A wheat ear length-related molecular marker and application thereof

Abstract

This invention discloses a molecular marker related to wheat spike length and its application. Genotypic data was obtained using a wheat 55K high-throughput gene chip. An allelic variation increasing spike length was detected on wheat chromosome 5B, originating from the spike length locus QSl.yaas-5B of Yangmai 12. Based on its closely linked marker information, a KASP marker primer set KASP_Q.5B was developed for efficient screening of spike length. Experiments demonstrated that the KASP molecular marker KASP_Q.5B described in this invention can be used for marker-assisted selection breeding of wheat spike length. The development and utilization of KASP_Q.5B provides a powerful tool for molecular breeding of wheat yield traits.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically a molecular marker related to wheat spike length and its application. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of the world's major food crops, and increasing wheat yield is particularly important given the international situation of rapid population growth and continuous reduction in arable land. Since the wheat ear is an important reproductive organ, some studies have shown that ear shape traits are significantly correlated with yield components (Gao et al., 2015; Hu et al., 2020). Therefore, understanding the genetic characteristics of wheat ear length is of great value for yield breeding. Wheat ear length is a complex quantitative trait controlled by multiple genes / QTLs, including Q, Tasg-D1, and AGO1d (Chen et al., 2020; Xu et al., 2022). QTL mapping is an effective way to reveal the genetic basis of these complex quantitative traits. (JI et al., 2021) used BSE-Seq analysis to genotype extreme pools and preliminarily mapped QSl.cib-5A on chromosome 5A, contributing 7.88%–26.60% of the phenotypic variation. (Zhou et al., 2017) detected nine spike length QTLs based on a wheat 90K SNP chip, explaining up to 23.60% of the contribution. (Li et al., 2021) used a wheat 55K SNP chip to identify and validate two major-effect QTLs controlling spike length and having pleiotropic effects on plant height, thousand-grain weight, and grain length. Related studies have shown that spike length QTLs not only exhibit pleiotropic effects on yield-related traits such as thousand-grain weight and grain length (Li et al., 2021), but also have a certain association with wheat scab (Lu et al., 2013). The Rht1 and Rht2 genes are insensitive to endogenous gibberellin (GA), producing shorter plants and smaller cells (Keyes et al., 1989; Rebetzke et al., 2004; Botwright et al., 2005), while Rht5, Rht8, Rht22, Rht24, and Rht25 are responsive to GA, exhibiting pleiotropic effects on spike density and spike length in addition to reducing plant height (Peng et al., 1999; Chen et al., 2014). Therefore, in addition to exploring new major spike length QTLs, understanding their pleiotropic effects on other related traits is also important. Yangmai 12 and Yanzhan 1 are two excellent wheat varieties released in the middle and lower reaches of the Yangtze River and the Huang-Huai River basins of China, respectively. Yangmai 12 has long spikes and is moderately resistant to Fusarium head blight. Therefore, the spike length loci of Yangmai 12 / Yanzhan 1 can be mined using the RIL (Recombinant inbred lines) population, providing genetic resources for high-yield wheat breeding.

[0003] KASP technology uses specific matching of primer terminal bases and universal fluorescent probes to genotype SNPs. Genotyping can be performed using a quantitative real-time PCR instrument or a combination of a regular PCR instrument and an ELISA reader. Similar to TaqMan (a fluorescent probe method for detecting oligonucleotides), KASP technology relies on reading and determining the terminal fluorescence signal. Each well reaction uses dual-color fluorescence to detect two genotypes of a single SNP site; different SNPs correspond to different fluorescence signals. However, it does not require the synthesis of specific fluorescent primers for each SNP site; all site detection can ultimately be performed using universal fluorescent primers. This reduces the reagent cost of KASP technology, making it more practical. In modern wheat, after many agronomic traits or disease resistance / stress-related genes / sites have been identified or finely mapped, researchers develop KASP markers based on the flanking sequences of tightly linked markers on both sides of the site, facilitating their use by breeders (Su et al., 2018; Zhang et al., 2020; Hu et al., 2022). KASP technology has advantages such as high throughput and convenient detection (Rasheed et al., 2019). Genotyping using SNP chips, and the SNPs linked to the target trait obtained through QTL mapping or GWAS analysis, can be converted into KASP marker sets for easy application in breeding. (Rasheed et al., 2019) developed a KASP marker set for 70 wheat functional genes, which has been widely used. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a linked molecular marker for marker-assisted selection in wheat breeding to increase spike length genotypes. Genotype data were obtained using a wheat 55K SNP high-throughput microarray, and the QTL region QSl.yaas-5B, significantly associated with spike length from the wheat variety Yangmai 12, was detected. Further, by screening the effectiveness and reliability of nearby SNP sequences, a KASP marker primer set was developed near this QTL peak for efficient screening of wheat germplasm with spike length-increasing genotypes.

