CN112779350A - Molecular marker closely linked with wheat spikelet grain number QTLQGns - Google Patents
Molecular marker closely linked with wheat spikelet grain number QTLQGns Download PDFInfo
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Abstract
The invention relates to the technical field of molecular marker assisted breeding, in particular to a molecular marker closely linked with wheat spikelet grain number QTL QGns. The molecular marker of the invention is KASP3, the nucleotide sequence of which is shown in SEQ ID NO.1, and the molecular marker is closely linked with wheat spikelet number QTL. The detection analysis shows that the molecular marker can accurately track the wheat spikelet grain number QTL, predict the wheat spikelet grain number characteristic and further facilitate molecular design breeding. The molecular marker KASP3 can improve the accuracy of wheat spikelet grain number prediction, so that a wheat variety or strain with the QTL for increasing the spikelet grain number can be quickly screened for breeding, and the breeding process of a wheat high-yield variety can be greatly accelerated.
Description
Technical Field
The invention relates to the technical field of molecular marker assisted breeding, in particular to a molecular marker KASP3 closely linked with wheat spikelet grain number QTL QGns.
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 plays an extremely important role in the fields of agricultural production and food processing, and is one of the most important food crops. The annual growth rate of global food production is currently 0.9%, which is much less than 2.4% of that required by the continuously growing world population for life in 2050. Therefore, the improvement of wheat yield is imminent. Due to the continuous reduction of the cultivated land area and climate change, the cultivation of high-yield wheat varieties becomes one of the main strategies for increasing the total yield of grains.
The number of spikelets is a key factor in determining crop yield. In recent years, the number of spikelets has become a focus of research. There are reports related to the study on the number of spikelets QTL in rice and barley, but the study on the number of spikelets of wheat is very little. Chen et al use Pubin 3504 and sting 4839 constructed F2:3The genetic population of (1) found that QTLs controlling spikelet counts are located on chromosomes 1A, 4A, 6D and 7A (Chen D, Wu X, Wu K, et al. novel and flexible genetic regions for spike related peptides in a what at plastic draft 3504with high grain number per spike unit variant environment. Journal of integrated agricultural 2017,16(11): 2386-. Sakuma et al mapped to a QTL controlling spikelet number on the 2A chromosome, accounting for 61% of phenotypic variation (Sak)uma S,Golan G,Guo Z,et al.Unleashing floret fertility in wheat through the mutation of a homeobox gene[J].Proceedings of the National Academy of Sciences,2019,116(11):5182–5187)。
Compared with the wheat material Chinese spring, the wheat material H461 has the characteristics of multiple small ear grain numbers, multiple small ear numbers, high thousand grain weight, high ear grain weight and the like. Meanwhile, the number of small ear grains of the wheat material in China spring is obviously lower than that of H461. Therefore, a genetic research group is constructed by utilizing H461 and Chinese spring, the spikelet grain number characteristic of wheat H461 is further verified, the spikelet grain number gene is positioned and controlled, a closely linked molecular marker is searched, the map-based cloning of the spikelet grain number gene is promoted, a new gene resource is provided for the creation and high-yield breeding of wheat specific spikelet grain number materials, the molecular marker is further utilized for auxiliary selection, the accuracy of spikelet grain number prediction is enhanced, the breeding efficiency is improved, and the aim of increasing the single yield of wheat is fulfilled at an accelerated speed.
The molecular marker assisted selection does not depend 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 small ear grain number QTL and is suitable for the KASP technology of the fluorescent quantitative PCR platform is screened out, the gene of the small ear grain number of the wheat can be selected, the ear grain number of the wheat can be effectively regulated and controlled, a reasonable population with a high ear grain number is shaped, the selection flux, the speed and the 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 the yield of the wheat breeding population on a large scale.
Disclosure of Invention
The invention aims to provide a molecular marker closely linked with wheat spikelet grain number QTL QGns.sicau-2D; another object of the present invention is to provide the use of the molecular marker.
