Primer combination for water conservation and quality screening of winter wheat and application of primer combination

Abstract

The invention discloses a primer combination for water saving and quality screening of winter wheat and application thereof, and relates to the technical field of molecular biology, the primer combination for water saving and quality screening of winter wheat comprises a primer pair P1 and a primer pair P2; according to the primer combination, the water-saving and drought-resistant characters are screened through PCR amplification, and SDS-PAGE electrophoresis is combined to detect grain protein subunit composition so as to screen quality characters. The efficient breeding method suitable for winter wheat is formed by designing a specific primer combination, constructing a molecular marker screening system and innovatively combining a cultivation management mode of water before drought and drought after water before winter wheat. According to the method, dependence on a single known gene is avoided, oriented improvement of water-saving drought resistance and quality is realized through collaborative screening and environment adaptability cultivation of a primer combination, and a new technical path is provided for wheat breeding in the arid region of North China.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a primer combination for water-saving and quality screening of winter wheat and an application thereof. Background Art

[0002] Hebei Province is one of the main wheat producing areas in my country, with an annual wheat planting area of ​​more than 33 million mu, and both the yield per unit area and the total yield are among the highest in the country. Its unique natural resource endowment has formed a significant industrial advantage - sufficient light and heat resources, light pests and diseases, and has bred high-quality medium-strong gluten wheat, making it a major high-quality wheat producing area in my country; but it also faces severe challenges - the per capita water resources are only 237m 3 , less than 12% of the national average. The extreme water shortage caused by over-exploitation of groundwater has made "water conservation" and "high quality" the core goals of wheat breeding in Hebei Province in the long term.

[0003] Traditional wheat breeding relies on field phenotypic screening, which has problems such as long cycles, low efficiency, and difficulty in aggregating target traits. Although the development of molecular marker technology has provided a means for the detection of water-saving and drought-resistant genes and quality-related subunits, existing methods mostly focus on the function of a single gene and lack a systematic solution for the drought stress environment and high-quality wheat quality requirements in Hebei Province. For example, it is difficult to take into account both field agronomic traits and regional adaptability by simply relying on marker screening of known gene sequences; conventional cultivation management has not fully combined the water regulation needs of arid areas, resulting in limited effects of synergistic improvement of water conservation and high yield.

[0004] With the advancement of agricultural water-saving policies in Hebei Province, developing a breeding method that can efficiently combine water-saving and drought-resistant characteristics with high-quality grain quality has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a primer combination for water-saving and quality screening of winter wheat and its application.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A primer combination for water-saving and quality screening of winter wheat, the primer combination comprising a primer pair P1 and a primer pair P2; the primer combination screens water-saving and drought-resistant traits through PCR amplification, and combines SDS-PAGE electrophoresis to detect grain protein subunit composition to screen quality traits.

[0008] As a preferred technical solution of the present invention, the primer pair P1 is the primer pair 1F and 1R composed of SEQ ID NO.1 and SEQ ID NO.2 in the sequence list; the primer pair P2 is the primer pair 2F and 2R composed of SEQ ID NO.3 and SEQ ID NO.4 in the sequence list.

[0009] A method for screening winter wheat germplasm resources based on the above primer combination comprises the following steps:

[0010] (1) Establish a winter wheat germplasm resource bank to collect locally approved or promoted wheat varieties;

[0011] (2) extracting genomic DNA from the germplasm resources, performing PCR amplification using the primer combination of claim 1, and screening for strains that meet the following conditions:

[0012] Target fragment A was amplified by primer pair P1;

[0013] Fragment B with a length of 677 bp was amplified by primer pair P2;

[0014] (3) for the strains selected in step (2), further detecting the grain protein subunit composition by SDS-PAGE electrophoresis to select strains that meet the high-quality protein standards;

[0015] (4) The strains that simultaneously meet the requirements of fragment A, fragment B, and high-quality protein subunits are included in the molecular marker screening library.