[0005] One of the objectives of this invention is to provide a KASP molecular marker related to wheat spike length, wherein the molecular marker is located on chromosome 5B in the wheat genome as shown in SEQ ID NO.4.

[0006] The second objective of this invention is to provide a KASP primer set for detecting the molecular marker. The KASP primer set is a combination of primers X1 for detecting whether the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the wheat genome is CC, TT, or both C and T. The primer set X1 contains two upstream primers and one downstream primer.

[0007] The upstream primer is designed based on the 36th deoxyribonucleotide of the sequence shown in SEQ ID NO.4 on chromosome 5B of the wheat genome and its upstream sequence, and the 3' end deoxyribonucleotide of one upstream primer is C and the 3' end deoxyribonucleotide of the other upstream primer is T;

[0008] The downstream primer was designed based on the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID NO.4 on chromosome 5B of the wheat genome.

[0009] Furthermore, the primer set X1 consists of upstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2, and downstream primers as shown in SEQ ID NO.3.

[0010] A third objective of this invention is to provide the application of the molecular marker or the KASP primer set in any of the following:

[0011] (A) To identify or assist in the identification of wheat spike length traits;

[0012] (B) Predict or compare the spike length trait of wheat to be tested;

[0013] (C) Selecting or screening wheat individual plants, lines, strains, or varieties with wheat spike length traits;

[0014] (D) Prepare products for identifying or assisting in the identification of wheat spike length traits;

[0015] (E) Prepare products for breeding or screening wheat individual plants, lines, strains or varieties with wheat spike length traits.

[0016] The fourth objective of this invention is to provide any of the following methods:

[0017] Method A: A method for comparing the spike length trait of wheat to be tested, comprising the following steps (A1) or (A2):

[0018] (A1) Detect whether the genotype at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the wheat genome is CC, TT, or CT;

[0019] (A2) The spike length of the wheat to be tested was determined as follows: the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is CC, and the wheat to be tested has an allelic variation that increases spike length;

[0020] Method B: A method for breeding or screening wheat individual plants, lines, strains, or varieties with the wheat spike length trait, comprising the following steps:

[0021] (B1) Detect whether the genotype at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the wheat genome is CC, TT, or CT;

[0022] (B2) Select the test wheat that is homozygous for C at the 36th deoxyribonucleic acid position of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the genome as the parent for breeding, and select the wheat that is homozygous for C at the 36th deoxyribonucleic acid position of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the genome in each generation of breeding, and finally obtain wheat single plants, lines, strains or varieties with relatively long wheat spike length;

[0023] (B3) Select the test wheat whose 36th deoxyribonucleic acid of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is T as the parent for breeding, and select wheat whose 36th deoxyribonucleic acid of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is T in each generation of breeding, and finally obtain wheat single plants, lines, strains or varieties with relatively short spike length.

[0024] Furthermore, the specific operation of step (A1) or step (B1) is as follows:

[0025] The above primer set was used to perform PCR amplification of the wheat genomic DNA to be tested. The amplified products were scanned for fluorescence signals, and the scan data were analyzed. Then, the type of deoxyribonucleotide at position 36 of chromosome 5B as shown in SEQ ID NO.4 in the wheat gene to be tested was determined as follows:

[0026] If the fluorescence signal data of the amplification product of the wheat to be tested is red, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the wheat genome to be tested is a homozygous C.

