome to be tested does not contain the specific fragment.

[0046] In this invention, the total number of spikelets refers to the total number of spikelets of the main spike.

[0047] Sixthly, the present invention claims protection for any of the following biological materials:

[0048] (F1) DNA fragment, which is a DNA fragment obtained by extending one or more nucleotides into or out of the 5' and 3' ends of a wheat 3D chromosome, containing at least the sequence shown in SEQ ID No. 5.

[0049] Furthermore, the nucleotide sequence of the DNA fragment is positions 36-167 of SEQ ID No. 1 or SEQ ID No. 1.

[0050] Specifically, positions 36-167 of SEQ ID No. 1 are the amplification products of the primer pair (SEQ ID No. 3 and SEQ ID No. 4) when the specific DNA fragment is not missing.

[0051] (F2) Primer pair, which is the primer pair described in the second aspect above (SEQ ID No. 3 and SEQ ID No. 4);

[0052] (F3) A reagent or kit containing the primer pair described in (F2).

[0053] If needed, the kit may also contain conventional PCR reagents, such as PCR reaction buffer, dNTPs, DNA polymerase, etc.

[0054] Seventhly, the present invention claims the use of the method described in the fourth or fifth aspect above, or the biological material described in the sixth aspect above, in any of the following:

[0055] (G1) Increases the number of grains per wheat ear;

[0056] (G2) Increase wheat yield;

[0057] (G3) Develop high-yield wheat varieties.

[0058] The number of grains per wheat ear can be the number of grains per main wheat ear.

[0059] In this invention, a relatively high total spikelet count means that, when the effects of other loci affecting the total spikelet count in the compared wheat genome are equal, the total spikelet count of the test wheat (homozygous) that does not contain the specific segment on the 3D chromosome (i.e., the "specific segment" is missing on the 3D chromosome) is higher than the total spikelet count of the test wheat that contains the specific segment on the 3D chromosome (i.e., the "specific segment" is not missing on the 3D chromosome). A relatively low total spikelet count means that, when the effects of other loci affecting the total spikelet count in the compared wheat genome are equal, the total spikelet count of the test wheat (homozygous) that contains the specific segment on the 3D chromosome (i.e., the "specific segment" is not missing on the 3D chromosome) is lower than the total spikelet count of the test wheat that does not contain the specific segment on the 3D chromosome (i.e., the "specific segment" is missing on the 3D chromosome).

[0060] In the foregoing, the wheat may be, but is not limited to, any one or more of the following varieties: Gao 8901, 3228, and hybrids of Gao 8901 and 3228. The hybrids of Gao 8901 and 3228 may be, for example, the PG-RIL genetic population or near-isogenic population of 3228 / Gao 8901.

[0061] Experiments have demonstrated that the molecular marker 3D-Ind7308 provided by this invention is a molecular marker tightly linked to a major QTL for the total number of spikelets in wheat. The major QTL locus for the total number of spikelets is located on the short arm of the wheat chromosome 3D, named QTss-3D, and is located in the AX-108807742–AX-111064903 region, corresponding to a 3.46 Mb physical region in the Chinese Spring Reference Genome (RefSeqv1.0). Using the molecular marker 3D-Ind7308 provided by this invention, it can be used to identify or assist in identifying the total number of spikelets in wheat, compare the total number of spikelets in wheat samples, and select wheat plants, lines, strains, or varieties with a relatively high number of spikelets. This invention provides a new tool for marker-assisted selection breeding of wheat, which can accelerate the breeding process of high-yielding wheat varieties. Attached Figure Description

[0062] Figure 1 Chromosomal localization of the locus QTss-3D, which controls the total spikelet number, was determined under four different environments. The total spikelet number QTL QTss-3D was located in the region between molecular markers AX-108807742 and AX-111064903.

[0063] Figure 2 This data represents the genotyping results of 3D-Ind7308 in the parents and a subset of the PG-RIL population. PG1, PG3, PG8, PG12, PG21, and PG23 are distinct lines within the PG-RIL population.

[0064] Figure 3 The difference in total spikelet number between families carrying two different allelic variants, 3D-Ind7308(P) and 3D-Ind7308(G), in the PG-RIL population. ** indicates extremely significant difference (P<0.01).

