CN110982933B - Molecular marker closely linked with major QTL (quantitative trait locus) of wheat grain length and application thereof - Google Patents
Molecular marker closely linked with major QTL (quantitative trait locus) of wheat grain length and application thereof Download PDFInfo
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Abstract
The inventionDisclosed is a molecular marker closely linked with major QTL of wheat grain length and its application, wherein the molecular marker is a DNA fragment obtained by PCR amplification using wheat Jing 411 genomic DNA as a template and an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 3. The molecular marker provided by the invention andqKl‑1BLthe close linkage can promote the transfer application of the locus in the promoted variety and is also beneficial to the polymerization breeding of the locus and other yield-related loci; the molecular marker provided by the invention is an Indel marker, has codominance, has the advantages of stable amplification, quick and efficient detection and the like of a primer pair, and can quickly determine whether wheat resources exist or notqKl‑ 1BLAnd presence pattern, and predicting grain length characteristics of wheat.
Description
Technical Field
The invention relates to a molecular marker and application thereof, in particular to a molecular marker closely linked with a major QTL (quantitative trait locus) of wheat grain length and application thereof, belonging to the technical field of crop seed selection and cultivation.
Background
Wheat (A), (B)Triticum aestivumL.) is one of the main grain crops in the world, and plays an important role in food safety in China. The available cultivated land area in China is small, and the contradiction between the cultivated land area and the grain demand is huge, so that the total grain yield needs to be increased by increasing the yield per unit in the limited land area. The effective number of ears, grains per ear and grain weight are the components of wheat yieldThree elements. The wheat grain type comprises the characteristics of grain length, grain width, grain thickness and the like, and is extremely obviously and positively correlated with the wheat grain weight. In addition, wheat grain type also affects processing and appearance commodity quality. Therefore, the genetic improvement of the wheat grain type character is particularly important in cultivating high-yield and high-quality wheat varieties.
In the traditional variety improvement practice, different varieties (lines) need to be subjected to a series of hybridization, and the phenotype identification of multiple points for many years not only wastes time and labor, but also has high difficulty and high cost. The molecular marker assisted breeding technology is a method for judging and detecting whether a target gene exists or not by utilizing a molecular marker closely linked with a target character gene. The target gene is selected in the early stage of breeding, so that the efficiency and the accuracy of target character improvement can be effectively improved, the breeding process is shortened, and the method is gradually and widely applied to agricultural biological breeding.
Like most agronomic traits, the grain trait is a typical quantitative trait, is regulated by multiple genes, and faces huge challenges in gene separation and identification, thereby limiting the development of wheat molecular markers and the application of molecular breeding. The QTL positioning result about the wheat grain length shows that the wheat grain length QTL is distributed on 21 chromosomes. Currently, several mapping populations have been used to detect some QTL loci associated with grain length on the 1BL chromosome. Due to the difference between the genetic population and the molecular marker used in the research, QTL intervals are large, and the linkage marker is far away from the target gene, so that the grain length molecular marker cannot be effectively used for molecular assisted breeding.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a molecular marker tightly linked with the major QTL of the wheat grain length and application thereof.
In order to achieve the above objects, the present invention provides, in the first place, a primer pair for identifying or assisting in identifying a wheat grain length trait, a method for preparing the primer pair, and a product for identifying or assisting in identifying a wheat grain length trait.
The primer pair for identifying or assisting in identifying the wheat grain length trait is characterized by consisting of two single-stranded DNAs shown as SEQ ID NO. 1 and SEQ ID NO. 2.
The method for preparing the primer pair provided by the invention is characterized by comprising the step of separately packaging the two single-stranded DNAs.
The product for identifying or assisting in identifying the wheat grain length character is characterized by comprising the primer pair.
In order to achieve the above object, the present invention also provides a method for identifying or assisting in identifying a wheat grain length trait.
The method for identifying or assisting in identifying the wheat grain length trait is characterized by comprising the following steps of:
step 1: extracting genome DNA of wheat to be detected;
step 2: taking the genome DNA of wheat to be detected as a template, and carrying out PCR amplification by using the primer pair;
and step 3: performing gel electrophoresis on the PCR product of the wheat to be detected, and determining the grain length property of the wheat to be detected according to the electrophoresis result: if the amplified fragment with the molecular weight of 344bp appears, the wheat variety or line is predicted to have the QTL locus for increasing the wheat grain length, otherwise, the wheat variety or line does not have the QTL locus for increasing the wheat grain length.