[0027] If the fluorescence signal data of the amplification product of the wheat to be tested is blue, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the wheat genome to be tested is a homozygous T.

[0028] Compared to existing technologies, this invention utilizes a wheat 55KSNP high-throughput gene chip to obtain genotype data, detecting the QTL region QSl.yaas-5B from the wheat variety Yangmai 12, which is significantly associated with wheat spike length. Furthermore, by screening the effectiveness and reliability of nearby SNP sequences, a KASP marker primer set was developed near this QTL peak for efficient screening of wheat germplasm with genotypes that increase spike length. This invention provides a valuable tool for the effective utilization of the wheat spike length locus QSl.yaas-5B in high-yield breeding. This KASP marker can rapidly screen for wheat spike length traits, facilitating the screening of wheat materials carrying superior allelic variations that increase spike length, thereby improving wheat breeding efficiency. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a partial genetic linkage map of chromosome 5B in Example 1 and a schematic diagram of the location of QSl.yaas-5B.

[0031] Figure 2 The results of the KASP primer set KASP_Q.5B of QSl.yaas-5B in Example 3 were obtained from 180 natural populations. Detailed Implementation

[0032] The embodiments of the technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. As a professional agricultural research institution, the applicant has long preserved relevant germplasm materials, and the relevant wheat varieties are all publicly available on the market or in existing germplasm banks.

[0033] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0034] Example 1: Screening of sites that are stable and significantly associated with wheat spike length

[0035] This embodiment uses 205 recombinant inbred lines (F) derived from “Yangmai 12 × Yanzhan 1”. 10 Using this material, the recombinant inbred line and two parents were planted for three consecutive growing seasons (2019, 2020, and 2021) at the Wanfu Experimental Base in the Lixiahe region of Jiangsu Province, two growing seasons (2019 and 2021) at the Yangtze University Experimental Base in Jingzhou, and one growing season (2020) at the Sihong Experimental Base for the investigation of spike length agronomic traits. A completely randomized design with two replicates was used. Each line was planted in two rows with 25 grains per row, with a row length of 1.5 m and a row spacing of 0.30 m. Field management practices were implemented according to local cultivation practices. During the late grain-filling stage of wheat, 20 representative plants with similar growth stages from each RIL were randomly selected. The spike length of the main spike was measured under six different conditions. The measurement method was as follows: from the base of the ear axis to the tip of the terminal spikelet, excluding the awn.

[0036] Genomic DNA was extracted from fresh leaf tissue using the CTAB method (Jiang et al., 2020), and DNA integrity and quantity were detected by gel electrophoresis.

[0037] Genotyping of parental and recombinant inbred line populations was performed using a wheat 55K single nucleotide polymorphism (SNP) array containing 53,063 SNPs provided by Zhongyu Jin Marker (Beijing) Biotechnology Co., Ltd. Then, genotyping was performed on parental and recombinant inbred line populations using 12 KASP or SSR markers associated with known genes, including Vrn-B1, Rht-B1, Rht-D1, and Rht8, to densify the genetic map (Rasheed et al., 2016; Zhang et al., 2019; Xu et al., 2020; Zhu et al., 2021). All polymorphic SNP flanking sequences were aligned with the Chinese Spring Reference Genome v2.1 (Ref v2.1) in the IWGSC Ensembl Plants database (http: / / plants.ensembl.org) to determine their physical locations, and the results were assigned as 1E. -10 The expected value (E) is used as the significance threshold.