[0016] A method for breeding water-saving high-quality winter wheat strains comprises the following steps:

[0017] (1) selecting a parent from the molecular marker screening library according to claim 2 for hybridization;

[0018] (2) Extracting DNA from individual plants in the F2 generation population, performing PCR amplification using the primer combination of claim 1, and screening homozygous plants that simultaneously meet the following conditions:

[0019] The amplified product of fragment A was verified by sequencing to be consistent with the target region;

[0020] Fragment B is 677 bp in length;

[0021] (3) for the individual plants selected in step (2), detecting the grain protein subunit composition by SDS-PAGE electrophoresis, and retaining the individual plants homozygous for high-quality protein subunits;

[0022] (4) The selected individual plants are cultivated in the field in the F3-F5 generations, using a water-first-then-dry management mode to obtain stable strains with strong drought resistance and excellent quality.

[0023] Preferably, the irrigation volume of the "water first, drought second" cultivation management in step (4) is 40-60m 3 / mu, no irrigation is required during the filling and maturity stages.

[0024] A screening kit for water-saving high-quality traits of winter wheat comprises: the above primer combination, PCR reaction buffer, standard reference substances for identifying fragments A and B, and protein detection reagents required for SDS-PAGE electrophoresis.

[0025] The beneficial effect of adopting the above technical solution is that the present invention forms a set of efficient breeding methods suitable for winter wheat by designing specific primer combinations, constructing a molecular marker screening system, and innovatively combining the "water first, drought later" cultivation management mode. This method avoids dependence on a single known gene, and instead achieves directional improvement of water-saving, drought resistance and quality through coordinated screening of primer combinations and environmental adaptability cultivation, providing a new technical path for wheat breeding in the arid areas of North China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the specific PCR amplification diagram of fragment A gene, where M: DL2000 marker; 1-12 different germplasm resources; 13: water;

[0027] Figure 2 The electrophoresis diagram of two allele types of fragment B;

[0028] Figure 3 This is the electrophoresis diagram of high-quality protein subunits of some wheat varieties. DETAILED DESCRIPTION

[0029] The following examples describe the present invention in detail. Various raw materials and equipment used in the present invention are conventional commercial products, and can be directly obtained by market purchase. In the description of the following examples, for the purpose of illustration rather than limitation, specific details such as specific system structures, technologies, etc. are proposed, so as to thoroughly understand the present application embodiments. However, it should be clear to those skilled in the art that the present application can also be realized in other embodiments without these specific details. In other cases, the detailed description of well-known systems, devices and methods is omitted to prevent unnecessary details from hindering the description of the present application.

[0030] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in the present specification and the appended claims refers to any combination of one or more of the items listed in association and all possible combinations, and includes these combinations. As used in the present specification and the appended claims, the term "if" can be interpreted as "when..." or "once" or "in response to determination" or "in response to detection" according to the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "once it is determined" or "in response to determination" or "once [described condition or event] is detected" or "in response to detection [described condition or event]" according to the context.

[0031] In addition, in the description of the present application specification and the attached claims, the terms "first", "second", "third", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance. The reference to "one embodiment" or "some embodiments" described in the present application specification means that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in the different parts of this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0032] Example 1 Detection of target fragments of germplasm resources

[0033] The breeding method for improving winter wheat water conservation and quality includes the following steps:

[0034] Primer design: By analyzing the target regions related to water conservation and drought resistance in the winter wheat genome, two pairs of specific primers were designed:

[0035] Primer pair P1 (F1: 5'-CGGACCCGTGAAGTGCGCA-3' (SEQ ID NO.1); R1: 5'-GCGCTTCGGCTCCGAAATC-3' (SEQ ID NO.2)): targeting the target region located on chromosome 4B, used to amplify fragment A (expected length: 262 bp), fragment A has allelic variation, wherein the genotype of fragment A being homozygous for CC is named C, and the genotype of fragment A being homozygous for TT is named c;

