CN111647677A - Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application - Google Patents
Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application Download PDFInfo
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Abstract
The invention provides a molecular marker closely linked with a wheat grain filling rate QTL QGfr. sicau-6D, wherein the molecular marker is K603, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the molecular marker K603 is closely linked with the wheat grain filling rate QTL. Detection and analysis show that the molecular marker can accurately track the wheat grain filling rate QTL and predict the grain filling rate characteristic of wheat, thereby facilitating molecular design breeding. The detection molecular marker K603 can enhance the accuracy of the prediction of the grain filling rate of the wheat, so that the wheat variety or line with the QTL for increasing the grain filling rate can be quickly screened for breeding, and the breeding process of the high-yield wheat variety can be greatly accelerated.
Description
Technical Field
The invention relates to the field of molecular biology and genetic breeding, in particular to a molecular marker K603 closely linked with a wheat grain filling rate QTL QGfr.
Background
Wheat (Triticum aestivum L.) is one of the world's important food crops, with over 40% of the population using wheat as a staple food material. In China, wheat occupies an important position in both agricultural production and food processing fields, and is one of the most important grain crops. The filling rate and filling duration of wheat grains directly influence the final size, quality and grain weight of the grains, and finally determine the yield of wheat. The filling property not only affects the grain weight, but also is closely related to the final size, the fullness index and the commodity of grains. The filling rate refers to the dry matter accumulation amount of grains in unit time, and the grains show the change rule of 'slow-fast-slow' and 'S' -shaped curve growth, and reflects the biochemical reaction efficiency of starch and protein synthesis (Shewry PR. wheel [ J ]. Journal of Experimental botanic, 2009,60(6): 1537-1553.). The research on the genetic mechanism of grain filling rate in the grain filling process of wheat has important significance for improving the yield of wheat.
Grain filling rate is Quantitative Trait (QTL), is controlled by multiple genes, and is greatly affected by environmental effects. In recent years, grain filling rate is becoming a focus of research. There are reports on the study of grain filling rate QTL in rice, corn and barley, but the wheat grain filling rate is very little. Kirigwi et al mapped a QTL controlling grain filling rate on the Xgwm 601-Xwmc 420 segments of the 4A chromosome, which could account for 33% of phenotypic variation (Kirigwi F, Van M, Brown G, et al. markers associated with a QTL for grain in moist under the bottom of the drug [ J ]. Molecular Breeding,2007,20(4): 401) 413.). Bhusal et al found that QTL controlling grain filling rate was located on chromosome 2A using the recombinant inbred line populations constructed by HD2808 and HUW510 (Bhusal N, Sarial A, Sharma P, et al mapping QTLs for grain based compositions in cohet under stress [ J ]. PloS One,2017,12(12): e 0189594.).
Compared with the wheat variety Chuannong 16 (national scrutiny variety), the wheat material H461 has the characteristics of rapid grain filling, few tillers, multiple spike grain numbers, high thousand grain weights, high spike grain weights and the like (Houyun, Zhengqing, Pu to en, Weiyuming, Liwei, Chuannong 16 as a new spike-type wheat variety and H461 as a big spike-type few tillers strain have initial reports on genetic difference research, university of Sichuan agriculture, 2003,21: 94-9). Meanwhile, the filling rate of the wheat variety Chuannong 16 is obviously lower than that of H461. Therefore, a genetic research group is constructed by using H461 and Chuannong 16, the grain filling rate characteristic of wheat H461 is further verified, the grain filling rate gene is positioned and controlled, closely linked molecular markers are searched, the map-based cloning of the grain filling rate gene is promoted, a new gene resource is provided for the creation and high-yield breeding of a wheat specific grain filling rate material, the accuracy of grain filling rate prediction is enhanced, the breeding efficiency is improved, and the aim of increasing the yield per unit of wheat is accelerated.