In step 2, the PCR amplification reaction system is 10 μ L, and comprises:
(1) 0.5 mu L of upstream primer shown in SEQ ID NO. 1 with the concentration of 10 mu mol/L;
(2) 0.5 mu L of downstream primer shown in SEQ ID NO. 2 with the concentration of 10 mu mol/L;
(3) 1 μ L of DNA template at a concentration of 50 ng/. mu.l;
（4）5μL 2×Taq PCR StarMix；
（5）3μL ddH2and O is used for complementing the reaction system.
In step 2, the PCR amplification procedure was as follows:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
In step 3, 6.0% native polyacrylamide gel was used as the gel, containing 5.85g acrylamide and 0.15g methylene acrylamide per 100mL of the gel solution.
Meanwhile, the invention also provides a molecular marker 1BL-16 related to the wheat grain length trait.
The molecular marker 1BL-16 related to the wheat grain length property is characterized in that the molecular marker 1BL-16 is a DNA fragment obtained by carrying out PCR amplification by using the primer pair with the genome DNA of wheat Jing 411 as a template, and the nucleotide sequence of the DNA fragment is shown as SEQ ID NO. 3.
The invention has the advantages that:
(1) the primer pair provided by the invention consists of two single-stranded DNAs shown as SEQ ID NO. 1 and SEQ ID NO. 2, has the advantages of stable amplification, quick and efficient detection and the like, and can promote the major QTL of the grain lengthqKl-1BLThe application in molecular breeding of high-yield and high-quality wheat;
(2) the molecular marker 1BL-16 provided by the invention, andqKl-1BLthe close linkage can promote the transfer application of the locus in the promoted variety and is also beneficial to the polymerization breeding of the locus and other yield-related loci;
(3) the molecular marker 1BL-16 provided by the invention is an Indel marker, has codominance, and the primer pair has the advantages of stable amplification, quick and efficient detection and the like, and whether wheat resources exist or not can be quickly determined by using the molecular marker 1BL-16qKl-1BLAnd the existence form, and the grain length characteristic of the wheat is predicted, so as to effectively screen the wheat containingqKl-1BLWheat varieties (lines) of the locus and for improvement of wheat varieties;
(4) by applying toqKl-1BLThe closely linked molecular marker 1BL-16 can greatly reduce the work of phenotype identification and can be utilized in the seedling stage of wheat, so that non-target plants are eliminated, the breeding cost is saved, and the breeding efficiency is improved.
Drawings
FIG. 1 shows the amplification band pattern of the molecular marker 1BL-16 provided by the present invention in part of KJ-RIL family, where M is 2000bpDNA Ladder, K is the amplification band pattern of wheat variety Kenong 9204, and J is the amplification band pattern of wheat variety Jing 411;
FIG. 2 is a QTL using MapQTL6.0 and IcMapping 4.1 for QTL-qKl-1BLPositioning the result graph of the analysis;
FIG. 3 shows major QTL-qKl-1BLIn a mapping interval on the chromosome 1B, a hollow rectangle represents a chromosome, the left side is the name of a molecular marker, the right side number is the position marked on the chromosome, the unit is MB, the left side bold molecular marker is 1BL-16, and black rectangles on the right side of the chromosome respectively represent QTL mapping intervals with the grain length under 9 environments;
FIG. 4 is a graph of the results of single-marker analysis of 188 KJ-RIL families based on grain length of 1BL-16 in 9 environments, AA representing the grain length reduction allele from Kenong 9204, BB representing the grain length increase allele from Jing 411, and "(S) ((S) ()) representing very significant differencesP<0.01) indicates significant difference (P<0.05）。
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Molecular marker closely linked with major QTL of wheat grain length
The wheat variety Kenong 9204 is a national examined variety in 2003, and the variety approval number is national examined wheat 2003037.
The wheat variety Jing 411 is also an approved variety and can be requested from a national crop germplasm resource library, and the national uniform number is ZM 020984.
