Molecular markers and methods for identifying wheat plant height and thousand kernel weight

Abstract

This invention discloses molecular markers and methods for identifying wheat plant height and thousand-grain weight. The molecular markers disclosed in this invention are nucleotides in the wheat genome corresponding to position 2184 of SEQ ID No. 1 in the sequence listing, which are either G or A. Experiments have shown that homozygous wheat with nucleotide G at position 2184 of SEQ ID No. 1 in the genome has a higher plant height than homozygous wheat with nucleotide A at the same position, and the thousand-grain weight of homozygous wheat with nucleotide A at the same position is higher than that of homozygous wheat with nucleotide G at the same position. The molecular markers of this invention are correlated with wheat plant height and thousand-grain weight, and can quickly and accurately identify wheat varieties with excellent plant height and thousand-grain weight. This invention has significant implications for the cultivation of stress-resistant, high-yielding wheat varieties or for research purposes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically molecular markers and methods for identifying wheat plant height and thousand-grain weight. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of my country's three major staple crops, and increasing wheat yield is crucial for ensuring national food security. Plant height is an important agronomical trait that determines crop yield, playing a significant role in plant morphology and yield improvement; thousand-grain weight is one of the factors constituting wheat yield, directly determining the yield level. With the development of molecular biology, molecular marker-assisted breeding provides a convenient and rapid method for selecting target traits in wheat. Utilizing molecular markers to discover and utilize superior gene resources provides a foundation for improving breeding efficiency and offers an efficient pathway for crop genetic improvement and germplasm innovation. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to determine the plant height and thousand-grain weight of wheat.

[0004] To solve the above-mentioned technical problems, the present invention first provides the application of substances that detect wheat molecular markers in the detection or auxiliary detection of wheat plant height or grain yield;

[0005] The wheat molecular marker is the nucleotide in the wheat genome corresponding to position 2184 of SEQ ID No. 1 in the sequence listing, which is either G or A.

[0006] In the above applications, the substance used to detect wheat molecular markers is a primer pair named TaTIFY11C-4A-Primer or a set of reagents for detecting wheat molecular markers.

[0007] The TaTIFY11C-4A-Primer consists of two single-stranded DNA molecules, as shown in SEQ ID No. 4 and SEQ ID No. 5 in the sequence listing.

[0008] The kit for detecting the wheat molecular marker includes a primer pair named TaTIFY11C-4A-Primer-SacI, which consists of two single-stranded DNA molecules as shown in SEQ ID No. 2 and SEQ ID No. 3 in the sequence listing.

[0009] The kit may also include the restriction endonuclease SacI.

[0010] The reagent kit may be TaTIFY11C-4A-Primer-SacI, or may be derived from the...

[0011] TaTIFY11C-4A-Primer-SacI is composed of SacI.

[0012] In the above applications, the plant height of homozygous wheat whose genome corresponds to nucleotide G at position 2184 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the plant height of homozygous wheat whose genome corresponds to nucleotide A at position 2184 of SEQ ID No. 1 in the sequence listing;

[0013] The grain yield of homozygous wheat with nucleotide A at position 2184 of SEQ ID No. 1 in the genome is higher than or candidate higher than that of homozygous wheat with nucleotide G at position 2184 of SEQ ID No. 1 in the genome.

[0014] This invention also provides a method for detecting or assisting in the detection of wheat plant height or grain yield, the method comprising detecting the wheat molecular markers and determining wheat plant height or grain yield according to the following method:

[0015] The plant height of homozygous wheat whose genome corresponds to nucleotide G at position 2184 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the plant height of homozygous wheat whose genome corresponds to nucleotide A at position 2184 of SEQ ID No. 1 in the sequence listing;

[0016] The grain yield of homozygous wheat with nucleotide A at position 2184 of SEQ ID No. 1 in the genome is higher than or candidate higher than that of homozygous wheat with nucleotide G at position 2184 of SEQ ID No. 1 in the genome.

[0017] In the above method, the detection of the wheat molecular marker can be performed using the substance for detecting wheat molecular markers.

[0018] In the above method, the detection of the wheat molecular marker using the TaTIFY11C-4A-Primer may include: performing PCR amplification on wheat genomic DNA using the TaTIFY11C-4A-Primer to obtain an amplification product; sequencing the amplification product to determine the nucleotide in the wheat genome corresponding to position 2184 of SEQ ID No. 1 in the sequence listing.

