CN117965801B - Molecular marker closely linked with thousand grain weight of wheat and application thereof - Google Patents
Molecular marker closely linked with thousand grain weight of wheat and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker closely linked with thousand grain weight of wheat and application thereof, belonging to the technical field of wheat molecular biotechnology and breeding. The molecular marker is T2A2, is positioned on wheat 2A chromosome, is amplified by primers shown in SEQ ID NO. 1 and SEQ ID NO. 2, and has a high band as a variation sequence. The molecular marker T2A2 provided by the invention can rapidly and accurately judge whether the wheat variety (line) has thousand grain weight hot spots, provides excellent gene resources and selection tools for molecular breeding of wheat yield traits, can greatly reduce phenotype identification work when used for molecular breeding of wheat, saves breeding cost and improves breeding efficiency.
Description
Technical Field
The invention relates to a molecular marker and application thereof, in particular to a molecular marker closely linked with thousand grain weight of wheat on a 2A chromosome and application thereof in auxiliary breeding and genetic improvement of wheat molecules, belonging to the technical field of wheat molecular biotechnology and breeding.
Background
Wheat is one of the global important food crops, and its three elements of yield include: spike number, spike number and thousand grain weight per unit area. Thousand kernel weight is a key factor influencing wheat yield, and is inherited stably, and the wheat yield is changed synchronously along with the thousand kernel weight. Therefore, the key hot spot section for controlling thousand grain weight is excavated, the functional molecular marker is developed, the breeding process of new varieties of high-yield wheat can be greatly accelerated, and excellent gene resources and selection tools are provided for molecular breeding of wheat yield traits.
Thousand grain weight of wheat is a complex quantitative trait controlled by micro-effect multiple genes, and has complex genetic basis and is easily influenced by environmental factors. Many reports on the thousand kernel weight QTL of wheat exist so far, because different mapping population materials have different genetic backgrounds, marking types, genetic maps and environmental conditions, the different QTLs are difficult to directly compare, the key hotspot stabilizing sections for controlling the thousand kernel weight are not clear, and the bottleneck exists in the efficient utilization of wheat molecular breeding.
Meta-analysis (meta-analysis) is a method that can combine different study data for statistical analysis and can comprehensively test actual data, and can effectively reduce the range of QTL intervals. With the advent of molecular technology and the development of various molecular markers, the construction of wheat molecular genetic patterns and the genetic research of yield traits have made breakthrough progress, and a plurality of high-density consistency integration patterns are constructed and related reports of yield trait meta-QTL analysis exist so far, but most of the integration patterns have insufficient marker density, the collinearity relationship between the physical position and the genetic position of the patterns is poor, and the analysis of thousand-grain weight meta-QTL information is incomplete. Therefore, by utilizing a high-density consistency integration map and through meta-analysis technology, effective markers related to thousand grain weight of wheat are researched and developed, and the method has important significance in breeding work.
Disclosure of Invention
The invention aims at: developing a molecular marker positioned in a thousand-grain weight hot spot section of a wheat 2A chromosome and clarifying an application method thereof, and detecting whether the wheat variety or strain has a site for increasing the thousand-grain weight of the wheat or not by the obtained molecular marker so as to accelerate the breeding process of a new wheat variety with high yield.
In order to achieve the above object, the present invention adopts the following technical scheme:
The molecular marker is T2A2, is positioned in a thousand grain weight high-frequency hot spot zone on a wheat 2A chromosome, is obtained by carrying out PCR amplification on wheat to be detected by an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, wherein 9 bases are inserted into a 209576685bp position in a variation site of a Chinese spring Refseqv1.0 reference genome sequence, a high band is a variation sequence, the nucleotide sequence of the high band is shown in SEQ ID NO. 3, the nucleotide sequence of the low band is shown in SEQ ID NO. 4, and the position of the thousand grain weight of the wheat corresponding to the high band is increased.
The application of the molecular marker T2A2 closely linked with the thousand grain weight of the wheat in breeding the wheat yield-related character, wherein the yield-related character is the thousand grain weight.