Specifically, the invention provides the following technical scheme:
the invention provides a molecular marker closely linked with wheat spikelet grain number QTL QGns. sicau-2D, which is a molecular marker KASP3 and is shown in a nucleotide sequence SEQ ID NO.1 (5 '-G CAAAGGAATCATTCAACGNTCACTCAAATAGGTGGCGTTCAGAT TT-3'; wherein N is A or G.). The polymorphism of the 20 th base of the sequence is A or G, the polymorphism is related to the number of wheat spikelets, and the 20 th base is G corresponding to the number of wheat spikelets.
The molecular marker KASP3 is closely linked with wheat spikelet number QTL QGns.sicau-2D, the molecular marker and the wheat spikelet number QTL QGns.sicau-2D are positioned in a marker AX-109316972-AX-110906716 section on a 2D chromosome, and the molecular marker is positioned in a confidence interval of the wheat spikelet number QTL QGns.sicau-2D. The wheat spikelet grain number QTL QGns. sicau-2D can obviously increase the wheat spikelet grain number, the LOD value is more than 3, and 26.57 percent of phenotypic variation is explained.
The invention also provides a specific primer group for amplifying the molecular marker.
Specifically, the invention provides a specific primer group for fluorescent quantitative PCR amplification of the molecular marker KASP 3.
One skilled in the art can design primers for amplifying the molecular marker KASP3 based on the KASP detection platform technology. Preferably, the specific primer group comprises primers with sequences shown as 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.
KASP3-1:5’-GAAGGTGACCAAGTTCATGCTGCAAAGGAATC ATTCAACGA-3’；(SEQ ID NO.2)；
KASP3-2:5’-GAAGGTCGGAGTCAACGGATTGCAAAGGAATC ATTCAACGG-3’；(SEQ ID NO.3)；
KASP3-3:5’-AAATCTGAACGCCACCTATTTG-3’；(SEQ ID NO.4)。
Furthermore, the 5' ends of the primers KASP3-1 and KASP3-2 are respectively connected with different fluorescent probes.
Specifically, 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 also provides the application of the molecular marker KASP3 or any one of the following specific primer groups:
(1) application in identifying wheat spikelet grain number QTL QGns.sicau-2D;
(2) the application in screening or identifying wheat varieties with multiple small ear grain numbers;
(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 spikelet grain number QTL QGns.sicau-2D, 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 10 μ L reaction system for the fluorescent quantitative PCR amplification comprises the following components: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains a primer with a nucleotide sequence shown as SEQ ID NO.2-4, and the molar ratio of the three primers is 2:2: 5.
Preferably, the reaction procedure of the fluorescent quantitative PCR amplification comprises: 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 spikelet grain number QTL QGns. sicau-2D provided by the invention is as follows: the wheat varieties containing the wheat multi-spike grain number QTL QGns.sicau-2D all have the same fluorescent signals as the fluorescent probes connected with the primers shown in SEQ ID NO.3, and the wheat varieties not containing the wheat multi-spike grain number QTL QGns.sicau-2D all have the fluorescent signals obviously different from the fluorescent probes connected with the primers shown in SEQ ID NO. 3.
In the invention, the wheat spikelet grain number QTL QGns. sicau-2D and the molecular marker KASP3 are obtained by the following method:
(1) hybridizing by using wheat H461 with multiple small grain number as female parent and wheat Chinese spring as male parent to obtain hybrid F1，F1Selfing the single plant to obtain F2F obtained by single particle propagation8And generating an RIL population, and randomly selecting 300 strains to form a genetic mapping population.
(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 DNA of Chinese spring as templates by using an Illumina 55K 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 Chinese spring is marked as B. F8The banding pattern of the colony strain is from H461 and recorded as A, and from Chinese spring and recorded as B.
Extracting said parent, F, by CTAB8DNA of each individual strain of the population is downloaded from a database http:// plants. ensemble. org/Triticum _ aestivum/Info/Index to obtain the sequence information of the scaffold in a target interval, and KASP molecular markers are carried out on differential sites by DNMAN 6.0 software according to the 55K SNP information in the interval to develop fluorescent quantitative primers. The band pattern of the obtained parent H461 is marked as A, and the band pattern of the parent Chinese spring is marked as B.
(3) Wheat field identification of said F8The number of spikelets of the plant population.