[0065] Figure 4 Differences in total spikelet number and grain number per spikelet were found among families carrying two different allelic variations, 3D-Ind7308(P) and 3D-Ind7308(G), in a near-isogenic population derived from the remaining heterozygous lines. ** indicates extremely significant difference (P<0.01), * indicates significant difference (P<0.05). Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] Wheat germplasm resource 3228 is described in “Wang J. et al. (2011) QTL mapping of yield-related traits in the wheat germplasm 3228. Euphatica 177:277-292.” It is available to the public from the applicant and may only be used to repeat the experiments of this invention and may not be used for other purposes.

[0069] Example 1: Discovery of InDel loci related to total spikelet number in wheat and development and validation of PCR markers.

[0070] I. Construction of Recombinant Inbred Line Population

[0071] Using wheat germplasm resource 3228 as the female parent and wheat variety Gao 8901 as the male parent, a hybridization was conducted, and the F1 generation containing 176 lines was obtained using the single-seed propagation method. 6:9 The population of recombinant inbred lines (PG-RIL).

[0072] II. Identification of total spikelet number trait in PG-RIL populations under multi-year environmental conditions

[0073] The PG-RIL population was planted at the Luancheng Agro-ecosystem Experimental Station of the Chinese Academy of Sciences (37°53'15″N, 114°40'47″E) for four years: 2013-2014, 2014-2015, 2015-2016, and 2016-2017. These four environments are referred to as LC13, LC14, LC15, and LC16, respectively. Each family was planted in two rows, 1.5 m long, with a row spacing of 0.25 m, and 30 seeds per row. Water, fertilizer, and other management practices in all field trials followed local standards. After wheat maturity, 10 plants were randomly selected from each line to investigate the total number of spikelets on the main ear, and the average value was taken.

[0074] III. Genotype Scanning and QTL Mapping

[0075] 1. Genomic DNA extraction

[0076] DNA was extracted from 176 lines and parents of the PG-RIL population using the CTAB method, and their concentrations were detected using NanoDrop2000.

[0077] 2. Wheat 660K SNP chip hybridization

[0078] Take the genomic DNA obtained in step 1 and mix it with a wheat 660K SNP chip ( The Wheat660 Genotyping Arrays were hybridized, and genotyping was performed on 176 lines to detect their genotypes.

[0079] 3. Localization of the stable major effect QTL QTss-3D for total spikelet number in wheat

[0080] Using a PG-RIL genetic mapping population, QTL mapping of the total spikelet number trait was conducted at the Luancheng Ecological Experimental Station of the Chinese Academy of Sciences over four years under four environmental conditions. A stable major-effect QTL, QTss-3D, controlling the total spikelet number was detected in the peak interval of chromosome 3D, AX-108807742–AX-111064903 (Table 1). Figure 1 This study explained 10.4–21.5% of the total spikelet number phenotypic variation, with the additive effect of increasing the total spikelet number originating from 3228, which increased the total spikelet number by an average of 0.545 (Table 1). The QTss-3D physical region corresponds to a physical region of 3.46 Mb, ranging from 121.98 to 125.44 Mb, on the 3D chromosome of the Chinese Spring reference genome (RefSeq v1.0).

[0081] Table 1. QTL information on the total number of spikelets in the QTss-3D segment.

[0082]

[0083] IV. Development of PCR Detection Markers

[0084] Using the resequencing results of genomes 3228 and Gao 8901, the genomic sequence differences between the parents within the target region were compared. It was found that compared to the Gao 8901 sequence (SEQ ID No. 1), the 3228 sequence (SEQ ID No. 2) contained a 42-base deletion at position 125387308 on chromosome 3D (physical location based on the Chinese spring reference genome (RefSeq v1.0), resulting in an insertion / deletion mutation. To address this insertion / deletion mutation, this invention developed the PCR marker 3D-Ind7308, with specific primer sequences SEQ ID No. 3 and SEQ ID No. 4. For the inserted DNA fragment, the amplified fragment length was 132 bp (positions 36-167 of SEQ ID No. 1), and for the deletion, the amplified fragment length was 90 bp (positions 36-125 of SEQ ID No. 2).

[0085] 3D-Ind7308-F: 5'-TTTCCTCCAATGTGTCGGCA-3' (SEQ ID No. 3);

[0086] 3D-Ind7308-R: 5'-CGGCTCATATACCGACCCAC-3' (SEQ ID No. 4).