[0038] First, preliminary quality control was performed on the wheat 55K SNP microarray results. IciMapping v4.1 software (http: / / www.isbreeding.net) was used to filter and remove redundancy from the original genotype data (the "BIN" function can simultaneously delete SNPs with a deletion rate greater than 20%, a minimum allele frequency less than 5%, and partial segregation (χ2 ≥ 33.3%), thus deleting redundant SNPs). The "MAP" function was then used to cluster the filtered markers (Li et al., 2007; 2008; Meng et al., 2015). The flanking sequences of the markers were then compared to their physical locations in the International Wheat Genome Sequencing Consortium (IWGSC) database v2.1 (Ma et al., 2021) (WheatOmics 1.0, http: / / 202.194.139.32 / blast / blast.html). Genetic distances for each population were calculated using the Kosambi mapping function in JoinMap 4.0 software (Kosambi et al., 1944; Van et al., 2006). Ear length QTLs were detected using the Complete Interval Mapping (ICIM) algorithm with the "BIP" function in IciMapping v4.1, with the LOD threshold set to 3.0 (Li et al., 2021). Genetic maps covering the QTL regions were plotted using MapChart v2.32 (Voorrips, 2002). QTLs located within overlapping confidence intervals were considered identical. The physical locations of QTL flanking markers were compared with previously reported gene / QTL pairs (http: / / 202.194.139.32 / blast / blast.html and http: / / wheatomics.sdau.edu.cn / genes / ).

[0039] In the Yangmai 12 / Yanzhan 1 population, the spike length locus QSl.yaas-5B, which was stable under four different environments and Mean values, was detected. Yangmai 12 provided an effect of increasing spike length, contributing 9.01–12.85% to the phenotype, with an additive effect of 4.02–9.47 (see appendix). Figure 1 (Table 1) This is a new wheat spike length locus after comparison with previous studies.

[0040] Table 1. QTL mapping results of spike length for Yangmai 12 / Yanzhan 1

[0041]

[0042] Note: aE1, E2, E3, and E4 represent the production nurseries in Yangzhou in 2019, Yangzhou in 2020, Sihong in 2020, and Jingzhou in 2021, respectively. Mean represents the average value of all environmental data. b This represents the physical location based on Chinese Spring version 2.1. c The commonly used logarithm to represent the maximum likelihood function. d Representative phenotypic contribution rate. e The values ​​represent additive effects (negative values ​​represent alleles that increase spike length from Yanzhan 1, and positive values ​​represent alleles that increase spike length from Yangmai 12).

[0043] Example 2 clarifies the relationship between panicle length QTL and panicle length, thousand-grain weight, and Fusarium head blight resistance.

[0044] The effects of the panicle length QTL on panicle length, thousand-grain weight, and Fusarium head blight resistance were analyzed using the mean values ​​of the QSl.yaas-5B flanking marker (AX110581004) and panicle length, thousand-grain weight, average number of diseased spikelets (PIS) in soil surface inoculation, and average number of diseased spikelets (PSS) in spray spore inoculation (mean values ​​of data from the Wanfu Experimental Base in Lixiahe District, Jiangsu Province in 2020 and 2021). The results showed that QSl.yaas-5B significantly increased panicle length, while also significantly increasing thousand-grain weight and decreasing PIS and PSS values, making it a panicle length locus with high utilization value (Table 2).

[0045] Table 2. Effects of QSl.yaas-5B on thousand-grain weight and Fusarium head blight resistance (Mean value)

[0046]

[0047] Note: Mean represents the average value. PIS: represents the average diseased spikelet percentage identified by the soil surface inoculation method; PSS: represents the average diseased spikelet percentage identified by the spore spray method. '*' and '**' represent P<0.05 and P<0.01, respectively.

[0048] Example 3: Development of KASP tags linked to QSl.yaas-5B

[0049] The above results indicate that the allelic variation of QSl.yaas-5B that increases spike length has a significant synergistic effect on thousand-grain weight and resistance to Fusarium head blight. Using wheat reference genome information, we converted the flanking SNP marker AX110581004 of this site's peak region into the KASP marker KASP_Q.5B. The primer sequences for KASP_Q.5B are shown in Table 3.

[0050] Table 3 Primer sequences for KASP_Q.5B

[0051]

[0052]

[0053] Note: F1 and F2 are forward primers, and R is the reverse primer. Competitive primers are underlined.

