CN113817862B - KASP-Flw-sau6198 molecular marker linked with wheat flag leaf width major QTL and application thereof - Google Patents
KASP-Flw-sau6198 molecular marker linked with wheat flag leaf width major QTL and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP-Flw-sau6198 molecular marker linked with a wheat flag leaf width major QTL and application thereof, belonging to the fields of molecular biology and crop genetic breeding. The molecular marker is an SNP molecular marker, the polymorphism is A/G, the molecular marker and the wheat flag leaf width QTL QFLW.sau-AM-4B.4 are co-positioned on the tetraploid wheat 4B chromosome long arm and are positioned in the QTL QFLW.sau-AM-4B.4 interval. Detection and analysis show that the molecular marker can accurately track the wheat flag leaf width QTLQFLW.sau-AM-4B.4, predict the flag leaf width characteristic of wheat, and is beneficial to quickly screening out wheat varieties or strains with wider flag leaves for breeding, thereby greatly accelerating the process of wheat breeding.
Description
Technical Field
The invention relates to the fields of molecular biology and crop genetic breeding, in particular to a KASP-Flw-sau6198 molecular marker linked with a wheat flag leaf width major QTL and application thereof.
Background
Wheat (Triticum sp.) is one of crops widely planted worldwide, and in the face of the ever-increasing world population, how to improve wheat yield becomes a key problem to the breeders.
Flag leaves are the main photosynthetic organs of wheat and can influence the accumulation of dry substances of crops and the formation of yield. Flag leaf size therefore has a great influence on the final yield of wheat. Flag leaf width is an important agronomic trait, is one of important constitutive factors of wheat plant type, is a main organ of plant photosynthesis, and plays an important role in yield potential in wheat. In the grouting period, half of the organic matter of the seeds is provided by flag leaves, and the yield of wheat is directly influenced. In addition, flag leaves determine the canopy structure of wheat, which affects flowering time, light exposure, photosynthesis, seed filling, and ultimately seed yield per plant. In view of the potential of flag leaves for increasing yield, the research on flag leaf characters is strengthened on the QTL level, and the main effect sites of flag leaf characters are mined, identified, analyzed and utilized, so that more understanding can be provided for the role of flag leaf characters in yield formation and new ways for providing high yield of wheat. However, in the process of improving common wheat, due to the utilization of a few backbone parent resources, the genetic diversity among varieties is reduced, the yield is limited by a narrow genetic base, and the adverse effect on the future wheat improvement breeding is obvious. However, a large amount of unexplored genetic resources in wheat kindreds can effectively help us to face the challenge of future wheat production, and the acceleration of the utilization of unique gene resources in the kindreds is very important for increasing the yield of common wheat.
The wheat flag leaf trait is a complex quantitative trait, is usually controlled by a gene network, is mostly a micro-effect gene and is also influenced by the environment. The difficulty of quantitative trait research is increased because the phenotype and the genotype cannot show a clear correspondence. The appearance of the third generation molecular marker, Single Nucleotide Polymorphism (SNP), provides a good platform for constructing a wheat high-density genetic linkage map and quantitative trait genetic research, and provides an effective way for analyzing such complex Quantitative Trait Loci (QTL).
Single Nucleotide Polymorphism (SNP) refers to a DNA sequence Polymorphism caused by a change such as a transition, a transversion, an insertion, or a deletion at a specific Nucleotide position in DNA in a genome. The technology is that known sequence information is utilized to compare and search SNP sites, specific primers are designed by utilizing the discovered variation sites to carry out PCR amplification on genome DNA or cDNA, specific polymorphic products based on the SNP sites are obtained, and finally, the electrophoresis technology is utilized to analyze the polymorphism of the products. The SNP markers have the advantages of large quantity and wide distribution; uneven distribution among individual genes and the entire genome; SNP allele frequencies are easily estimated.