The invention has the advantages that:
(1) The molecular marker T2A2 provided by the invention can be used for rapidly detecting whether a wheat variety (line) contains a thousand-grain-weight hot spot section, taking genomic DNA of the wheat variety (line) to be detected as a template, and carrying out PCR amplification on the wheat variety (line) by using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, so that whether the wheat variety (line) has a thousand-grain-weight hot spot site can be rapidly and accurately judged, and excellent gene resources and selection tools are provided for molecular breeding of wheat yield traits;
(2) The molecular marker T2A2 positioned in the wheat thousand seed weight hot spot section provided by the invention is used for wheat molecular breeding, so that the phenotype identification work can be greatly reduced, and the wheat can be utilized in the wheat seedling stage, so that non-target plants are eliminated, the breeding cost is saved, the breeding efficiency is improved, and more genetic resources are developed for wheat breeding.
Drawings
FIG. 1 is a plot of a wheat thousand kernel weight hot spot segment on a 2A chromosome, wherein the open rectangle represents the chromosome, the black solid is the thousand kernel weight high frequency hot spot segment on the wheat 2A chromosome, the right side of the chromosome is the name of the molecular marker, the left side of the chromosome is the position of the molecular marker on the chromosome, and the units are Mb; T2A2 is a thousand-grain weight InDel mark;
FIG. 2 is a diagram of PCR amplification results of a molecular marker T2A2 in a natural population part family, wherein M represents a 50bp DNA Ladder, and numerals 1 to 44 represent natural population part family amplification results;
Fig. 3 is a graph of single marker analysis results of thousand kernel weights of 293 families (heterozygous and deleted) of natural populations based on the molecular marker T2A2, wherein black bars are individuals with high amplified bands, white bars are individuals with low amplified bands, and x represents extremely significant differences (P < 0.01) and x represents significant differences (P < 0.05).
Detailed Description
The genetic resource related to the invention is wheat Triticum aestivum l, given by teacher of national academy of agricultural sciences.
The present invention will be described in detail with reference to the drawings and examples.
1. Thousand grain weight QTL element analysis
Carrying out statistics and arrangement on reported thousand grain weight related QTLs, integrating the QTLs into an ultra-high density consistency integration map according to a marker name, carrying out statistics on the QTLs integrated into the map with 1cM as a step length, defining a section with a site appearance more than 15 times as a high-frequency hot spot section, re-dividing the QTLs with 10cM as a step length by the obtained hot spot section, carrying out statistics on the physical position of the hot spot section in China spring Refseqv1.0 by combining genetic physical position information of the integration map, and selecting a section with a physical interval less than 30Mb on a 2A chromosome to carry out next InDel mutation site screening.
The information of the thousand-grain weight high-frequency hot spot region obtained by screening on the 2A chromosome is specifically shown in table 1.
TABLE 1 high frequency hot spot segment information for thousand kernel weight on 2A chromosome
2. Sequence difference analysis between target regions
And carrying out sequence difference analysis in a region of a high-frequency hot spot section (< 30 Mb) of thousands of weights of the screened 2A chromosome on a wheat genome variation combined database website (http:// heat. Cau. Edu. Cn/WheatUnion/b_17 /), screening sample libraries to select NG, NC and MP (containing 625 samples in total), selecting InDel as variation types, and selecting variation sites with the sample detection rate of more than 40% and the variation bases of more than or equal to 5 as the next step of specific primer screening sites.
174 Mutation sites meeting the requirements are screened out.
3. Thousand-grain weight hot spot segment molecular marker development
The above-screened 174 mutation sites are designed by utilizing PRIMERSERVER (http:// writes.sdau.edu.cn/PRIMERSERVER /) of WheatOmics to screen out specific primers with similar annealing temperatures of upstream and downstream primers and amplified fragment sizes of 200bp-500bp for further verification. The nucleotide sequence of the specific primers finally screened is as follows:
An upstream primer: AGGGGGACAGATGTATAGCT (SEQ ID NO: 1)
A downstream primer: GCGTAGTAGGAGTAGTCTGC (SEQ ID NO: 2)
And (3) carrying out PCR amplification on the DNA of each strain of the natural wheat population by using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, wherein the obtained molecular marker is marked as T2A2.
4. Performing field planting and phenotype identification in different test environments on natural population
And (3) carrying out field planting and phenotype identification of 7 test environments on natural populations, and respectively examining thousand seed weights of the strains in different years and at different places.