[0054] Preparation of KASP primers and PCR reaction

[0055] Using wheat reference genome information, flanking SNPs in the peak region of spike length loci with breeding value were converted into KASP markers. Primer design was performed using the online software Primer 3.0. Based on the KASP design principle, two specific primers (F1 / F2) and one universal primer (R) were designed for each marker. A specific sequence binding to FAM fluorescence was added to the tail of F1 primers, and a specific sequence binding to HEX fluorescence was added to the tail of F2 primers. The primers were synthesized by Beijing Jiacheng Biotechnology Co., Ltd.

[0056] The total KASP reaction volume was 6 μL, containing 3.5 μL of 2×KASP Master Mix, 0.1 μL of KASP primer mixing working solution, and 20 ng / μL of [unspecified reagent]. -1 2.4 μL of template DNA.

[0057] The KASP reaction procedure consisted of three steps: Step 1: 94℃ for 15 min; Step 2: 94℃ for 25 s, then 61–55℃ for 1 min, decreasing the temperature by 0.6℃ per cycle, for a total of 10 cycles; Step 3: 94℃ for 20 s, then 55℃ for 45 s, for a total of 29 cycles. KASP genotyping results were analyzed using a KASP fluorescence analyzer (LGC, Pherastar plus).

[0058] The RIL population, along with its parents, underwent KASP_Q.5B amplification using the method described above. Analysis of the KASP_Q.5B amplification products using KlusterCaller software showed that the fluorescence signal data clustered near the Y-axis (red) in the genotyping results fluorescence signal coordinate system, identical to that of Yangmai 12. This indicates that the genotype of these wheat varieties at the 36th base (SNP site) flanking the QSl.yaas-5B locus (e.g., SEQ ID NO. 4) is C. Conversely, analysis of the amplification products using KlusterCaller software showed that the fluorescence signal data clustered near the X-axis (blue) in the coordinate system, different from the genotyping of Yangmai 12. This indicates that the genotype of these wheat varieties at the QSl.yaas-5B locus is T. The material genotyping was successful, and the KASP primer set was designed successfully.

[0059] Table 4. t-test results of RIL families carrying different genotypes.

[0060]

[0061] Note: a Different lowercase letters following the numbers indicate highly significant differences (P<0.01); b The ** after the t-value indicates a highly significant difference (P<0.01).

[0062] Table 4 shows the results of a two-sample t-test using Excel 2019. It can be seen that the pedigree of KASP_Q.5B with CC genotype had a significantly longer ear length by 5.14% compared to the TT pedigree (P<0.01). (The statistical method used is a standard method in this field, and for details, please refer to the literature "Gai Junyi, Experimental Statistical Methods, China Agricultural Press, September 2000").

[0063] The aforementioned KASP primer set and genotype detection system were applied individually or simultaneously to the selection of wheat spike length. The effect of QSl.yaas-5B was significant, indicating that the KASP marker was successfully developed and can be further used for the detection of breeding materials.

[0064] Example 4: Application of KASP primer set in breeding

[0065] Field Trial: This study used 180 wheat lines from across China as research subjects. These 180 lines were planted in the yield evaluation nursery of the Wanfu Experimental Base in the Lixiahe region of Jiangsu Province for two consecutive growing seasons in 2020 and 2021. A randomized block design was used, with each line planted in two rows of 25 grains each, with a row length of 1.5m and a row spacing of 0.30m. The experiment was replicated twice, and field management followed local high-yield cultivation practices. During the late grain-filling stage, 20 representative plants with similar growth stages from each RIL were randomly selected. The spike length was measured under six different environmental conditions. The measurement method was from the base of the ear axis to the tip of the terminal spikelet, excluding the awn (basal sterile spikelets were not included).

[0066] The KASP primer set obtained in Example 1 was used to genotype 180 wheat varieties (lines). The genotyping results are as follows: Figure 2 As shown.

[0067] The fluorescence signal data of the KASP_Q.5B amplification products, analyzed by Kluster Caller software, clustered near the Y-axis (red) in the fluorescence signal coordinate system of the genotyping results, which is the same as that of Yangmai 12. This proves that the genotype of these wheat varieties at the 36th base (SNP site) of the flanking nucleotide sequence (such as SEQ ID NO.4) of the molecular marker KASP_Q.5B is C. The fluorescence signal data of the amplification products, analyzed by Kluster Caller software, clustered near the X-axis (blue) in the coordinate system, which is different from the genotyping of Yangmai 12. This proves that the genotype of these wheat varieties at this SNP site is T.