The molecular marker assisted selection is not dependent on phenotype selection, namely is not influenced by various factors such as environmental conditions, gene interaction, genotype and environment interaction and the like, but directly selects the genotype, so that the breeding efficiency can be greatly improved. Competitive Allele-Specific PCR (Kompetitive Allele Specific PCR) allows for accurate biallelic detection of SNPs and indels at Specific sites in a wide range of genomic DNA samples. The detection method has the advantages of simplicity and convenience in operation, good specificity, high throughput, rapidness, low detection cost, accurate result and the like, and realizes real closed-tube operation, so that the detection method is generally concerned. Therefore, the molecular marker which is closely linked with the grain filling rate QTL and is suitable for the KASP technology of the fluorescence quantitative PCR platform is screened out, so that the wheat grain filling rate gene can be selected, the wheat grain morphogenesis can be effectively regulated and controlled, a reasonable grain morphogenesis group can be shaped, the selection flux, speed and accuracy are improved, the technical bottleneck of large-scale popularization and application is solved, and the molecular marker has important significance for improving the quality and yield of the wheat breeding group in a large scale.
Disclosure of Invention
The invention aims to provide a molecular marker closely linked with a wheat grain filling rate QTL QGfr.
The invention also aims to provide application of the molecular marker in wheat breeding.
In order to achieve the purpose, the molecular marker closely linked with the wheat grain filling rate QTL QGfr. sicau-6D provided by the invention is a molecular marker K603, and the nucleotide sequence SEQ ID No.1 shows (5 '-CAGTAAACTAAAATGTTGTTTTGCANATATATCTATAAACTAAAAACAAAAATTCTGATAGTAGCA-3'; wherein N is C or A). The polymorphism of the 26 th base of the sequence is C or A, and the polymorphism is related to the filling rate of wheat grains.
The molecular marker K603 is closely linked with the wheat grain filling rate QTL QGfr. sicau-6D, the molecular marker K603 and the wheat grain filling rate QTL QGfr. sicau-6D are positioned in a marker SNP 494-SNP 3370 section on a 6D chromosome together, the wheat grain filling rate QTL QGfr. sicau-6D can obviously increase the wheat grain filling rate, the LOD value is more than 4, and 8.42 percent of phenotypic variation is explained.
The invention also provides a specific primer pair for amplifying the molecular marker K603 by fluorescent quantitative PCR.
The primers for amplifying the molecular marker K603 can be designed by the skilled person based on the KASP detection platform technology, and preferably, the specific primer pair comprises primers with the sequences shown in SEQ ID NO. 2-4. Wherein, the 5' ends of the primers shown in SEQ ID NO.2 and SEQ ID NO.3 are respectively connected with different fluorescent probes.
K603-1:5’-GAAGGTGACCAAGTTCATGCTCAGTAAACTAAAATGTTGTTTTGCAC-3’；(SEQ IDNO.2)
K603-2:5’-GAAGGTCGGAGTCAACGGATTCAGTAAACTAAAATGTTGTTTTGCAA-3’；(SEQ IDNO.3)
K603-3:5’-TGCTACTATCAGAATTTTTGTTTTTAG-3’；(SEQ ID NO.4)
Moreover, the 5' ends of the primers K603-1 and K603-2 are respectively connected with different fluorescent probes;
the sequence of the fluorescent probe is as follows:
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.5), which in the examples of the present invention bind to FAM fluorophore.
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.6) in an embodiment of the invention the probe binds to a HEX fluorophore.
The invention provides any one of the following applications of molecular marker K603 or the specific primers:
(1) the application in identifying the wheat grain filling rate QTL QGfr. sicau-6D;
(2) the application in screening or identifying wheat varieties with quickly grouted grains;
(3) the application in wheat molecular marker assisted breeding;
(4) application in improving wheat germplasm resources.
The invention also provides a method for identifying the wheat grain filling rate QTL QGfr. sicau-6D, which takes the genome DNA of the wheat to be detected as a template, adopts the specific primer group to carry out fluorescence quantitative PCR amplification, and carries out genotyping on the wheat to be detected according to the PCR amplification result.