[0019] The PCR amplification reaction system can be as follows: 12.2 μL ddH2O, 4.0 μL 5×PCR buffer, 0.4 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL transfastpfu enzyme (5 U), and 1 μL template DNA (20 ng / μL). Both the 5×PCR buffer and transfastpfu enzyme (5 U) are products of Beijing TransGen Biotech Co., Ltd.

[0020] The PCR amplification reaction conditions can be as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1.5 min, 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0021] The above method, using the aforementioned reagent kit to detect the wheat molecular marker, may include: performing PCR amplification of wheat genomic DNA using the TaTIFY11C-4A-Primer-SacI to obtain an amplification product; digesting the amplification product with SacI to obtain a digestion product; detecting the size of the digestion product; and determining the nucleotide at position 2184 of SEQ ID No. 1 in the wheat genome according to the following method:

[0022] If the enzyme digestion product contains only one DNA fragment of size 288 bp and does not contain DNA fragments of size 265 bp and 23 bp respectively, then the nucleotide at position 2184 of SEQ ID No. 1 in the wheat genomic DNA is A, and the wheat is homozygous for this position.

[0023] If the enzyme digestion products are two DNA fragments of 265bp and 23bp in size and do not contain a DNA fragment of 288bp in size, then the nucleotide at position 2184 of SEQ ID No.1 in the wheat genomic DNA is G, and the wheat is homozygous for this position.

[0024] If the enzyme digestion product contains three DNA fragments of sizes 288bp, 265bp, and 23bp, then the nucleotide at position 2184 of SEQ ID No. 1 in the wheat genomic DNA corresponds to G and A (i.e., a heterozygous type where the position is G on one chromosome and A on the other chromosome).

[0025] The substance used to detect wheat molecular markers is also within the scope of protection of this invention.

[0026] The wheat molecular markers mentioned above are also within the scope of protection of this invention.

[0027] This invention also provides any of the following applications:

[0028] The application of wheat molecular markers described in X1) in wheat breeding;

[0029] X2) The application of the wheat molecular markers described herein in the detection or auxiliary detection of wheat plant height or grain yield;

[0030] X3) The application of the substances used to detect wheat molecular markers in wheat breeding;

[0031] X4) The application of the substance for detecting wheat molecular markers in the preparation of wheat breeding products;

[0032] X5) The application of the substance for detecting wheat molecular markers in the preparation of products for detecting or assisting in the detection of wheat plant height or grain yield;

[0033] Application of the method described in X6) in wheat breeding;

[0034] X7) The application of detecting substances in the wheat genome corresponding to nucleotide 2184 of SEQ ID No. 1 in the sequence listing in the breeding of wheat with excellent plant height or grain yield;

[0035] X8) The application of detecting substances in the wheat genome corresponding to nucleotide 2184 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent plant height or grain yield.

[0036] The present invention also provides a wheat breeding method, the method comprising: detecting the 2184th nucleotide in the wheat genome corresponding to SEQ ID No.1 in the sequence listing, and selecting wheat whose nucleotide at the 2184th nucleotide in the wheat genome corresponding to SEQ ID No.1 in the sequence listing is A as a parent for breeding.

[0037] In this invention, the grain yield can be expressed as the weight of a thousand grains.

[0038] In this invention, the wheat can be any of the 32 wheat varieties and any wheat material in Table 1 or its descendants. The 32 wheat varieties are PANDAS, An85zhong124-1, Yanzhan 1, Bawangbian, Beijing 10, Beijing 14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali 1, DanR8093, Fengkang 13, Jimai 41, Jimai 6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle 5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 22 9th-25, Zigan Baimangxian, Jingpin 10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, and White Rough Wheat.

[0039] Experiments have shown that homozygous wheat varieties with nucleotide G at position 2184 of SEQ ID No. 1 in the genome have higher plant heights than homozygous wheat varieties with nucleotide A at the same position, and the thousand-grain weight of homozygous wheat with nucleotide A at the same position is higher than that of homozygous wheat with nucleotide G at the same position. By detecting the wheat molecular markers of this invention, wheat varieties with superior plant height and thousand-grain weight can be quickly and accurately identified. This invention provides a new method for marker-assisted selection breeding of wheat and has significant implications for the cultivation of stress-resistant, high-yielding wheat varieties or for related research.

[0040] 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. Attached Figure Description

[0041] Figure 1 This is a partial result of wheat detection using molecular markers for wheat plant height and yield, as presented in this invention. The electrophoretic bands in lane G are 265 bp and 23 bp in length, respectively, while the band in lane A is 288 bp. Due to the long electrophoresis time, the 23 bp fragment is somewhat blurry. M represents the DNA molecular weight standard.