Different environments include: 2019-2020 Shandong tobacco stage waterfall valley (E1), 2019-2020 Shandong tobacco stage eastern Shandong university experiment base (E2), 2020-2021 Shandong tobacco stage waterfall valley (E3), 2020-2021 Hebei mountain village (E4), 2020-2021 Shandong tobacco stage eastern Shandong university experiment base (E5), 2021-2022 Shandong tobacco stage waterfall valley (E6), 2021-2022 Shandong tobacco stage bath village (E7). And finally, solving BLUE values of thousand grain weight characters of 7 environments by using an Ime4 package of R, and performing subsequent banding typing analysis.
The wheat planting method comprises the following steps: 2 rows are planted in each line, and 40 grains are sown in each row; the row length is 3m, the plant spacing is 7.5cm, the row spacing is 25cm, and the plant is grown and harvested normally.
Thousand grain weight determination method: after the wheat is mature, threshing the single plant, randomly selecting 100-200 normal grains on a single spike of the single plant main stem, detecting by using a tobranchlet agricultural seed tester, and repeating the average value for three times.
5. Verifying the molecular marker T2A2 in the natural wheat population
1. Extraction of DNA from each strain of wheat natural population by modified CTAB method
The method for extracting the DNA of each strain of the natural wheat population by using the modified CTAB (cetyl trimethyl ammonium bromide) method comprises the following steps:
(1) Placing steel balls and 0.2g of fresh wheat leaves into a 2.0mL centrifuge tube, rapidly placing the centrifuge tube into liquid nitrogen, and shaking the centrifuge tube to grind the wheat leaves into fine powder;
(2) Adding 0.8mL of CTAB extract into the centrifuge tube, shaking uniformly, and carrying out water bath at 65 ℃ for 60min, wherein the shaking uniformly is reversed for 4 times at intervals;
(3) Cooling the centrifuge tube at room temperature, adding equal volume of chloroform-isoamyl alcohol (volume ratio of 24:1), shaking vigorously for 1min, mixing, and centrifuging at 8000rpm for 10min;
(4) Sucking 600. Mu.L of the supernatant into another 1.5mL centrifuge tube, adding 0.8 times of pre-cooled isopropanol (-20 DEG pre-cooling) to precipitate DNA, and centrifuging at 12000rpm for 6min;
(5) Pouring out the supernatant, adding a proper amount of 70% ethanol into the centrifuge tube to wash the precipitate for 2 times, then obliquely placing the centrifuge tube in a fume hood to blow dry, adding 400 mu L of TE to dissolve after no alcohol smell exists, and storing in a refrigerator at the temperature of minus 20 ℃ for a long time.
2. Genotyping wheat natural population using molecular marker T2A2
(1) PCR amplification of DNA from wheat natural population
The PCR amplification system was 10. Mu.L, and specifically: 1. Mu.L of DNA template, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 5. Mu.L of 2 XTaq PCR premix, 3. Mu.L of ddH 2 O.
The PCR amplification procedure adopts a common amplification procedure, and specifically: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, and circulation 32 times; extending at 72 ℃ for 5min; and (5) finishing amplification and storing at 12 ℃.
(2) Electrophoresis of PCR amplified products
The PCR amplified products are subjected to electrophoresis by using non-denaturing polyacrylamide gel with the mass concentration of 6.0%, an electrophoresis buffer is 1 XTBE, and the constant pressure electrophoresis is carried out for 2 hours at 147V, and the gel preparation and electrophoresis processes are as follows:
placing the glass plate horizontally, placing a flat plate below the glass plate, placing a concave plate above the glass plate, placing a seal between the flat plate and the concave plate, and inserting a comb until the size is proper; 50mL of non-modified polyacrylamide gel with the mass concentration of 6.0% is needed for each plate of the small plate, 20 mu L of tetramethyl ethylenediamine (TEMED) and 200 mu L of ammonium persulfate aqueous solution with the mass concentration of 10% are added into each 50mL of non-modified polyacrylamide gel, and the mixture is stirred uniformly; draining and pouring glue by using a glass rod, and inserting a comb after glue pouring is finished; after the gel is solidified, the comb is pulled out, and the gel hole is washed by deionized water; placing the coagulated glass plate in an electrophoresis tank, clamping the groove inwards by using a clamp, filling electrophoresis buffer solution 1 XTBE up and down, spotting 0.25 mu L, and then carrying out constant-pressure electrophoresis for 2h at 147V; taking down the gel block after electrophoresis, fixing in a fixing solution for 3min, and rapidly washing with deionized water for 3 times for 30s each time; dyeing in silver dye liquor for 6min, and rapidly washing with water (about 10s, not more than 20 s); developing in a developing solution until an amplified band appears (when Marker appears and the color is no longer deepened); and (5) placing the film in deionized water, stopping imaging, and taking a picture to obtain the non-denaturing polyacrylamide gel electrophoresis picture.