KASP (Kompetitive Allle-Specific PCR), namely competitive Allele-Specific PCR, carries out accurate double Allele typing on SNP and In Del sites through Specific matching of terminal basic groups of primers, and is widely applied to molecular marker-assisted selection of crops such as rice, wheat, soybean and the like as a flexible, economic and accurate SNP detection method.
At present, QTL positioning related reports are carried out on flag leaf width, and QTL related to the flag leaf width is found to be widely existed in wheat and distributed on different chromosomes of the wheat. However, there are not many closely linked molecular markers that are currently associated with the wheat flag leaf wide trait and can be used for practical molecular breeding. Therefore, the research of obtaining the QTL or the gene related to the flag leaf width has important guiding significance for selecting wheat plants with proper flag leaf width and breeding wheat by utilizing a molecular biology technology.
Disclosure of Invention
The invention aims to provide a KASP-Flw-sau6198 molecular marker linked with a wheat flag leaf width major QTL and application thereof, and aims to solve the problems in the prior art, wherein the molecular marker KASP-Flw-sau6198 is extremely obviously related with the flag leaf width QTL QFLW.sau-AM-4B.4, presents the characteristics of a tightly linked marker, has high accuracy for molecular marker auxiliary selection, can obviously improve the selection and identification efficiency of longer flag leaf wheat varieties in different environments, and has high success rate.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a KASP-Flw-sau6198 molecular marker linked with QTL QFLW.sau-AM-4B.4, wherein the molecular marker is an SNP molecular marker with polymorphism A/G, is co-located on a tetraploid wheat 4B chromosome long arm with wheat flag leaf width QTL QFLW.sau-AM-4B.4 and is located in a QTL QFLW.sau-AM-4B.4 interval.
The invention also provides a primer composition, which comprises the nucleotide sequence shown as SEQ ID NO: 1-2 and the sequence shown in SEQ ID NO: 3, and the primer combination is used for amplifying the KASP-Flw-sau6198 molecular marker.
Furthermore, different fluorescent modifying groups are respectively added at the 5 'end or different fluorescent modifying groups are respectively added at the 3' end of the primer sequence shown in SEQ ID NO. 1-2.
Further, fluorescence modifying groups include, but are not limited to, FIFC, FAM, TET, HEX, JOE, TAMRA, BHQ.
The invention also provides a chip for identifying wheat genes or gene fragments thereof, which comprises the KASP-Flw-sau6198 molecular marker and/or the primer composition.
The invention also provides a kit for identifying wheat genes or gene fragments thereof, which comprises the KASP-Flw-sau6198 molecular marker and/or the primer composition.
The invention also provides application of the wheat flag leaf width QTL QFLW.sau-AM-4B.4 or wheat KASP-Flw-sau6198 molecular marker in regulation and control of wheat flag leaf width characters.
The invention also provides an application of the KASP-Flw-sau6198 molecular marker or the primer composition or the chip or the kit, which is used for any one of the following applications:
(1) the application in molecular breeding of crops;
(2) the application in cultivating transgenic wheat;
(3) the application in the improvement of wheat germ plasm resources;
(4) the application in screening wheat varieties or lines with proper flag leaf width;
(5) the application in wheat flag leaf wide gene genetic analysis and fine positioning.
The invention provides a method for screening a wheat line containing flag leaf width QTL QFLW.sau-AM-4B.4, which comprises the following steps:
and (2) performing fluorescent quantitative PCR amplification by using the genome DNA of a plant sample to be detected as a template and using the primer composition, and performing genotype typing according to an obtained amplification product so as to screen out a wheat strain containing the flag leaf width QTL QFLW.sau-AM-4 B.4.
In the primer composition, different fluorescent modifying groups are respectively added at 5 'or 3' to the primer sequence shown in SEQ ID NO.1-2, and a plant capable of reading the fluorescent group marked by SEQ ID NO.1 is identified as a plant containing wheat flag leaf width QTL QFLW.sau-AM-4 B.4.