(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. Complete Interval Mapping (Inclusive Composite Interval Mapping) using software IciMapping 4.2, in combination with F8Population spikelet number phenotype data maps spikelet number QTL QGns. sicau-2D to a segment AX-109316972-AX-110906716 on the 2D chromosome.
(5) And converting SNP sites in the target section into fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 4 pairs (table 1) were designed using DNAMAN 6.0 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: amplifying the primer sequence;
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (to which FAM fluorophores can be bound);
h, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached);
TABLE 14 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 4 pairs of primers designed above are selected, and the DNA of parent H461 and Chinese spring are used as templates for PCR amplification, so as to obtain 1 pair of molecular marker primers with good effect, which are named as KASP3-1/2/3 (the nucleotide sequences are respectively shown as SEQ ID NO. 2-4). The amplified product is a molecular marker KASP3 with polymorphism, and the nucleotide sequence is shown in SEQ ID NO. 1.
b)F8KASP analysis of the population: using the PCR primer of the molecular marker KASP3 with polymorphism obtained in the above steps to amplify parent H461, Chinese spring and F8And (3) carrying out genotype identification on DNA of the population plants to obtain molecular marker data. The parent H461 is marked as A, the length of the amplified fragment is 47bp, and the single base difference site is G. The parent Chinese spring is marked as B, the length of the amplified fragment is 47bp, and the single base difference site is A. F8The population strain type is from H461 as A and from Chinese spring as B.
The invention has the beneficial effects that: the invention discloses a wheat H461 spikelet number QTL QGns. sicau-2D, which is located on a wheat 2D chromosome and obviously increases the spikelet number of wheat. The QTL has higher utilization value in wheat yield (regulating and controlling the number of spikelets) breeding. The invention discloses a molecular marker KASP3 for accurately detecting new spike grain number QGns.sicau-2D of wheat H461 based on a fluorescent quantitative PCR platform for the first time, and the molecular marker KASP3 is a codominant marker, and has the advantages of accurate and efficient detection and convenient and stable amplification. The molecular marker KASP3 disclosed by the invention is obviously related to the panicle grain number QTL QGns.
Drawings
FIG. 1 is a linkage genetic map between the position of wheat H461 panicle number QTL QGns. sicau-2D on 2D chromosome and molecular marker KASP3 in example 1 of the present invention.
FIG. 2 shows the result of genotyping DNA of leaves in the trefoil stage of H461, Chinese spring, Chuanmai 107 using fluorescent quantitative PCR primers in example 2 of the present invention.
FIG. 3 shows H461X Chuanmai 107F in example 2 of the present invention8And carrying out genotyping on the progeny of the RIL group by using fluorescent quantitative PCR primers.
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 spikelet number QTL QGns. sicau-2D and molecular marker KASP3
In the invention, the wheat spikelet number QTL QGns. sicau-2D and the molecular marker KASP3 are obtained by the following method:
(1) the hybrid F1, F is obtained by hybridizing the wheat H461 with multiple small grain number as female parent and the wheat Chinese spring as male parent1Selfing the single plant to obtain F2F obtained by single particle propagation8And generating an RIL population, and randomly selecting 300 strains to form a genetic mapping population.
(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 DNA of Chinese spring as templates by using a 55K 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 Chinese spring is marked as B. F8The banding pattern of the colony strain is from H461 and recorded as A, and from Chinese spring and recorded as B.
Extracting said parent, F, by CTAB8DNA of each individual strain of the population is downloaded from a database http:// plants. ensemble. org/Triticum _ aestivum/Info/Index to obtain the sequence information of the scaffold in a target interval, and KASP molecular markers are carried out on differential sites by DNMAN 6.0 software according to the 55K SNP information in the interval to develop fluorescent quantitative primers. The band pattern of the obtained parent H461 is marked as A, and the band pattern of the parent Chinese spring is marked as B.
(3) Wheat field identification of said F8The number of spikelets of the plant population.
(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. Complete Interval Mapping (Inclusive Composite Interval Mapping) using software IciMapping 4.2, in combination with F8Population spikelet number phenotype data mapped spikelet number QTL QGns. sicau-2D to a 6.68cM (flanking markers AX-109316972 and AX-110906716) segment on the 2D chromosome.