[0036] The genomic DNA of 356 wheat germplasm resources was extracted as PCR template. The PCR amplification system was 20 μL, including 2 μL DNA template (50 ng μL-1), 1 μL forward and reverse primers (10 μmol L-1), 10 μL 2× Taq master mix (Shanghai, China), ddH 2 O 6μL. PCR reaction program: 94℃ pre-denaturation for 5min; 94℃ denaturation for 40s, 61.1℃ annealing for 40s, 72℃ extension for 40s, 35 cycles; 72℃ extension for 10min. PCR products were detected by agarose gel electrophoresis. 12 PCR products were randomly selected for sequencing, and fragment A was successfully amplified specifically ( Figure 1 ).

[0037] Primer pair P2 (F2: 5'-GGAGCATGCTTACAAAACCTATG-3' (SEQ ID NO. 3); R2: 5'-GACCCACCTGTCAGAGACTTT-3' (SEQ ID NO. 4)): targeting the target region of chromosome 2A, specifically amplifying fragment B with a length of 677 bp;

[0038] The genomic DNA of 356 wheat germplasm resources was extracted as PCR template. The PCR amplification system was 20 μL, including 2 μL DNA template (50 ng μL-1), 1 μL forward and reverse primers (10 μmol L-1), 10 μL 2× Taq master mix (Shanghai, China), ddH 2 O 6μL. PCR reaction program: 94℃ pre-denaturation for 5min; 94℃ denaturation for 40s, 56.0℃ annealing for 40s, 72℃ extension for 90s, 35 cycles; 72℃ extension for 10min. PCR products were detected by agarose gel electrophoresis ( Figure 2 ), the fragment sizes were 677 bp (D) and 1378 bp (d).

[0039] Verified by agarose gel electrophoresis and sequencing, all 12 samples randomly sequenced for fragment A showed consistency ≥99%;

[0040] The 677 bp band in fragment B accounted for 75.8% (270 / 356).

[0041] According to the composition characteristics of fragment A and fragment B, 356 germplasm resources were divided into four haplotypes, namely CD, Cd, cD and cd (Table 1).

[0042] Table 1 Types of water-saving and drought-resistant genes

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] According to Mendel’s law of independent assortment, there are 16 offspring types after configuration and combination (Table 2). According to the breeding goal, the offspring materials are expected to contain two excellent allele variations of fragment A and fragment B. Among them, 9 parent combinations meet the requirements, namely CD×CD, CD×Cd, CD×cD, CD×cd, Cd×CD, Cd×cD, cD×CD, cD×Cd, and cd×CD.

[0053] Table 2 Parental types of water-saving and drought-resistant genes

[0054]

[0055] Example 2: Detection of high-quality protein subunits

[0056] (1) SDS-PAGE electrophoresis

[0057] Take 2 grains of each germplasm resource and grind them into powder independently. Perform SDS-PAGE electrophoresis. According to the electrophoresis band position of the reference material Shi Luan 02-1, read the high-quality protein bands of various germplasm resources and determine the subunit composition (such as Figure 3 );

[0058] (2) Composition of high-quality protein subunits in germplasm resources:

[0059] The results of high-quality protein detection of 356 germplasm resources showed that 217 germplasm resources contained 7+8 high-quality subunits (Table 2). It is known that the proportion of 7+8 high-quality subunits at the B1 locus of wheat in the Huanghuai wheat region is more than 60%, which is the dominant subunit. In order to improve breeding efficiency, high-quality subunits at the A1 and D1 loci were screened on the basis of 217 materials containing 7+8 subunits. A total of 11 types were detected, namely 1,7+8,5+10, 1,7+8,5+12, 1,7+8,2+12, 1,7+8,4+12, n,7+8,5+10, n,7+8,5+12, n,7+8,2+12, n,7+8,4+12, 2*,7+8,5+10, 2*,7+8,5+12, 2*,7+8,4+12 (Table 3).