Preferably, the fluorescent quantitative PCR reaction system comprises: 10 μ L Master Mix, 2.8 μ L mixed primer, 10ng template DNA, double distilled water to total amount of 20 μ L; wherein the mixed primer consists of SEQ ID No: 1-SEQ ID No: 3 to 150. mu.L, 150. mu.L and 350. mu.L of each of the three primers shown in FIG. 3 at a concentration of 10 ng/. mu.L, and ddH was added2O450 mu L of the mixture.
Preferably, the reaction procedure of the fluorescent quantitative PCR includes: pre-denaturation at 94 ℃ for 12 min; denaturation at 94 ℃ for 20s, renaturation/elongation at 59 ℃ for 55s, for 10 cycles; denaturation at 94 ℃ for 20s, renaturation/elongation at 56 ℃ for 65s, for 30 cycles; after completion, fluorescence readings were taken.
The invention discloses the following technical effects:
(1) the invention discloses a flag leaf width QTL QFLW.sau-AM-4B.4 from tetraploid local wheat Ailanmai for the first time, which is positioned on the long arm of a tetraploid wheat 4B chromosome and can obviously increase the flag leaf width of the wheat. The QTL has higher utilization value in wheat yield (flag leaf width regulation) breeding.
(2) The invention discloses a molecular marker KASP-Flw-sau6198 for accurately detecting flag leaf width QTL QFLW.sau-AM-4B.4 of wheat Ailanmai' based on a fluorescence quantitative PCR platform, which is a codominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification.
(3) The molecular marker KASP-Flw-sau6198 disclosed by the invention is remarkably related to flag leaf width QTL QFLW.sau-AM-4B.4, presents the characteristic of a close linkage marker, has high accuracy for the auxiliary selection of the molecular marker, can remarkably improve the selection and identification efficiency of wheat wide flag leaf varieties suitable for different environments, and has high success rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a diagram showing the location of the small wheat flag leaf width QTL QFLW.sau-AM-4B.4 on the 4B chromosome in example 1 of the present invention;
FIG. 2 shows the fluorescence reading results of the molecular marker KASP-Flw-sau6198 of the plant of the recombinant inbred line strain of 'Ailanmai' x 'LM 001' in example 1 of the present invention; wherein, FAM (round, 'Ailanmai') fluorescence is a strain with wider flag leaves, and HAX (square, 'LM 001') fluorescence is a strain with narrower flag leaves; the triangle fluorescence is a heterozygous strain; diamond fluorescence was blank control;
FIG. 3 shows the fluorescence reading results of the molecular marker KASP-Flw-sau6198 of the recombinant inbred line strain of wheat 'Ailanmai' X wheat variety 'PI 503554' in example 2 of the present invention; wherein, FAM (round, 'Ailanmai') fluorescence is a strain with wider flag leaves, and HAX (square, 'PI 503554') fluorescence is a strain with narrower flag leaves; the diamond fluorescence is blank.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The wheat lines 'Ailanmai', 'LM 001', 'PI 503554' used in the examples below are all provided by Sichuan university of agriculture wheat.
Example 1 obtaining of wheat flag leaf width QTL QFLW. sau-AM-4B.4 and its molecular marker KASP-Flw-sau6198
(1) Hybridizing by using wheat strain 'Ailanmai' as female parent and wheat strain 'LM 001' as male parent to obtain hybrid F1，F1Selfing the single plant to obtain F2At F2Using single ear propagation up to F8And (4) obtaining a recombinant inbred line containing 121 lines to form a genetic mapping population.
(2) Identifying the wide phenotype of flag leaves of the recombinant inbred line population: analyzing and identifying the flag leaf width of the recombinant inbred line in the mature period of the wheat, removing the single plants at the two ends of each row, collecting five single plants with consistent growth vigor respectively, calculating the flag leaf width, and obtaining an average value which represents the flag leaf width of the plant line.