(5) SNP markers AX-109316972 and AX-110906716 are converted into fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 4 pairs (table 1) were designed using DNAMAN 6.0 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: amplifying the primer sequence;
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (to which FAM fluorophores can be bound);
h, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached).
(6) Competitive allele specific PCR (KASP) assay
a) Screening of polymorphic molecular markers between parents: the 4 pairs of primers designed above are selected, and the DNA of parent H461 and Chinese spring are used as templates for PCR amplification, so as to obtain 1 pair of molecular marker primers with good effect, which are named as KASP3-1/2/3 (the nucleotide sequences are respectively shown as SEQ ID NO. 2-4). The amplification product is a molecular marker KASP3 with polymorphism, the nucleotide sequence is shown in SEQ ID NO.1, and n in the sequence is a or g.
b)F8KASP analysis of the population: using the PCR primer of the molecular marker KASP3 with polymorphism obtained in the above steps to amplify parent H461, Chinese spring and F8And (3) carrying out genotype identification on DNA of the population plants to obtain molecular marker data. The parent H461 is marked as A, the length of the amplified fragment is 47bp, and the single base difference site is G. The parent Chinese spring is marked as B, the length of the amplified fragment is 47bp, and the single base difference site is A. F8The population strain type is from H461 as A and from Chinese spring as B.
c) Complete Interval Mapping (Inclusive Composite Interval Mapping) using software IciMapping 4.2, in combination with F8The phenotypic data of the population spikelet particle number shows that the molecular marker KASP3 is closely linked with the spikelet particle number QTL QGns. As can be seen from FIG. 1, spikelet number QTL QGns. sicau-2D is located in the 6.68cM segment between markers AX-109316972 and AX-110906716, while molecular marker KASP3 cosegregates with AX-109316972 and is tightly linked to QTL.
The linkage genetic map of the position of the wheat multi-spikelet number QTL QGns. sicau-2D on the 2D chromosome and the molecular marker KASP3 is shown in figure 1.
Example 2 application of molecular marker KASP3 closely linked with wheat spikelet grain number QTL QGns
1. DNA extraction
The test material is selected from H461, Chinese spring and Sichuan wheat 107, wherein the Chinese spring and Sichuan wheat 107 is a wheat material with small spike grain number, and H461 is a wheat material with large spike grain number. 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 spikelet grain number QTL QGns
2.1 primer design
SNP markers AX-109316972 and AX-110906716 were converted to design fluorescent quantitative PCR primers for subsequent screening. The fluorescent quantitative PCR primer 4 pairs were designed using DNAMAN 6.0 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: amplifying the primer sequence;
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (to which FAM fluorophores can be bound);
h, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached).
2.2 fluorescent quantitative PCR platform test primers and differences between their parents
(1) Extracting the DNA of the leaves of H461, Chinese spring and Chuanmai in the 107 trilobate 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:
KASP3-1:5’-GAAGGTGACCAAGTTCATGCTGCAAAGGAATC ATTCAACGA-3’；
KASP3-2:5’-GAAGGTCGGAGTCAACGGATTGCAAAGGAATC ATTCAACGG-3’；
KASP3-3:5’-AAATCTGAACGCCACCTATTTG-3’；
moreover, the 5' ends of the primers KASP3-1 and KASP3-2 are respectively connected with different fluorescent probes;
the sequence of the fluorescent probe is as follows:
f, probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (to which FAM fluorophores can be bound);
h, probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached).
(3) A fluorescent quantitative PCR amplification reaction system: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers KASP3-1, KASP3-2 and KASP3-3, and the volume ratio is 2:2: 5. That is, in KASP Assay Mix, primers KASP3-1, KASP3-2 and KASP3-3 at a concentration of 100. mu.M were mixed at 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 wheat multi-spike grain number QTL QGns.sicau-2D all have the same genotype as the wheat H461 and are marked as type A, and wheat varieties without the wheat multi-spike grain number QTL QGns.sicau-2D all have fluorescence signals obviously different from the wheat H461, such as Chinese spring and Sichuan wheat 107 and are marked as type B. The results of genotyping H461, Chinese spring, Chuanmai 107 using KASP primer are shown in FIG. 2.