[0042] Figure 2 This figure shows the statistical results of plant height and thousand-grain weight for different homozygous genotypes of the TaTIFY11C-4A gene in a natural wheat population. In the results figure, the left side of each environment represents the results of homozygous wheat with position G at SEQ ID No. 1 in the sequence listing, and the right side represents the results of homozygous wheat with position A at that position. E1: 15-SY-DS; E2: 15-SY-DS-HS; E3: 15-SY-WW; E4: 15-SY-WW-HS; E5: 16-CP-DS; E6: 16-SY-DS; E7: 16-SY-DS-HS; E8: 16-SY-WW; E9: 16-SY-WW-HS; E10: 17-CP-WW; E11: 17-SY-DS; E12: 17-SY-DS-HS. 15: 2015; 16: 2016; 17: 2017. SY: Shunyi; CP: Changping; WW: Abundant Water; DS: Drought Stress; HS: High Temperature Stress. Detailed Implementation

[0043] 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, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0044] Example 1: Obtaining molecular markers related to plant height and thousand-grain weight

[0045] I. Obtaining the polymorphic site of the TaTIFY11C-4A gene

[0046] (1) Based on the characteristics of the wheat TaTIFY11C-4A genome DNA sequence, specific primers for the genome were designed. The primer sequences are as follows:

[0047] TaTIFY11C-4A-Primer-F (forward primer): 5′-ACCAAGGATAGCAACACTC-3′ (SEQ ID No. 4 in the sequence listing);

[0048] TaTIFY11C-4A-Primer-R (reverse primer): 5′-AACGGCAACAATCAAGGA-3′ (SEQ ID No. 5 in the sequence listing).

[0049] The recognition sequences of TaTIFY11C-4A-Primer-F and TaTIFY11C-4A-Primer-R are located upstream and downstream of the TaTIFY11C-4A gene promoter, respectively.

[0050] (2) Using the genomic DNA of 32 wheat materials (all from the National Germplasm Resource Bank) as templates, PCR amplification was performed using the primers in step (1) to obtain PCR amplification products. Then, the obtained PCR amplification products were sequenced and sequence aligned.

[0051] The PCR amplification system used (20 μL) consisted of: 12.2 μL ddH2O, 4.0 μL 5×PCR buffer, 0.4 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 1.6 μL dNTPs (2.5 mmol / L), 0.4 μL transfastpfu enzyme (5 U), and 1 μL template DNA (20 ng / μL). Both the 5×PCR buffer and transfastpfu enzyme (5 U) were products of Beijing TransGen Biotech Co., Ltd.

[0052] PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1.5 min, 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0053] Sequence analysis revealed one SNP site in the TaTIFY11C-4A genomic DNA (SEQ ID No. 1), located at position 2184 of the TaTIFY11C-4A gene (exhibiting G and A polymorphisms), denoted as molecular marker TaTIFY11C-4A-2184.

[0054] The names of the 32 wheat materials used are as follows: PANDAS, An85zhong124-1, Yanzhan No.1, Bawangbian, Beijing No.10, Beijing No.14, Cangzhou Wheat, Changwu 131, Chang 6878, Dali No.1, DanR8093, Fengkang 13, Jimai 41, Jimai No.6, Jin 2148-7, Jinghe 8922, Linkang 5108, Baiqi Wheat, Changle No.5, Hongheshang, Beijing 8686, 04-044, 04-030, Chun 229th-25, Zigan Baimangxian, Jingpin No.10, Chun 04 9th-5-1, Chun 45 9th-50-1, Neixiang 188, Jing 411, China Spring, and White Rough Wheat.

[0055] II. Detection of Molecular Markers

[0056] (1) For the molecular marker at position 2184 of the TaTIFY11C-4A gene shown in SEQ ID No.1 of the sequence listing, primers capable of detecting the marker were designed, and the primer sequences are as follows:

[0057] TaTIFY11C-4A-Primer-SacI-F (forward primer): 5′-ACGAAGTCTCAATGGACGGAGACCTAG-3′ (SEQ ID No. 2);

[0058] TaTIFY11C-4A-Primer-SacI-R (reverse primer): 5′-GGGGGTGGTGGAAGAAATGCCTGAG-3′ (SEQ ID No. 3).