[0087] Using genomic DNA from 3228 and Gao8901 as templates, PCR amplification was performed on the templates using genome-specific primers 3D-Ind7308-F / R.

[0088] The PCR reaction system and amplification procedure are shown in Table 2.

[0089] Table 2. PCR reaction system and amplification procedure

[0090] Element Volume (μL) 2×PCR Mastermix 12.5 3D-Ind7308-F (10μM) 0.5 3D-Ind7308-R (10μM) 0.5 DNA template 2.5 <![CDATA[ddH2O]]> 9 Total volume (μL) 25

[0091] The PCR reaction procedure is shown in Table 3.

[0092] Table 3. PCR reaction procedure

[0093]

[0094] After the PCR amplification products were separated by electrophoresis on a 2.5% agarose gel, they were detected using a gel imaging system.

[0095] If the same target band as the parent *Gynostemma pentaphyllum* 8901 (132 bp, positions 36-167 of SEQ ID No. 1) is amplified, the allele genotype is named 3D-Ind7308(G). If the same target band as the parent 3228 (90 bp, positions 36-125 of SEQ ID No. 2) is amplified, the allele genotype is named 3D-Ind7308(P). In the case of heterozygosity, two bands will be amplified, one 132 bp and the other 90 bp.

[0096] V. Genotyping and Validation Results

[0097] Following the method described in step four, the PG-RIL population constructed in step one was scanned using 3D-Ind7308 markers. The results showed that 84 families amplified a 90bp band corresponding to the superior allele 3D-Ind7308(P), and 90 families amplified a 132bp band corresponding to the 3D-Ind7308(G) allele (the other two families amplified heterozygous bands). Some results are shown below. Figure 2As shown. Two-tailed t-test results indicated that, under LC13, LC14, LC15, and LC16 conditions, the genotyping results of the 3D-Ind7308 marker in the PG-RIL population were highly significantly correlated with the total spikelet number (P < 0.01). Families carrying the superior allelic variation of 3D-Ind7308(P) showed an increase in the total spikelet number by 1.23, 0.86, 0.94, and 1.26 spikelets respectively compared to the allelic site 3D-Ind7308(G) under the four conditions. Figure 3 ).

[0098] Example 2: Application of PCR molecular markers in the identification and screening of wheat QTss-3D near-isogenic lines

[0099] Following the method described in Example 1, the 3D-Ind7308 marker was used to detect 176 families (F7) of the PG-RIL population, and a residual heterozygous line PG-3D1 (with amplification products showing both 132bp and 90bp bands) was screened. After self-pollination, a secondary segregating population (F2) containing 265 individual plants derived from this residual heterozygous line was constructed. The genotypes of individual plants in this population were detected using the 3D-Ind7308 marker. Specifically, genomic DNA was extracted from leaves at the seedling stage; using the genomic DNA from the 265 materials as templates, PCR amplification and genotype detection were performed using the 3D-Ind7308 marker primers developed in Example 1. Near-isogenic (NIL) populations were constructed by self-pollination of individual plants with homozygous genotypes in all target regions (at least 50 plants per genotype), and phenotypic identification was performed at the Luancheng Ecological Experimental Station of the Chinese Academy of Sciences (see step two of Example 1). The results showed that, compared with families carrying the 3228 allele (3D-Ind7308(G)), families carrying the 3228 allele (3D-Ind7308(P)) had significantly increased total spikelet number and grain number per spike by 0.95 and 2.64 grains, respectively. Figure 4 The above results indicate that the major-effect QTL QTss-3D can increase the number of grains per spike by increasing the total number of spikelets. This locus and its 3D-Ind7308 marker have important application value in molecular marker-assisted breeding for high-yield wheat.

[0100] The molecular marker 3D-Ind7308 of this invention is also applicable in the PG-RIL genetic population and near-isogenic lines derived from the remaining heterozygous lines. The experimental results are consistent with expectations, indicating that the marker 3D-Ind7308, which is closely linked to the locus controlling the total spikelet number, does indeed have an auxiliary selection effect on the total spikelet number in wheat. Since the total spikelet number is an important factor determining the number of grains per spike in wheat, this invention is also of great significance for breeding wheat varieties with a high number of grains per spike. The primer sequences and physical locations of the molecular marker of this invention are well-defined, the results are accurate, and the process is efficient. Based on case studies, it can be directly used for marker-assisted selection breeding.