(3)55K SNP chip analysis
a) DNA extraction: the DNA of parent 'Ailanmai', 'LM 001' and the plant of the recombinant inbred line population is extracted by a CTAB method.
b) The extracted DNA is subjected to quality detection by using an ultramicro spectrophotometer, and is sent to a company for genotype analysis after being qualified, and the genotype analysis of the parents and mapping group in the research is completed by a 55K SNP chip developed by combining Beijing Boao crystal and classical biotechnology limited (http:// www.capitalbiotech.com) and Jia Suo Jian project.
c) Constructing a linkage map: according to 55K SNP chip data, a genetic map is constructed by using JoinMap4.0. Combining flag leaf width phenotype data of a population, using an integral complex Interval Mapping-ADD (ICIM-ADD) in QTL IciMapping 4.0, setting a threshold LOD to be more than or equal to 3.28, using BLUP (best linear unbiased prediction) values of 6 ecological points and 6 ecological point flag leaf widths in 2017 + 2021 to detect the QTL, positioning the wheat flag leaf width QTL LW.sau-AM-4B.4, and calculating the position of the QTL LW.sau-AM-4B.4 and the genetic distance between molecular markers.
d) Comparison of flag leaf sites and acquisition of molecular markers: the previous reports that there are more QTL or genes associated with flag leaves. Jianan Ma et al used the RIL population containing 199 strains of common wheat "20828" and "CN 16" to locate 1 QTL site for stably controlling the leaf width of flag, which is located on 2D chromosome, and explain 6.85% -21.35% of the phenotypic variation rate; kaiye Liu et al used the combined RIL population containing 213 lines of semi-wild wheat "Tibetan 1817" and common wheat "Ningdong 3331" to locate 3 QTL sites controlling flag leaf width, which are located on 1B and 4B chromosomes, respectively, and the interpretation rate of phenotypic variation of a single site is 6.06% -14.70%; yang Tu et al used the RIL population containing 128 lines of common wheat "20828" and "SY 95-71" to locate 2 stably controlled flag leaf wide QTL sites on 2B and 2D chromosomes, respectively, with an interpretation rate of phenotypic variation at individual sites of 8.89% -48.89%; wright et al created a new parental population using wheat in a number of tetraploid loci that differ in physiological traits and identified a flag leaf width QTL on the 5B chromosome in conjunction with a wheat 35K SNP chip. The QTLs located on chromosome 4B were rare and all were far from qflw. sau-AM-4b.4, indicating that qflw. sau-AM-4b.4 is likely to be a new stable QTL.
In order to further densify the map and obtain molecular markers closely linked with the flag leaf width QTL QFLW. sau-AM-4B.4, the flanking markers are physically positioned and genes positioned in the interval are screened by utilizing the data positioning result of the 55K SNP chip. Further sequencing the gene and excavating polymorphic sites, thereby developing and obtaining the high-efficiency KASP molecular marker.
One of the difficulties is: although there is currently a Durum Wheat genome V1.0 reference sequence, the gene sequence structure and polymorphic site of different Wheat genotypes may be greatly different due to the possible chromosome structure variation in the Wheat evolution process, and the simplest method is homologous sequence cloning in order to obtain the gene sequence of a specific Wheat genotype efficiently, quickly and at low cost. Since tetraploid wheat has two homologous chromosome sets, it is difficult to isolate a sequence specific to one of the homologous chromosomes, and it is necessary to design a primer specific to one of the chromosome sets. On the basis of skillfully mastering comparative genomics technology and bioinformatics technology, sequence interception, comparison and analysis are carried out on reference genomes such as tetraploid wild emmer wheat and Durum wheat to obtain a polymorphic site specific to a certain chromosome, so that a specific primer for amplifying a target region is designed. After the primers are designed, the primers are further analyzed by means of comparative genomics technology for specificity, annealing temperature, amplification length and the like, so that the usability of the primers is determined. In summary, although the KASP labeling technique has been widely applied to diploid species, it is extremely difficult for those skilled in the art to obtain a highly efficient KASP label in tetraploid wheat.