[0060] The specific method to identify the parents of high-quality protein and high-quality subunits is as follows: A1 site is 1, n or 2*, 1 subunit is named E, and the rest are e; D1 site is 5+10 and 5+12 is named F, and the rest are f. A1 and D1 sites are located on chromosomes 1A and 1D, respectively, which conforms to the law of free combination. There are 16 types of offspring (Table 4). According to the breeding goal, the offspring materials are expected to contain two excellent alleles of 1 and 5+10 or 1 and 5+12. Among them, 9 parent combinations meet the requirements, namely EF×EF, EF×Ef, EF×eF, EF×ef, Ef×EF, Ef×eF, eF×EF, eF×Ef, ef×EF, ef×EF.

[0061] Table 3 High-quality protein subunit composition

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Table 4 High-quality protein parent types

[0068]

[0069] Example 3: Configuration combination

[0070] (1) The combination is based on the principle that the two parents have complementary genes in water-saving and drought-resistant and quality-related genes. There are 171 materials in the germplasm resources that contain excellent alleles of both fragment A and fragment B water-saving and drought-resistant genes. When used as parents, only the high-quality subunit configuration combination determined in Example 2 needs to be referred to; there are 160 materials in the germplasm resources that contain only one excellent allele of the water-saving and drought-resistant gene, and the configuration combination of the water-saving and drought-resistant gene and the high-quality subunit in Example 1 needs to be referred to at the same time; 25 materials in the germplasm resources do not contain excellent alleles of the water-saving and drought-resistant gene, and can only be used as parents with water-saving and drought-resistant gene CD type materials, and the high-quality subunit configuration combination determined in Example 2 needs to be referred to at the same time.

[0071] (2) Wheat water-saving, drought-resistance and quality are complex agronomic traits controlled by multiple genes. Aggregating more water-saving and drought-resistance genes and quality-related loci is expected to improve wheat water-saving, drought-resistance and quality. In order to breed water-saving, drought-resistant and high-quality wheat varieties, it is also necessary to comprehensively consider field phenotypes, such as the complementary configuration of agronomic traits such as yield, yellowing, disease resistance and cold resistance. The combination of molecular design breeding and conventional selection is the basis for breeding new varieties with water-saving, drought-resistance and good quality.

[0072] Example 4: Molecular marker tracking detection

[0073] (1) The specific method for molecular marker tracking detection of water-saving and drought-resistant genes is as follows: select individual plants with excellent agronomic traits from each combination F2 generation, extract leaf gDNA after marking, use the primers in Example 1 to perform PCR amplification on fragment A and fragment B respectively, and detect by agarose gel electrophoresis. The number of F2 generation lines detected for each combination is not less than 16;

[0074] (2) The specific method for tracking and detecting high-quality subunits is as follows: single plants homozygous for the excellent allele variants of fragments A and B in the F2 generation are screened and harvested (CCDD); two seeds are randomly selected from each strain, ground, and the seed protein is extracted for SDS-PAGE electrophoresis detection to identify the strains homozygous for the high-quality subunits at the A1 and D1 sites (EEFF).

[0075] Example 5: Cultivation management with water first and drought later

[0076] The specific method of cultivation management with water before drought is: sow the selected homozygous strains F3-F5 generations with sufficient moisture to ensure the formation of full and strong seedlings, give full play to the individual tillering potential, apply topdressing and water at the jointing stage of wheat in spring, and no longer water until maturity (generally, water is applied once during the grain filling period of wheat fields). The drought treatment in the later stage is easy to select drought-resistant offspring materials in a drought environment. During the growth period, phenotypic identification of conventional agronomic traits such as field luxuriance, cold resistance, plant height, yellowing, disease resistance, and grain fullness is carried out.