[0059] (2) The steps for detecting the molecular markers of the wheat to be tested using the primers from step (1) are as follows:

[0060] Using the wheat genomic DNA to be tested as a template, PCR amplification was performed using the primers from step (1), and PCR amplification product A was obtained. The nucleotide sequence of PCR amplification product A is positions 1922 to 2209 of SEQ ID No. 1.

[0061] The PCR amplification system (10 μL) consisted of: 3.6 μL ddH2O, 0.2 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 5 μL 2×PCRMix, and 1 μL template DNA (20 ng / μL). The 2×PCRMix was a product of Zhuangmeng Biotechnology Co., Ltd., catalog number ZT201A.

[0062] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ for 10 min, and stored at 4℃.

[0063] The PCR amplification product A obtained in step (1) was digested with the restriction endonuclease SacI to obtain digested product B. The electrophoretic detection results of part of the digested product B are as follows: Figure 1 As shown. If the enzyme digestion product B consists of two DNA fragments of 265 bp and 23 bp in size, it indicates that the nucleotide corresponding to position 2184 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested is G, and the wheat being tested is homozygous. If the enzyme digestion product B consists of only one DNA fragment of 288 bp in size, it indicates that the nucleotide corresponding to position 2184 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested is A, and the wheat being tested is homozygous. If the enzyme digestion product B consists of three DNA fragments of 288 bp, 265 bp, and 23 bp in size, it indicates that the nucleotides corresponding to position 2184 of SEQ ID No. 1 in the sequence listing of the wheat genomic DNA being tested are G and A, and the wheat being tested is heterozygous.

[0064] Example 2: Association analysis of wheat molecular markers with plant height and thousand-grain weight

[0065] I. Genotyping of natural populations and its association with plant height and thousand-grain weight

[0066] The molecular markers from Example 1 were used to genotype the natural population (Table 1), and association analysis was performed between the molecular markers and plant height and thousand-grain weight. The specific steps are as follows:

[0067] 1. Detection of molecular markers

[0068] In a natural population of 323 hexaploid wheat accessions, each wheat variety was selected as the test wheat and its molecular markers were detected according to the method described in step two of Example 1. The nucleotide corresponding to position 2184 of SEQ ID No. 1 in the sequence listing was determined for each individual wheat. All wheat varieties in the natural population were sourced from the National Germplasm Resource Bank.

[0069] The detection results of the molecular markers are shown in Table 1. "G" indicates that the nucleotide at position 2184 of SEQ ID No. 1 in the sequence listing is homozygous for G, "A" indicates that the nucleotide at position 2184 of SEQ ID No. 1 in the sequence listing is homozygous for A, and "-" indicates that the nucleotide at position 2184 of SEQ ID No. 1 in the sequence listing is heterozygous for both G and A.

[0070] Table 1. Detection results of various molecular markers in wheat from natural populations

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] 2. Association analysis between molecular markers and plant height and thousand-grain weight

[0077] In 2015, 2016 and 2017, under 12 different environmental conditions, natural wheat populations were planted at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, in Shunyi (SY) (116°56′E; 40°23′N) and Changping (CP) (116°13′E; 40°13′E). After the wheat was harvested, the plant height and thousand-grain weight of different wheat varieties were measured, and the average plant height and thousand-grain weight for each year were calculated.

[0078] The molecular marker (i.e., the nucleotide at position 2184 of SEQ ID No. 1) in Example 1 was correlated with plant height and thousand-grain weight using the GLM model with Tassel 5.0 software.

[0079] The statistical results of plant height and thousand-grain weight of various wheat species are shown in Tables 2 and 3. The homozygous wheat with position G at SEQ ID No. 1 (2184th position) in the sequence listing showed significantly higher plant heights under normal irrigation, drought, and high temperature conditions compared to the homozygous wheat with position A at the same locus. Conversely, the thousand-grain weight of the homozygous wheat with position A at SEQ ID No. 1 (2184th position) in the sequence listing was significantly higher than that of the homozygous wheat with position G at the same locus. This indicates that the molecular markers of this invention are correlated with wheat plant height and thousand-grain weight under normal irrigation, drought, and high temperature conditions, and can be used to breed wheat varieties with superior plant height and thousand-grain weight under these conditions.

[0080] Table 2. Results of plant height (cm) for different homozygous types in natural wheat populations

[0081]

[0082] In Table 2, 15: 2015; 16: 2016; 17: 2017. WW: Sufficient water was provided, and irrigation was carried out during three periods: before winter, flowering, and grain filling, with an irrigation volume of 750 m³. 3 ha -1 (75mm); DS: Drought stress, with no irrigation throughout the growing season and relying solely on natural rainfall, with total rainfall of 161, 173, and 143 mm for the three growing seasons, respectively; HS: High temperature stress, with high temperatures simulated by a greenhouse covered with polyethylene one week after flowering. * indicates p-value < 0.05 for significance analysis, ** indicates p-value < 0.01 for significance analysis.