3. Applicability of primer sequence KASP3-1/2/3 in population detection process
(1) Hybridizing H461 as female parent and Chuanmai 107 as male parent to obtain F1，F1Selfing to obtain F2Addition to F by single seed descent8Generation RIL validation populations. 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, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains primers KASP3-1, KASP3-2 and KASP3-3, and the volume ratio is 2:2: 5. That is, in KASP Assay Mix, primers KASP3-1, KASP3-2 and KASP3-3 were mixed at a concentration of 100. mu.M in 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 multi-spike grain number QTL QGns.sicau-2D all showed the same genotype as wheat H461 and are marked as type A, while wheat varieties without the wheat multi-spike grain number QTL QGns.sicau-2D all showed fluorescence signals which are obviously different from wheat H461 and are marked as type B, and the results are shown in figure 3. Randomly spot-inspecting 107 strains and 51 strains to amplify segments with the same type as H461, wherein the segments are wheat spikelet grain number QTL QGns. The 56 plants can amplify B-type fragments which are the same as those of the Chinese spring and Sichuan wheat 107, are plants which do not contain the wheat multi-spikelet grain number QTL QGns.
(6) Field identification of the 107F8The number of spikelets of the plants is shown in the table 2, and the results of predicting the number of spikelets of the genetic population by using a molecular marker KASP3 of QTL QGns. The average number of spikelets of the plants with the same type as H461 is 5.48, which is obviously higher than that of the plants with the type of 107 of Chinese spring and Sichuan wheat by 5.10. The actual result is consistent with the expected result, which shows that the panicle number QTL QGns.sicau-2D of the invention has the effect of remarkably increasing the panicle number, and the molecular marker KASP3 can be used for tracking and identifying the panicle number QTL QGns.sicau-2D.
TABLE 2 results of KASP3 typing in genetic populations
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 spikelet grain number QTL QGns. sicau-2D and application thereof
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<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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gcaaaggaat cattcaacgn tcactcaaat aggtggcgtt cagattt 47
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<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaaggtgacc aagttcatgc tgcaaaggaa tcattcaacg a 41
<210> 3
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaaggtcgga gtcaacggat tgcaaaggaa tcattcaacg g 41
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaatctgaac gccacctatt tg 22
<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> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaaggtgacc aagttcatgc tgaacgccac ctatttgagt gac 43
<210> 8
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gaaggtcgga gtcaacggat tgaacgccac ctatttgagt gat 43
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caatcgcaaa ggaatcattc 20
<210> 10
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gaaggtgacc aagttcatgc tcacgtaagt aaccggaata agataga 47
<210> 11
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gaaggtcgga gtcaacggat tcacgtaagt aaccggaata agatagc 47
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
catcgagcag ttccatatca ttatag 26
<210> 13
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaaggtgacc aagttcatgc tcgtaagtaa ccggaataag ataga 45
<210> 14
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaaggtcgga gtcaacggat tcgtaagtaa ccggaataag atagc 45
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tcgagcagtt ccatatcatt atag 24
Claims (10)
1. The molecular marker is closely linked with wheat spikelet number QTL QGns. sicau-2D, and is characterized in that the molecular marker is KASP3, the molecular marker and the wheat spikelet number QTL QGns. sicau-2D are co-located in a marker AX-109316972-AX-110906716 section on a wheat 2D chromosome, and the molecular marker is located in a wheat spikelet number QTL QGns. sicau-2D confidence interval.
2. The molecular marker according to claim 1, wherein the nucleotide sequence is represented by SEQ ID No.1, and the 20 th base polymorphism in the sequence is A or G.
3. The molecular marker of claim 1 or 2, wherein the wheat spikelet number QTL qgns. sicau-2D is capable of significantly increasing wheat spikelet number with a LOD value greater than 3 accounting for 26.57% of phenotypic variation.
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) application in identifying wheat spikelet grain number QTL QGns.sicau-2D;
(2) the application in screening or identifying wheat varieties with multiple small ear grain numbers;
(3) the application in wheat molecular marker assisted breeding;
(4) application in improving wheat germplasm resources.