[0077] Example 6: Participating in national or central and southern Hebei variety trials

[0078] Participating in the successful regional and biological trials of wheat varieties in central and southern Hebei Province:

[0079] (1) Among the water-saving and drought-resistant germplasm resources, Jimai 22 is a wheat variety that has been widely promoted and applied, and has the characteristics of wide adaptability; Liangxing 99 has a good yellowing and is well-liked by the people. Both have been used for comparison in the northern area of ​​the Yellow Sea; Shiyou 17 is a high-quality variety bred by the Shijiazhuang Academy of Agricultural Sciences. This variety has a strong wheat flavor and is very suitable for making noodles and dumplings. Shiyou 17, Jimai 22, and Liangxing 99 are complementary in agronomic traits and quality, and meet the conditions for selecting conventional breeding parents.

[0080] (2) The water-saving and drought-resistant fragments and high-quality protein subunit compositions of Shiyou 17, Jimai 22, and Liangxing 99 were tested, and it was found that the genotypes of their water-saving and drought-resistant fragments were Cd, cD, and cD, respectively, and the genotypes of their high-quality subunits were EF, eF, and ef, respectively, and they were all complementary. The genotype of Shiyou 17 was CdEF, the genotype of Jimai 22 was cDeF, and the genotype of Liangxing 99 was cDef (Table 5). Shiyou 17 was configured with Jimai 22 and Liangxing 99 in the CdEF×cDeF and CdEF×cDef combinations, respectively. Among them, the probability of CDEF in the F2 generation of the CdEF×cDeF combination was 1 / 8, and the probability of CDEF in the F2 generation of the CdEF×cDef combination was 1 / 64. Therefore, the Shiyou 17 / Jimai 22 combination screened no less than 8 F2 plants, and the Liangxing 99 / Shiyou 17 combination screened no less than 64 F2 plants. The marker detection was carried out until the F2 homozygous CDEF strains and the plants with excellent field performance were obtained.

[0081] Table 5 Breeding varieties and their parental genotypes

[0082]

[0083] (3) F3-F5 continued to select strains with excellent agronomic traits under the water-first-dry-last cultivation mode to participate in regional trials in the northern Huanghuai area or central and southern Hebei. Shi 4366 and Shimai 26 successfully completed the trial process and obtained variety certification.

[0084] Example 7: Quality Inspection

[0085] Protein properties are closely related to the quality of steamed bread, affecting the appearance, volume, specific volume, internal structure, etc. of steamed bread. The specific quality testing is as follows:

[0086] (1) The protein content of flour of the approved varieties Shi 4366 and Shimai 26, the high-quality parent donor Shiyou 17 and the water-saving control variety Shimai 22 was determined by Kjeldahl nitrogen determination. The relative content of different molecular weight proteins in flour was determined by Agilent 1200 high performance liquid chromatography (Agilent Technologies Co., Ltd.), including SDS-insoluble protein macropolymers (UPP), large molecular weight polymeric proteins (LPP), large molecular weight monomeric proteins (LMP), small molecular weight monomeric proteins (SMP) and LPP / LMP. The data were from 3 replicates, and were sorted and plotted using Excel 2016. SPSS 18.0 was used to perform analysis of significant differences (ANOVA).

[0087] (2) Statistical analysis showed that among the six protein content indicators, the PC and LMP contents of Shimai 26 were significantly higher than those of Shiyou 17, and the PC, UPP, LPP and LPP / LMP of Shi 4366 were significantly higher than those of Shiyou 17 (Table 6). The results of the texture parameter analysis of steamed bread showed that the hardness, elasticity and resilience of Shimai 26 were significantly better than those of Shiyou 17. Except for resilience, Shi 4366 had no significant difference with Shiyou 17. Its resilience was significantly better than that of the water-saving control variety Shimai 22 (Table 7).