[0083] Table 3. Results of 1000-grain weight (g) for different homozygous types in natural wheat populations

[0084]

[0085] In Table 3, * indicates a p-value of <0.05, ** indicates a p-value of <0.01, and *** indicates a p-value of <0.001.

[0086] The present invention has been described in detail above. Those skilled in the art will recognize that 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. While specific embodiments have been provided, 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.

Claims

1. Application of substances that detect molecular markers in wheat in the detection or auxiliary detection of wheat plant height or grain yield; The wheat molecular marker is the nucleotide in the wheat genome corresponding to position 2184 of SEQ ID No. 1 in the sequence listing, which is either G or A.

2. The application according to claim 1, characterized in that: The substance used to detect wheat molecular markers is named […]. TaTIFY11C-4A - Primer pairs for the Primer or a kit of reagents for detecting the wheat molecular markers; The TaTIFY11C-4A -Primer consists of two single-stranded DNA molecules, as shown in SEQ ID No. 4 and SEQ ID No. 5 in the sequence listing; The complete set of reagents for detecting the wheat molecular markers includes the following name: TaTIFY11C-4A -Primer- Sac The primer pair of I, the TaTIFY11C-4A -Primer- Sac I consists of two single-stranded DNA sequences shown in SEQ ID No. 2 and SEQ ID No. 3 of the sequence listing.

3. The application according to claim 2, characterized in that: The kit also includes restriction endonucleases. Sac I.

4. The application according to any one of claims 1-3, characterized in that: The plant height of wheat whose genome corresponds to nucleotide G at position 2184 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the plant height of wheat whose genome corresponds to nucleotide A at position 2184 of SEQ ID No. 1 in the sequence listing; Wheat with nucleotide A at position 2184 of SEQ ID No. 1 in the genome has a higher or candidate higher grain yield than wheat with nucleotide G at position 2184 of SEQ ID No. 1 in the genome.

5. A method for detecting or assisting in the detection of wheat plant height or grain yield, characterized in that: The method includes detecting the wheat molecular markers described in claim 1 and determining wheat plant height or grain yield according to the following method: The plant height of wheat whose genome corresponds to nucleotide G at position 2184 of SEQ ID No. 1 in the sequence listing is greater than or candidate greater than the plant height of wheat whose genome corresponds to nucleotide A at position 2184 of SEQ ID No. 1 in the sequence listing; Wheat with nucleotide A at position 2184 of SEQ ID No. 1 in the genome has a higher or candidate higher grain yield than wheat with nucleotide G at position 2184 of SEQ ID No. 1 in the genome.

6. The method according to claim 5, characterized in that: The detection of the wheat molecular markers described in claim 1 is performed using any of the substances described in claims 1-3 for detecting wheat molecular markers.

7. Any of the following applications: X1) The application of the wheat molecular markers described in claim 1 in breeding wheat plant height or grain yield; X2) The application of the wheat molecular markers described in claim 1 in the detection or auxiliary detection of wheat plant height or grain yield; X3) The application of any of the substances for detecting wheat molecular markers as described in claims 1-3 in breeding wheat plant height or grain yield; X4) The use of any of the substances for detecting wheat molecular markers as described in claims 1-3 in the preparation of wheat plant height or grain yield breeding products; X5) The use of any of the substances for detecting wheat molecular markers as described in claims 1-3 in the preparation of products for detecting or assisting in the detection of wheat plant height or grain yield; X6) Application of the method according to claim 5 or 6 in breeding wheat plant height or grain yield; X7) The application of detecting substances in the wheat genome corresponding to nucleotide 2184 of SEQ ID No. 1 in the sequence listing in the breeding of wheat with excellent plant height or grain yield; X8) The application of detecting substances in the wheat genome corresponding to nucleotide 2184 of SEQ ID No. 1 in the sequence listing in the preparation and breeding of wheat products with excellent plant height or grain yield.

8. Breeding methods for wheat plant height or grain yield, including: Nucleotide 2184 of the wheat genome corresponding to SEQ ID No. 1 in the sequence listing was detected, and wheat with nucleotide A corresponding to SEQ ID No. 1 in the sequence listing was selected as the parent for breeding.

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