Preferably, the reaction system for the fluorescent quantitative PCR amplification comprises: 2 XKASP Mastermix 5. mu.L, KASP Assaymix 0.14. mu.L, template DNA50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the nucleotide sequence of the primer contained in the KASP AssayMix is shown in SEQ ID NO.2-4, and the volume ratio of the three primers is 2:2: 5.
In the examples of the present invention, the fluorescent quantitative PCR procedure: activating at 95 deg.C for 15 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 30 times; fluorescence signals were collected at 37 ℃ for 60 s.
The judgment standard of the method for identifying the wheat grain filling rate QTL QGfr. sicau-6D provided by the invention is as follows: wheat varieties containing the rapid wheat grain filling QTL QGfr. sicau-6D all have fluorescence signals which are the same as the fluorescence signals connected with the primer shown in SEQ ID NO.2, and wheat varieties containing no wheat grain filling rate QTL QGfr. sicau-6D all have fluorescence signals which are obviously different from the fluorescence signals connected with the primer shown in SEQ ID NO. 2.
In the invention, the wheat grain filling rate QTL QGfr. sicau-6D and the molecular marker K603 are obtained by the following method:
(1) the method comprises the steps of utilizing grain fast-grouted wheat H461 as a female parent and wheat Chuannon 16 as a male parent to perform hybridization to obtain a hybrid F1, performing F1 generation individual plant selfing to obtain F2, obtaining an F8 generation RIL group containing 249 lines by adopting a single seed transmission method, and randomly selecting 188 lines to form a genetic mapping group.
(2) Extracting DNA of each strain of the genetic mapping population by using a CTAB method, and carrying out genotyping by using DNA of a parent H461 and Chuannong 16 as templates by using an Illumina90K SNP chip technology to obtain genotype data of the RIL population. The band pattern of the parent H461 is marked as A, and the band pattern of the parent Chuannon 16 is marked as B. The banding pattern of the strain of the F8 colony is from H461 and recorded as A, and from Chuannong 16 and recorded as B.
Extracting DNA of each individual plant of the parent F8 population by using a CTAB method, downloading and obtaining the sequence information of scaffold in a target interval from a database http:// plants. ensemble. org/Triticum _ aestivum/Info/Index, and carrying out KASP molecular marking on differential sites by using DNAMAN6.0 software according to 90K SNP information in the interval to develop and design fluorescent quantitative primers. The band pattern of the obtained parent H461 is marked as A, and the band pattern of the parent Chuannon 16 is marked as B.
(3) And (3) identifying the grain filling rate of the F8 population plants in the field in the wheat filling stage.
(4) And constructing a wheat molecular linkage map by using the obtained genetic type data of the RIL population by using JoinMap4.0 mapping software, searching the optimal marker number and the optimal marker sequence, and determining a linkage population for subsequent use. The grain filling rate QTLQGfr. sicau-6D is positioned in a section of SNP 494-SNP 3370 on a 6D chromosome by using a complete Interval Mapping method (Inclusive Composite Interval Mapping) of software IcMapping 4.1 and combining with grain filling rate phenotype data of an F8 population.
(5) And converting SNP sites in the target section into fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 7 pair (table 1) was designed using DNAMAN6.0 software. Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-30 bp, the length of the amplification product is 45-70bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%. The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
TABLE 17 pairs of KASP primer sequences and amplified fragment lengths
(6) Competitive allele specific PCR (KASP) assay
a) Screening of polymorphic molecular markers between parents: the 7 pairs of primers designed above are selected, and the DNA of parent H461 and Chuannong 16 are used as templates for PCR amplification, so as to obtain 1 pair of molecular marker primers with good effect, which are named as K603-1/2/3 (the nucleotide sequences are respectively shown in SEQ ID NO. 2-4). The amplification product is a molecular marker K603 with polymorphism, and the nucleotide sequence is shown in SEQ ID NO. 1.
b) KASP analysis of the F8 population: and amplifying DNA of parent H461, Chuannong 16 and F8 population plants by using the PCR primer of the molecular marker K603 with polymorphism obtained in the step, and carrying out genotype identification to obtain molecular marker data. The type of the parent H461 is marked as A, the length of the amplified fragment is 66bp, and the single base difference site is C. The type of the parent Chuannon 16 is marked as B, the length of the amplified fragment is 66bp, and the single base difference site is A. The strain type of the F8 population is from H461 and recorded as A, and from Chuannong 16 and recorded as B.