Finally, through multiple cloning and sequencing, primer design and amplification, a KASP primer 10 pair (table 1) is designed in total, and finally, a marker KASP-Flw-sau6198 (polymorphism A/G) is obtained and is closely linked with flag leaf width QTL QFLW.
TABLE 110 pairs of KASP primer sequences
e) And (6) analyzing. The designed 10 pairs of KASP primers finally obtain 1 molecular marker KASP-Flw-sau6198, which is closely linked with flag leaf width QTL QFLW.sau-AM-4B.4, and the results are shown in FIG. 1 and FIG. 2.
Example 2 application of molecular marker KASP-Flw-sau6198 to selection of control flag leaf Width QTL QFLW. sau-AM-4B.4
(1) A tetraploid wheat 'PI 503554' with narrower flag leaves is used as a male parent, a tetraploid local wheat 'Ailanmai' with wider flag leaves is used as a female parent to construct a recombinant inbred line, and 54 lines are randomly selected from the progeny lines.
(2) The obtained 54 strains are subjected to KASP-Flw-sau6198 marker detection, and the specific method comprises the following steps: extracting DNA of 54 strains; taking the DNA fragment as a template, taking a specific primer pair of a molecular marker KASP-Flw-sau6198 as a primer to carry out PCR amplification and carry out fluorescence reading, wherein the primer is as follows:
primer on FAM tag: (FAM tag sequence underlined) 5-GAAGGTGACCAAGTTCATGCTTCGACAGCCGGACTTCTCGA-3’(SEQ ID No.1)
Primers on HEX tag: (the wave line part is the HEX tag sequence) 5-
A universal downstream primer: 5'-TGAATATGCGTCCGTCCGG-3' (SEQ ID No.3)
The amplification system of PCR amplification is: mu.L Master Mix, 2.8. mu.L of mixed primers, 10ng of template DNA, and double distilled water to a total amount of 20. mu.L, while at least 3 independent blanks were added in which the DNA template was replaced by double distilled water. The mixed primers are prepared by adding 150. mu.L, 150. mu.L and 350. mu.L of three primers of SEQ ID No.1-3 at a concentration of 10 ng/. mu.L, respectively, and adding ddH2O450 mu L is mixed to prepare the product.
The reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 12 min; denaturation at 94 ℃ for 20s, renaturation/elongation at 59 ℃ for 55s, for 10 cycles; 30 cycles of denaturation at 94 ℃ for 20s and renaturation/elongation at 56 ℃ for 65 s; after completion, fluorescence readings were taken.
As a result of the fluorescence reading (as shown in FIG. 3), the genotype of the plant in which FAM (circular) fluorescence was detected in accordance with ` Ailanmai ` was designated as "A", the genotype of the plant in which broad flag leaf type fluorescence was detected as "A", the genotype of the plant in which HAX (square) fluorescence was detected as "PI 503554" was designated as "B", and the genotype of the plant in which narrow flag leaf type fluorescence was detected as "B". The genotype and flag leaf width field phenotype values of each line are shown in table 2.