8. A method for identifying wheat spikelet number QTL QGns.sicau-2D is characterized in that genome DNA of wheat to be detected is taken as a template, a specific primer group of 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 10 μ L reaction system for the fluorescent quantitative PCR amplification comprises the following components: 2 XKASP Mastermix 5. mu.L, KASP Assay Mix 0.14. mu.L, template DNA 50ng, DNase/RNase-free deionized water to a total amount of 10. mu.L; wherein, the KASP Assay Mix contains a primer with a nucleotide sequence shown as SEQ ID NO.2-4, and the molar ratio of the three primers is 2:2: 5;
and/or the reaction program of the fluorescent quantitative PCR amplification comprises: 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 of claim 8 or 9, wherein the wheat varieties having the wheat multi-ear number QTL qgns. sicau-2D all exhibit the same fluorescent signal as the fluorescent probe attached to the primer set forth in SEQ ID No.3, and the wheat varieties not having the wheat multi-ear number QTL qgns. sicau-2D all exhibit a fluorescent signal that is significantly different from the fluorescent probe attached to the primer set forth in SEQ ID No. 3.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116769961A (en) * | 2023-07-28 | 2023-09-19 | 四川农业大学 | Wheat spike number per spike QTL linkage molecular marker developed by multi-sieve-mixing-determining four-step method and application |
Citations (7)
| 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 |
| CN108513545A (en) * | 2015-11-20 | 2018-09-07 | 4D制药研究有限公司 | Include the composition of bacterium bacterial strain |
| CN109825639A (en) * | 2019-04-24 | 2019-05-31 | 鲁东大学 | Molecular labeling and its application with wheat grains per spike main effect QTL compact linkage |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN110184381A (en) * | 2019-06-25 | 2019-08-30 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | One kind SNP site relevant to the every fringe spikelet number of wheat and its application |
| CN111471790A (en) * | 2020-04-26 | 2020-07-31 | 四川农业大学 | Molecular marker closely linked with wheat grain filling rate QT L QGfr. sicau-7D.1 and application thereof |
| US20200362366A1 (en) * | 2018-01-12 | 2020-11-19 | Basf Se | Gene underlying the number of spikelets per spike qtl in wheat on chromosome 7a |
-
2021
- 2021-02-07 CN CN202110182817.5A patent/CN112779350A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108513545A (en) * | 2015-11-20 | 2018-09-07 | 4D制药研究有限公司 | Include the composition of bacterium bacterial strain |
| 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 |
| US20200362366A1 (en) * | 2018-01-12 | 2020-11-19 | Basf Se | Gene underlying the number of spikelets per spike qtl in wheat on chromosome 7a |
| CN109825621A (en) * | 2019-02-22 | 2019-05-31 | 四川农业大学 | Wheat spikelet number QTL chain SNP marker and its application |
| CN109825639A (en) * | 2019-04-24 | 2019-05-31 | 鲁东大学 | Molecular labeling and its application with wheat grains per spike main effect QTL compact linkage |
| CN110184381A (en) * | 2019-06-25 | 2019-08-30 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | One kind SNP site relevant to the every fringe spikelet number of wheat and its application |
| CN111471790A (en) * | 2020-04-26 | 2020-07-31 | 四川农业大学 | Molecular marker closely linked with wheat grain filling rate QT L QGfr. sicau-7D.1 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| JIAN MA等: ""Identification and validation of a major and stably expressed QTL for spikelet number per spike in bread wheat"", 《THEORETICAL AND APPLIED GENETICS》 * |
| YU LIN等: ""QTL mapping for grain number per spikelet in wheat using a high-density genetic map"", 《THE CROP JOURNAL》 * |
| 张坤普等: ""小麦籽粒产量及穗部相关性状的QTL定位"", 《作物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116769961A (en) * | 2023-07-28 | 2023-09-19 | 四川农业大学 | Wheat spike number per spike QTL linkage molecular marker developed by multi-sieve-mixing-determining four-step method and application |
| CN116769961B (en) * | 2023-07-28 | 2024-01-26 | 四川农业大学 | Wheat spike number per spike QTL linkage molecular marker developed by multi-sieve-mixing-determining four-step method and application |
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