The invention has the following advantages: the invention discloses a grain filling rate QTLQGfr. sicau-6D from wheat H461 for the first time, which is positioned on a wheat 6D chromosome and remarkably increases the grain filling rate of wheat. The QTL has higher utilization value in wheat yield (seed filling rate regulation) breeding. The invention discloses a molecular marker K603 for accurately detecting the new grain filling rate QGfr. sicau-6D of wheat H461 based on a fluorescent quantitative PCR platform for the first time, and the marker is a codominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification. The molecular marker K603 disclosed by the invention is obviously related to the grain filling rate QTL QGfr. sicau-6D, presents coseparation marker characteristics, has high accuracy for molecular marker-assisted selection, improves the selection and identification efficiency of wheat specific grain filling rate varieties suitable for different environments, and has high success rate.
Drawings
FIG. 1 is a linkage genetic map of the wheat H461 grain filling rate QTL QGfr. sicau-6D on the 6D chromosome and the molecular marker K603.
FIG. 2 shows the result of genotyping DNA of leaves in the trefoil stage of H461, Chuannon 16, Chuanmai 107 using fluorescent quantitative PCR primers in example 2 of the present invention.
FIG. 3 shows the results of genotyping the F8RIL population progeny of H461 × Chuanmai 107 using fluorescent quantitative PCR primers in example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 obtaining of wheat grain filling rate QTL QGfr. sicau-6D and molecular marker K603
In the invention, the wheat grain filling rate QTL QGfr. sicau-6D and the molecular marker K603 are obtained by the following method:
(1) the method comprises the steps of utilizing grain fast-grouted wheat H461 as a female parent and wheat Chuannon 16 as a male parent to perform hybridization to obtain a hybrid F1, performing F1 generation individual plant selfing to obtain F2, obtaining an F8 generation RIL group containing 249 lines by adopting a single seed transmission method, and randomly selecting 188 lines to form a genetic mapping group.
(2) Extracting DNA of each strain of the genetic mapping population by using a CTAB method, and carrying out genotyping by using DNA of a parent H461 and Chuannong 16 as templates by using an Illumina90K SNP chip technology to obtain genotype data of the RIL population. The band pattern of the parent H461 is marked as A, and the band pattern of the parent Chuannon 16 is marked as B. The banding pattern of the strain of the F8 colony is from H461 and recorded as A, and from Chuannong 16 and recorded as B.
Extracting DNA of each individual plant of the parent F8 population by using a CTAB method, downloading and obtaining the sequence information of scaffold in a target interval from a database http:// plants. ensemble. org/Triticum _ aestivum/Info/Index, and carrying out KASP molecular marking on differential sites by using DNAMAN6.0 software according to 90K SNP information in the interval to develop and design fluorescent quantitative primers. The band pattern of the obtained parent H461 is marked as A, and the band pattern of the parent Chuannon 16 is marked as B.
(3) And (3) identifying the grain filling rate of the F8 population plants in the field in the wheat filling stage.
(4) And constructing a wheat molecular linkage map by using the obtained genetic type data of the RIL population by using JoinMap4.0 mapping software, searching the optimal marker number and the optimal marker sequence, and determining a linkage population for subsequent use. The kernel filling rate qtlqgfr. sicau-6D was located within a 16.24cM (flanking markers SNP494 and SNP3370) segment on the 6D chromosome using the complete Interval Mapping method of software IciMapping 4.1 (Inclusive Composite Interval Mapping) in combination with the F8 population kernel filling rate phenotypic data.