The DNA of each strain of the wheat natural population is amplified by PCR by using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and the PCR amplification result of part of families is shown in figure 2.
(3) Analyzing the electrophoresis result
In the Chinese spring refseqv1.0 reference genome sequence, the mutation site of the molecular marker T2A2 is 9 bases inserted at 209576685bp, the high band is a mutation sequence, the size of the PCR amplified fragment of the high band is 272bp (SEQ ID NO: 3), and the size of the PCR amplified fragment of the low band is 263bp (SEQ ID NO: 4). Of the 318 families in the natural wheat population, there are 175 bands with only one high band and no low band (variation, denoted as a), 118 bands with only one low band and no high band (usual, denoted as B), and 9 bands with both high and low bands (heterozygous, denoted as H), and 16 bands with no band (deletion, denoted as /).
3. Genotype and thousand grain weight correlation analysis obtained by molecular marker T2A2 identification
The PCR read results and thousand kernel weight data of 318 families of wheat natural population under 7 test environments (E1, E2, E3, E4, E5, E6 and E7) are shown in Table 2.
TABLE 2 PCR read results and thousand kernel weight data for 318 families of wheat natural population
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Note that: a represents only one high band and no low band, B represents only one low band and no high band, H represents two high and low bands simultaneously, and/represents no band.
Heterozygosity (H) and deletion (/) were removed and the remaining wheat was genotyped and thousand kernel weight analyzed. The correlation analysis results of genotypes and thousand-grain weights obtained by the identification of the molecular marker T2A2 are shown in Table 3.
TABLE 3 correlation analysis results of genotypes and thousand-grain weights obtained by molecular marker T2A2 identification
Note that: BLUE represents the best linear unbiased estimate of each trait under 7 circumstances, P < 0.05, P < 0.01.
Table 3 is plotted to obtain the thousand grain weight single marker analysis results shown in fig. 3. In combination with the previous thousand-grain weight BLUE value, the average thousand-grain weight of individuals with high band type is 43.8g, the average thousand-grain weight of individuals with low band type is 41.8g, the average thousand-grain weight of individuals with high band type is increased by 2.0g compared with that of individuals with low band type, and the individuals with high band type have extremely obvious difference after T test.
It can be seen that the detection using the molecular marker T2A2 shows a high band, i.e. a site of increased thousand kernel weight.
It should be noted that the above examples are only examples for clearly illustrating the present invention, and are not limiting to the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which are obvious from the technical proposal of the invention are still within the protection scope of the invention.
Claims (1)
1. The application of the molecular marker T2A2 closely linked with the thousand grain weight of wheat in breeding the wheat yield-related characters is characterized in that:
The molecular marker T2A2 is positioned in a thousand grain weight high-frequency hot spot zone on a wheat 2A chromosome, the high-frequency hot spot zone is obtained by carrying out PCR amplification on wheat to be detected by an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, a variation site in a China spring refseqv1.0 reference genome sequence is 9 bases inserted at a 209576685bp position, a high band is a variation sequence, a site for increasing thousand grain weight exists in the wheat corresponding to the high band, the nucleotide sequence of the high band is shown in SEQ ID NO. 3, and the nucleotide sequence of the low band is shown in SEQ ID NO. 4;
The yield-related traits are thousand kernel weight.
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| CN117403003A (en) * | 2023-12-12 | 2024-01-16 | 鲁东大学 | Molecular marker of wheat TaCHLI-7B gene and application thereof |
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| JP2005229850A (en) * | 2004-02-17 | 2005-09-02 | Japan Science & Technology Agency | Gene marker connected to gene locus participating on thousand-kernel weight and its utilization |
| EP4111855A1 (en) * | 2021-06-29 | 2023-01-04 | INIAV - Instituto Nacional de Invesigação Agrária E Veterinária, I.P. | Snp based panel for mediterranean wheat plant selection and breeding |
| CN117385098A (en) * | 2023-12-12 | 2024-01-12 | 鲁东大学 | Molecular marker related to wheat grain density and application thereof |
| CN117403003A (en) * | 2023-12-12 | 2024-01-16 | 鲁东大学 | Molecular marker of wheat TaCHLI-7B gene and application thereof |
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| Title |
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