[0101] 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. Application of specific segments on wheat 3D chromosomes as molecular markers in any of the following: (A1) To identify or assist in the identification of the total number of spikelets in wheat; (A2) Prepare products for identifying or assisting in the identification of the total number of spikelets in wheat; (A3) Compare the total number of spikelets of the wheat samples being tested; (A4) Prepare a product for comparing the total number of spikelets in the wheat being tested; (A5) Select wheat plants, lines, strains, or varieties with a relatively large total number of spikelets; (A6) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively large total number of spikelets; The specific fragment is the DNA fragment shown in SEQ ID No.

5.

2. The application of a substance used to detect the presence of a specific segment on wheat 3D chromosomes in any of the following: (A1) To identify or assist in the identification of the total number of spikelets in wheat; (A2) Prepare products for identifying or assisting in the identification of the total number of spikelets in wheat; (A3) Compare the total number of spikelets of the wheat samples being tested; (A4) Prepare a product for comparing the total number of spikelets in the wheat being tested; (A5) Select wheat plants, lines, strains, or varieties with a relatively large total number of spikelets; (A6) To prepare products for breeding wheat single plants, lines, strains or varieties with a relatively large total number of spikelets; The specific fragment is the DNA fragment shown in SEQ ID No.

5.

3. Use according to claim 2, characterized in that: The substance used to detect whether a specific segment is present on the wheat 3D chromosome is a primer pair used to amplify the specific segment.

4. Use according to claim 3, characterized in that: The upstream primer in the primer pair is designed based on a sequence in the wheat 3D chromosome located upstream of the specific fragment, and the downstream primer is designed based on a sequence in the wheat 3D chromosome located downstream of the specific fragment.

5. Use according to claim 4, characterized in that: The primer pair consists of a single-stranded DNA molecule as shown in SEQ ID No. 3 and a single-stranded DNA molecule as shown in SEQ ID No.

4.

6. Use according to any one of claims 1 to 5, characterized in that: The total number of spikelets refers to the total number of spikelets of the main spike.

7. A method for identifying or assisting in the identification of the total number of spikelets in wheat, comprising the following steps: (B1) Detect whether a specific segment is present in the 3D chromosome of the wheat being tested; (B2) Based on the results of (B1), the total number of spikelets of the wheat to be tested is determined as follows: the total number of spikelets of the wheat to be tested that does not contain the specific segment in the 3D chromosome is greater than or more candidates than the total number of spikelets of the wheat to be tested that contains the specific segment in the 3D chromosome; The specific fragment is the DNA fragment shown in SEQ ID No.

5.

8. A method for breeding wheat single plants, lines, strains, or varieties with a relatively large total number of spikelets, comprising the following steps (C1) and (C2): (C1) Detect whether a specific segment is present in the 3D chromosome of the wheat sample; (C2) Based on the results of (C1), select wheat plants that do not contain the specific segment in the 3D chromosome as parents for breeding, and select wheat plants that do not contain the specific segment in the 3D chromosome in each generation of breeding, so as to finally obtain wheat plants, lines, strains or varieties with a relatively large total number of spikelets. The specific fragment is the DNA fragment shown in SEQ ID No.

5.

9. The method according to claim 7 or 8, characterized in that: The method for detecting whether the wheat 3D chromosome to be tested contains a specific segment is as follows (E1) or (E2): (E1) Direct sequencing; (E2) Using the genome of the wheat to be tested as a template, perform PCR amplification using any of the primer pairs described in claims 3-5, and determine whether the specific fragment is contained in the 3D chromosome of the wheat to be tested based on the amplification results.

10. The method of claim 7 or 8, wherein: The total number of spikelets refers to the total number of spikelets of the main spike.

11. Any of the following biological materials: (F1) A pair of DNA fragments, wherein the DNA fragments are the DNA fragment shown at positions 36-167 of SEQ ID No. 1 and the DNA fragment shown at positions 36-125 of SEQ ID No. 2; (F2) Primer pair, which is the primer pair described in claim 4; (F3) A reagent or kit containing the primer pair described in (F2).

12. The application of the method of claim 7 or 8 in any of the following: (G1) Increase the number of grains per wheat ear; (G2) Increase wheat yield; (G3) Develop high-yield wheat varieties.

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