(5) SNP494 and SNP3370 are converted into fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 7 pair (table 1) was designed using DNAMAN6.0 software. Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-30 bp, the length of the amplification product is 45-70bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%. The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
(6) Competitive allele specific PCR (KASP) assay
a) Screening of polymorphic molecular markers between parents: the 7 pairs of primers designed above are selected, and the DNA of parent H461 and Chuannong 16 are used as templates for PCR amplification, so as to obtain 1 pair of molecular marker primers with good effect, which are named as K603-1/2/3 (the nucleotide sequences are respectively shown in SEQ ID NO. 2-4). The amplification product is a molecular marker K603 with polymorphism, the nucleotide sequence is shown as SEQ ID NO.1, and n in the sequence is c or a.
b) KASP analysis of the F8 population: and amplifying DNA of parent H461, Chuannong 16 and F8 population plants by using the PCR primer of the molecular marker K603 with polymorphism obtained in the step, and carrying out genotype identification to obtain molecular marker data. The type of the parent H461 is marked as A, the length of the amplified fragment is 66bp, and the single base difference site is C. The type of the parent Chuannon 16 is marked as B, the length of the amplified fragment is 66bp, and the single base difference site is A. The strain type of the F8 population is from H461 and recorded as A, and from Chuannong 16 and recorded as B.
c) By using a complete interval mapping method (contained Composite IntervalMapping) of software IcMapping 4.1 and combining grain filling rate phenotype data of an F8 population, a molecular marker K603 is found to be closely linked with a grain filling rate QTLQGfr. As can be seen from fig. 1, the grain filling rate QTL qgfr. sicau-6D is located in the 16.24cM segment between markers SNP494 and SNP3370, while molecular marker K603 cosegregates with SNP3370 and is in close linkage with QTL.
The linkage genetic map of the position of the wheat grain rapid filling QTL QGfr. sicau-6D on the 6D chromosome and the molecular marker K603 is shown in figure 1.
Example 2 application of molecular marker K603 closely linked with wheat grain filling QTL QGfr. sicau-6D
1. DNA extraction
The test material is selected from H461, Chuannong 16 and Chuanmai 107, wherein the Chuannong 16 and the Chuanmai 107 are varieties with slow grain filling rate, and H461 is a variety with fast grain filling rate. And (3) extracting the leaf DNA of the wheat sample in the trefoil stage by adopting a CTAB method.
2. Screening of primers for detecting wheat grain filling rate QTL QGfr
2.1 primer design
And (3) converting the SNP markers SNP494 and SNP3370 to design fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 7 pairs were designed using DNAMAN6.0 software (see table 1). Design standard of fluorescent quantitative PCR primer: the length of the amplification primer is 18-30 bp, the length of the amplification product is 45-70bp, the annealing temperature is 57-62 ℃, and the GC content is 40-60%. The sequence of the synthetic primer is as follows:
forward primer 1: f probe + amplification primer sequence
Forward primer 2: h probe + amplification primer sequence
Reverse primer: amplification primer sequences
F, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
2.2 fluorescent quantitative PCR platform test primers and differences between their parents
(1) Extracting the DNA of the leaves of H461, Chuannong 16 and Chuanmai 107 in the trefoil stage.
(2) Using genome DNA of wheat to be detected as a template, designing a primer based on a KASP detection platform technology, and carrying out fluorescent quantitative PCR amplification;
wherein, the primer sequence of the step 2 is as follows:
K603-1:5’-GAAGGTGACCAAGTTCATGCTCAGTAAACTAAAATGTTGTTTTGCAC-3’；
K603-2:5’-GAAGGTCGGAGTCAACGGATTCAGTAAACTAAAATGTTGTTTTGCAA-3’；
K603-3:5’-TGCTACTATCAGAATTTTTGTTTTTAG-3’；
moreover, the 5' ends of the primers K603-1 and K603-2 are respectively connected with different fluorescent probes;
the sequence of the fluorescent probe is as follows:
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)
H, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (bindable HEX fluorophore)
(3) A fluorescent quantitative PCR amplification reaction system: 2 XKASP Mastermix 5. mu.L, KASPAssay Mix 0.14. mu.L, template DNA50ng, Dnase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers K603-1, K603-2 and K603-3 in a volume ratio of 2:2: 5. That is, in KASP Assay Mix, primers K603-1, K603-2 and K603-3 were mixed at a concentration of 100. mu.M in a volume ratio of 2:2: 5.