[0088] Table 6 Relative content of proteins with different molecular weights

[0089]

[0090] Note: Protein content (PC), SDS-insoluble protein macropolymer (UPP), large molecular weight polymer protein (LPP), large molecular weight monomer protein (LMP), small molecular weight monomer protein (SMP) and LPP / LMP.

[0091] Table 7 Texture parameters of steamed bread of wheat varieties

[0092] hardness Adhesion elasticity Chewing degree Responsiveness Shimai 26 2406±86a -22.326±6.736a 0.868±0.015a 1346±60a 0.397±0.005a Stone 4366 1048±117c -20.859±8.683a 0.876±0.004a 592±64c 0.373±0.003b Shiyou17 988±13c -19.908±5.725a 0.882±0.005a 563±7c 0.389±0.004a Stone Wheat 22 1544±28b -30.224±1.237a 0.869±0.004a 805±18b 0.336±0.004c

[0093] The present invention forms a set of efficient breeding methods suitable for winter wheat by designing specific primer combinations, constructing a molecular marker screening system, and innovatively combining the "water first, drought later" cultivation management model. This method avoids dependence on a single known gene, and instead achieves directional improvement of water-saving, drought resistance and quality through coordinated screening of primer combinations and environmental adaptability cultivation, providing a new technical path for wheat breeding in the arid areas of North China.

[0094] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0095] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A primer combination for water-saving and quality screening of winter wheat, characterized in that: The primer combination comprises a primer pair P1 and a primer pair P2; the primer combination is used to screen water-saving and drought-resistant traits through PCR amplification, and is combined with SDS-PAGE electrophoresis to detect grain protein subunit composition to screen quality traits.

2. A primer combination for water-saving and quality screening of winter wheat according to claim 1, characterized in that: The primer pair P1 is the primer pair 1F and 1R consisting of SEQ ID NO.1 and SEQ ID NO.2 in the sequence list; the primer pair P2 is the primer pair 2F and 2R consisting of SEQ ID NO.3 and SEQ ID NO.4 in the sequence list.

3. A method for screening winter wheat germplasm resources based on the primer combination of claim 1, characterized in that: The following steps are involved: (1) Establish a winter wheat germplasm resource bank to collect locally approved or promoted wheat varieties; (2) Extracting genomic DNA from the germplasm resources, performing PCR amplification using the primer combination of claim 1, and screening strains that meet the following conditions: Target fragment A was amplified by primer pair P1; Fragment B with a length of 677 bp was amplified by primer pair P2; (3) For the strains selected in step (2), further detecting the grain protein subunit composition by SDS-PAGE electrophoresis to select strains that meet the high-quality protein standards; (4) The strains that simultaneously meet the requirements of fragment A, fragment B, and high-quality protein subunits are included in the molecular marker screening library.

4. A method for breeding water-saving high-quality winter wheat strains, characterized in that: The following steps are involved: (1) selecting a parent from the molecular marker screening library described in claim 2 for hybridization; (2) Extracting DNA from individual plants in the F2 generation population, performing PCR amplification using the primer combination of claim 1, and screening homozygous plants that simultaneously meet the following conditions: The amplified product of fragment A was verified by sequencing to be consistent with the target region; Fragment B is 677 bp in length; (3) For the individual plants selected in step (2), the grain protein subunit composition is detected by SDS-PAGE electrophoresis, and the individual plants homozygous for high-quality protein subunits are retained; (4) The selected individual plants are cultivated in the field in the F3-F5 generations, using a water-first-then-dry management model to obtain stable strains with strong drought resistance and excellent quality.

5. The breeding method according to claim 4, characterized in that: In step (4), the irrigation amount for the "water first, drought later" cultivation management is 40-60 m³ / mu, and no irrigation is required during the filling and maturity stages.

6. A screening kit for water-saving and high-quality traits of winter wheat, characterized in that: The method comprises: the primer combination according to claim 1, a PCR reaction buffer, a standard control substance for identifying fragments A and B, and a protein detection reagent required for SDS-PAGE electrophoresis.

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