TABLE 2 'Ailanmai' x 'PI 503554' recombinant inbred line KASP-Flw-sau6198 genotype and phenotype correspondence results
As can be seen from table 2, the mean flag leaf width of plants of the same type of 'Ailanmai' containing flag leaf width QTL qflw. sau-AM-4b.4 was 1.63, significantly higher than the flag leaf width of plants of the type of 'PI 503554' (mean 1.52). The actual result is consistent with the expected result, which shows that the flag leaf width QTL QFLW.sau-AM-4B.4 of the invention really has the effect of remarkably increasing the flag leaf width; meanwhile, the molecular marker KASP-Flw-sau6198 of the invention can be used for tracing and identifying the flag leaf width QTL QFLW. sau-AM-4 B.4.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
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<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gaaggtgacc aagttcatgc tttctatttt ccaaagccat g 41
<210> 23
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gaaggtcgga gtcaacggat tttctatttt ccaaagccat t 41
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tgaacactgc tcttttaa 18
<210> 25
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gaaggtgacc aagttcatgc tccatggacc agaagttcc 39
<210> 26
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gaaggtcgga gtcaacggat tccatggacc agaagttct 39
<210> 27
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tcggcagtat actgttgg 18
<210> 28
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaaggtgacc aagttcatgc ttaaaggaaa aaacaattaa 40
<210> 29
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaaggtcgga gtcaacggat ttaaaggaaa aaacaattag 40
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctcccgctaa aacttagc 18
Claims (8)
1. A KASP-Flw-sau6198 molecular marker linked with QTL QFLW.sau-AM-4B.4 is characterized in that the molecular marker is an SNP molecular marker with polymorphism A/G, is co-located on the tetraploid wheat 4B chromosome long arm with wheat flag leaf width QTL QFLW.sau-AM-4B.4 and is located in the QTL QFLW.sau-AM-4B.4 interval; the molecular marker is represented by SEQ ID NO: 1-3, and performing amplification.
2. A primer composition comprising a primer as set forth in SEQ ID NO: 1-2 and the sequence shown in SEQ ID NO: 3, said primer combination for amplifying the KASP-Flw-sau6198 molecular marker of claim 1.
3. A kit for identifying a wheat gene or a gene fragment thereof, comprising the primer composition of claim 2.
4. An application of wheat flag leaf width QTL QFLW. sau-AM-4B.4 or wheat KASP-Flw-sau6198 molecular marker in the regulation and control of wheat flag leaf width characters.
5. Use of the molecular marker KASP-Flw-sau6198 of claim 1, or the primer composition of claim 2, or the kit of claim 3, in genetic analysis and fine mapping of the wheat flag leaf width gene.
6. A method for screening a wheat line containing flag leaf width QTL QFLW.sau-AM-4B.4 is characterized by comprising the following steps: performing fluorescent quantitative PCR amplification by using genome DNA of a plant sample to be detected as a template and the primer composition of claim 2, parting according to the fluorescent detection result genotype of the obtained amplification product, recording the genotype of the plant in which the fluorescence consistent with the female parent of the wider flag leaf is detected as A, a wider flag leaf strain, the genotype of the plant in which the fluorescence consistent with the male parent of the narrower flag leaf is detected as B, and a narrower flag leaf strain, and further screening out the wheat strain containing the flag leaf width QTL QFLW.sau-AM-4 B.4.
7. The method of claim 6, wherein the fluorescent quantitative PCR reaction system comprises: 10 μ L Master Mix, 2.8 μ L mixed primer, 10ng template DNA, double distilled water to total amount of 20 μ L; wherein, the mixed primer is composed of SEQ ID No: 1-SEQ ID No: 3 to 150. mu.L, 150. mu.L and 350. mu.L of each of the three primers shown in FIG. 3 at a concentration of 10 ng/. mu.L, and ddH was added2O450 mu L of the mixture.
8. The method of claim 6, wherein the reaction sequence of the fluorescent quantitative PCR comprises: pre-denaturation at 94 ℃ for 12 min; denaturation at 94 ℃ for 20s, renaturation/elongation at 59 ℃ for 55s, for 10 cycles; denaturation at 94 ℃ for 20s, renaturation/elongation at 56 ℃ for 65s, for 30 cycles; after completion, fluorescence readings were taken.
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| CN110499387B (en) * | 2019-09-27 | 2021-12-03 | 四川农业大学 | Wheat flag leaf length QTL linked molecular marker and application thereof |
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