(4) Fluorescent quantitative PCR procedure: activating at 95 deg.C for 15 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 30 times; fluorescence signals were collected at 37 ℃ for 60 s.
(5) The specific method for analyzing PCR products is as follows: wheat samples containing the quick wheat grain filling QTLQGfr. sicau-6D have the same genotype as that of wheat H461 and are marked as type A, and wheat varieties without the quick wheat grain filling QTLQGfr. sicau-6D have fluorescence signals obviously different from that of wheat H461, such as Chuannong 16 and Chuannong 107 and are marked as type B. The results of genotyping H461, Chuannong 16, Chuanmai 107 using KASP primers are shown in FIG. 2.
3. Applicability of primer sequence K603-1/2/3 in population detection process
(1) H461 is used as a female parent, Chuanmai 107 is used as a male parent to obtain F1, F1 is selfed to obtain F2, and generation is added to the RIL of the F8 generation through a single-seed-transmission method to verify the population. And extracting the leaf DNA of each strain in the three-leaf stage in the population.
(2) And (2) performing fluorescent quantitative PCR amplification by using the DNA obtained in the step (1) as a template and using the primer provided by the invention, wherein the fluorescent dye is SsoFast EvaGreen.
(3) A fluorescent quantitative PCR amplification reaction system: 2 XKASP Mastermix 5. mu.L, KASPAssay Mix 0.14. mu.L, template DNA50ng, Dnase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers K603-1, K603-2 and K603-3 in a volume ratio of 2:2: 5. That is, in KASP Assay Mix, primers K603-1, K603-2 and K603-3 were mixed at a concentration of 100. mu.M at a volume ratio of 2:2:5, and then 0.14. mu.L of the mixture was aspirated.
(4) Fluorescent quantitative PCR procedure: activating at 95 deg.C for 15 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 30 times; fluorescence signals were collected at 37 ℃ for 60 s.
(5) The specific method for analyzing PCR products is as follows: wheat samples containing the wheat grain filling rate QTLQGfr. sicau-6D all have genotypes which are the same as those of wheat H461 and are marked as type A, while wheat varieties without the wheat grain filling rate QTLQGfr. sicau-6D all have fluorescence signals which are obviously different from those of wheat H461 and are marked as type B, and the results are shown in figure 3. And randomly sampling 96 strains, 47 strains can amplify segments with the same type as H461, and the segments are the plants of the wheat grain filling QTLQGfr. 49 plants can amplify B-type segments which are the same as 16 and 107 of Chuannong, are plants which do not contain QTL QGfr. sicau-6D of wheat grain filling rate, and are predicted to have slower grain filling rate in the plant of the prediction line in the filling period.
(6) The grain filling rate of the 96F 8 plants is identified in the field during the wheat filling period, the result is shown in table 2, and the result of predicting the grain filling rate of the genetic population by the molecular marker K603 of the grain filling rate QTL QGfr. The average grain filling rate of the plants with the same type as H461 is 100.5 mg-100 grains-1Sky-1The average grain filling rate of the seeds is obviously higher than that of the plants of Chuannong 16 and Chuanmai 107 types and is 86.5 mg.100 grains-1Sky-1. The actual result is consistent with the expected result, which shows that the grain filling rate QTL QGfr. sicau-6D of the invention has the effect of remarkably increasing the grain filling rate, and the molecular marker K603 can be used for tracking and identifying the grain filling rate QTL QGfr. sicau-6D.