[0068] Table 5. t-test results of tested varieties (lines) carrying different genotypes.

[0069]

[0070] Note: The asterisk (*) after the number indicates a significant difference relative to “TT” at P<0.05.

[0071] Table 5 shows that a two-sample t-test using Excel 2019 revealed that the presence of the Yangmai 12 allelic variant (CC) at the QSl.yaas-5B locus significantly increased spike length by 3.61% (P<0.05), consistent with the results from the RIL population. The aforementioned KASP primer set and genotype detection system, applied individually or simultaneously, demonstrated a significant effect of QSl.yaas-5B in the selection of materials to validate spike length traits in wheat populations, indicating the successful development of the KASP marker and its potential for further use in breeding material testing.

[0072] It is easy to conclude from the above experimental results that: using the KASP primer set of the present invention to amplify wheat genomic DNA by PCR, and determining whether it carries the genotype of Yangmai 12 that increases spike length by KASP typing, the detection method is simple to operate, and the results are intuitive and effective. Using this KASP primer set can greatly improve the efficiency of molecular marker-assisted selection breeding of wheat with increased spike length.

[0073] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A KASP molecular marker associated with wheat spike length, characterized in that, The molecular marker is located on chromosome 5B in the wheat genome, as shown in SEQ ID NO.4, where N is C or T.

2. A KASP primer set for detecting the molecular marker of claim 1, characterized in that, The KASP primer set consists of upstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2, and downstream primers as shown in SEQ ID NO.

3.

3. The use of the molecular marker of claim 1 or the KASP primer set of claim 2 in any of the following: (A) To identify or assist in the identification of wheat spike length traits; (B) Predict or compare the spike length trait of wheat to be tested; (C) Selecting or screening wheat individual plants, lines, strains, or varieties with wheat spike length traits; (D) Prepare products for identifying or assisting in the identification of wheat spike length traits; (E) Prepare products for breeding or screening wheat individual plants, lines, strains or varieties with wheat spike length traits.

4. Any of the following methods: Method A: A method for comparing the spike length trait of wheat to be tested, comprising the following steps (A1) or (A2): (A1) Detect whether the genotype at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the wheat genome is CC, TT, or CT; (A2) The spike length of the wheat to be tested was determined as follows: the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is CC, and the wheat to be tested has an allelic variation that increases spike length; Method B: A method for breeding or screening wheat individual plants, lines, strains, or varieties with the wheat spike length trait, comprising the following steps: (B1) Detect whether the genotype at position 36 of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the wheat genome is CC, TT, or CT; (B2) Select the test wheat that is homozygous for C at the 36th deoxyribonucleotide position of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the genome as the parent for breeding, and select the wheat that is homozygous for C at the 36th deoxyribonucleotide position of the molecular marker shown in SEQ ID NO.4 on chromosome 5B in the genome in each generation of breeding, and finally obtain wheat single plants, lines, strains or varieties with relatively long wheat spikes; (B3) Select the test wheat whose 36th deoxyribonucleic acid of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is T as the parent for breeding, and select wheat whose 36th deoxyribonucleic acid of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the genome is T in each generation of breeding, and finally obtain wheat single plants, lines, strains or varieties with relatively short spike length.

5. The method according to claim 4, characterized in that, The specific operation of step (A1) or step (B1) is as follows: The wheat genomic DNA to be tested was amplified by PCR using the primer set as described in claim 2. The amplified product was scanned for fluorescence signals, and the scan data was analyzed. Then, the type of deoxyribonucleotide at position 36 of chromosome 5B as shown in SEQ ID NO.4 in the wheat gene to be tested was determined as follows: If the fluorescence signal data of the amplification product of the wheat to be tested is red, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the wheat genome to be tested is a homozygous C. If the fluorescence signal data of the amplification product of the wheat to be tested is blue, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID NO.4 on chromosome 5B of the wheat genome to be tested is a homozygous T.

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