TABLE 2
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Sichuan university of agriculture
<120> molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application
<130>KHP201111453.8
<160>24
<170>SIPOSequenceListing 1.0
<210>1
<211>66
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>misc_feature
<222>(26)..(26)
<223>n is c or a
<400>1
cagtaaacta aaatgttgtt ttgcanatat atctataaac taaaaacaaa aattctgata 60
gtagca 66
<210>2
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gaaggtgacc aagttcatgc tcagtaaact aaaatgttgt tttgcac 47
<210>3
<211>47
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gaaggtcgga gtcaacggat tcagtaaact aaaatgttgt tttgcaa 47
<210>4
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tgctactatc agaatttttg tttttag 27
<210>5
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
gaaggtgacc aagttcatgc t 21
<210>6
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gaaggtcgga gtcaacggat t 21
<210>7
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gaaggtgacc aagttcatgc tgttgggtag tcctatttga 40
<210>8
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gaaggtcgga gtcaacggat tgttgggtag tcctatttgg 40
<210>9
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
tgctttcaac atactatag 19
<210>10
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gaaggtgacc aagttcatgc tgctttcaac atactataga tacaatcaat 50
<210>11
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
gaaggtcgga gtcaacggat tgctttcaac atactataga tacaatcaac 50
<210>12
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
caatctgttg ggtagtccta tttg 24
<210>13
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gaaggtgacc aagttcatgc ttcaacatac tatagataca atcaat 46
<210>14
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gaaggtcgga gtcaacggat ttcaacatac tatagataca atcaac 46
<210>15
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
caatctgttg ggtagtccta t 21
<210>16
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
gaaggtgacc aagttcatgc tcaacatact atagatacaa tcaat 45
<210>17
<211>45
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gaaggtcgga gtcaacggat tcaacatact atagatacaa tcaac 45
<210>18
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caatctgttg ggtagtccta tttg 24
<210>19
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
gaaggtgacc aagttcatgc tacatactat agatacaatc aac 43
<210>20
<211>43
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
gaaggtcgga gtcaacggat tacatactat agatacaatc aat 43
<210>21
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
tccaatctgt tgggtagtcc t 21
<210>22
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
gaaggtgacc aagttcatgc ttactataga tacaatcaat 40
<210>23
<211>40
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
gaaggtcgga gtcaacggat ttactataga tacaatcaac 40
<210>24
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
ctgttgggta gtcctatttg20
Claims (10)
1. The molecular marker closely linked with the wheat grain filling rate QTL QGfr. sicau-6D is named as K603, and is characterized in that the molecular marker K603 and the wheat grain filling rate QTL QGfr. sicau-6D are co-located in a marker SNP 494-SNP 3370 section on a wheat 6D chromosome, and the molecular marker K603 is located in a confidence interval of the wheat grain filling rate QTL QGfr. sicau-6D.
2. The molecular marker according to claim 1, wherein the nucleotide sequence thereof is represented by SEQ ID No.1, and the 26 th base polymorphism in the sequence is C or A.
3. The molecular marker of claim 1 or 2, wherein the wheat grain filling rate QTLQGfr. sicau-6D can significantly increase the wheat grain filling rate, the LOD value is greater than 4, and 8.42% of phenotypic variation is explained.
4. A specific primer set for amplifying the molecular marker of any one of claims 1 to 3.
5. The specific primer group of claim 4, wherein the specific primer group comprises primers with sequences shown as SEQ ID NO. 2-4.
6. The specific primer set of claim 5, wherein the primers shown in SEQ ID NO.2 and SEQ ID NO.3 have different fluorescent probes attached to their 5' ends, respectively.
7. The molecular marker of any one of claims 1 to 3 or the specific primer set of any one of claims 4 to 6, wherein any one of the following applications:
(1) the application in identifying the wheat grain filling rate QTL QGfr. sicau-6D;
(2) the application in screening or identifying wheat varieties with quickly grouted grains;
(3) the application in wheat molecular marker assisted breeding;
(4) application in improving wheat germplasm resources.
8. A method for identifying a wheat grain filling rate QTL QGfr. sicau-6D is characterized in that genome DNA of wheat to be detected is taken as a template, a specific primer group in any one of claims 4-6 is adopted for carrying out fluorescence quantitative PCR amplification, and the wheat to be detected is subjected to genotyping according to the PCR amplification result.
9. The method of claim 8, wherein the reaction system for the fluorescent quantitative PCR amplification comprises: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the nucleotide sequence of the primer contained in the KASP Assay Mix is shown in SEQ ID NO.2-4, and the volume ratio of the three primers is 2:2: 5; and/or
Fluorescent quantitative PCR procedure: activating at 95 deg.C for 15 min; denaturation at 95 ℃ for 20s, annealing and extension at 65 ℃ for 60s, and circulating for 10 times, wherein the annealing and extension temperature is reduced by 1 ℃ every time; denaturation at 94 ℃ for 20s, annealing and extension at 57 ℃ for 60s, and circulating for 30 times; fluorescence signals were collected at 37 ℃ for 60 s.
10. The method according to claim 8 or 9, wherein the wheat varieties containing the rapid wheat grain filling QTL QGfr. sicau-6D all have the same fluorescent signals as the fluorescent probes connected with the primers shown in SEQ ID No.2, and the wheat varieties not containing the rapid wheat grain filling QTL QGfr. sicau-6D all have the fluorescent signals which are obviously different from the fluorescent probes connected with the primers shown in SEQ ID No. 2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010340304.8A CN111647677B (en) | 2020-04-26 | 2020-04-26 | Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010340304.8A CN111647677B (en) | 2020-04-26 | 2020-04-26 | Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application |
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| Publication Number | Publication Date |
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| CN111647677A true CN111647677A (en) | 2020-09-11 |
| CN111647677B CN111647677B (en) | 2022-07-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202010340304.8A Active CN111647677B (en) | 2020-04-26 | 2020-04-26 | Molecular marker closely linked with wheat grain filling rate QTL QGfr. sicau-6D and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708691A (en) * | 2021-01-29 | 2021-04-27 | 江苏里下河地区农业科学研究所 | KASP primer group for identifying filling rate of Yangmai 16 and derivative varieties thereof and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755465A (en) * | 2017-01-12 | 2017-05-31 | 四川农业大学 | The molecular labeling of QTL QFll.sicau 2D close linkage long with wheat flag leaf and application |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN110295251A (en) * | 2019-07-18 | 2019-10-01 | 四川农业大学 | Chain SNP marker and its application with wheat available tillering QTL |
-
2020
- 2020-04-26 CN CN202010340304.8A patent/CN111647677B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755465A (en) * | 2017-01-12 | 2017-05-31 | 四川农业大学 | The molecular labeling of QTL QFll.sicau 2D close linkage long with wheat flag leaf and application |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN110295251A (en) * | 2019-07-18 | 2019-10-01 | 四川农业大学 | Chain SNP marker and its application with wheat available tillering QTL |
Non-Patent Citations (1)
| Title |
|---|
| R. X. WANG ET AL.: "QTL mapping for grain filling rate and yield-related traits in RILs of the Chinese winter wheat population Heshangmai 3 Yu8679", 《THEORETICAL AND APPLIED GENETICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708691A (en) * | 2021-01-29 | 2021-04-27 | 江苏里下河地区农业科学研究所 | KASP primer group for identifying filling rate of Yangmai 16 and derivative varieties thereof and application |
| CN112708691B (en) * | 2021-01-29 | 2022-05-20 | 江苏里下河地区农业科学研究所 | KASP primer group for identifying filling rate of Yangmai 16 and derivative varieties thereof and application |
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|---|---|
| CN111647677B (en) | 2022-07-08 |
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