Utilizes QTL positioning analysis to analyze the major QTL of grain lengthqKl-1BLLocated in the interval from the long arm AX-109370525 to AX-110579468 of chromosome 1B, and subjected to re-sequencing by utilizing the data of Kenong 9204 and Jing 411qKl-1BLAn InDel molecular marker 1BL-16 with polymorphism among parents is designed and developed near a prediction peak of the LOD of the segment. The sequence of the upstream primer of the molecular marker 1BL-16 is as follows: GGCCGCACAAAGGACAACAAA (SEQ ID NO: 1), the sequence of the downstream primer is: TCGGCCATAATAAACAACATCTCC（SEQ ID NO:2）。
The genome DNA of the wheat variety Jing 411 is taken as a template, and the upstream primer shown by SEQ ID NO. 1 and the downstream primer shown by SEQ ID NO. 2 are used for PCR amplification.
The PCR amplification reaction system is 10 mu L and comprises:
(1) 0.5 mu L of upstream primer shown in SEQ ID NO. 1 with the concentration of 10 mu mol/L;
(2) 0.5 mu L of downstream primer shown in SEQ ID NO. 2 with the concentration of 10 mu mol/L;
(3) 1 μ L of DNA template at a concentration of 50 ng/. mu.l;
（4）5μlL2×Taq PCR StarMix；
（5）3μLddH2and O is used for complementing the reaction system.
The PCR amplification procedure was as follows:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
The model of the PCR instrument: t100TMThermal Cycler。
The resulting amplification product was electrophoretically separated on a 6.0% native polyacrylamide gel (5.85 g acrylamide and 0.15g methylene acrylamide per 100mL of gel solution) and the molecular weight of the final amplification product was 631bp (shown in SEQ ID NO: 3).
Second, method for obtaining molecular marker 1BL-16
The method for acquiring the molecular marker 1BL-16 specifically comprises the following steps:
step 1: form F containing 188 families6Generation RIL population
Hybridizing the wheat variety Kenong 9204 with short grain length as female parent and the wheat variety Jing 411 with long grain length as male parent to obtain hybrid F1Hybrid F1Selfing to produce F2，F2Selfing generation by generation to form F containing 188 families6A surrogate RIL population;
step 2: extraction of KJ-RIL population leaf DNA of wheat by CTAB method
Placing steel balls and about 0.2g of fresh leaves into a 2.0mL centrifuge tube, and centrifugingPlacing the tube in liquid nitrogen, oscillating to make the leaf into powder, adding 0.8mL of CTAB extract, carrying out 65 ℃ water bath for 30min, reversing for 4 times, shaking up, placing the centrifugal tube at room temperature, cooling, adding equal volume of chloroform-isoamyl alcohol (volume ratio 24: 1), oscillating vigorously for 1min, centrifuging at 120000rpm for 10min, sucking supernatant 500uL to 1.5mL, adding equal volume of precooled isopropanol (precooling at-20 ℃) to precipitate DNA, centrifuging at 12000rpm for 5min, pouring off the supernatant, adding appropriate amount of 70% ethanol to wash and precipitate for 2 times, reversing the centrifugal tube to suck water on paper, drying the DNA in the air, and finally adding 400uL ddH2Dissolving O, and storing in a refrigerator at-20 deg.C.
All the DNAs extracted above were subjected to PCR amplification using the upstream primer shown by SEQ ID NO. 1 and the downstream primer shown by SEQ ID NO. 2.
The PCR amplification reaction system is 10 mu L and comprises:
(1) 0.5 mu L of upstream primer shown in SEQ ID NO. 1 with the concentration of 10 mu mol/L;
(2) 0.5 mu L of downstream primer shown in SEQ ID NO. 2 with the concentration of 10 mu mol/L;
(3) 1 μ L of DNA template at a concentration of 50 ng/. mu.l;
（4）5μL 2×Taq PCR StarMix；
（5）3μL ddH2and O is used for complementing the reaction system.
The PCR amplification procedure was as follows:
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40s, for 34 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
The model of the PCR instrument: t100TMThermal Cycler。
The amplification product was electrophoresed on 6.0% native polyacrylamide gel with electrophoresis buffer of 1 × TBE at 150V for 2 h.
Silver nitrate dyeing: after electrophoresis, the stripping gel block is fixed in a fixing solution for 3min, rapidly washed by deionized water for 3 times, dyed in a silver dye solution for 8min, washed by the deionized water for 2 times, transferred into a developing solution for development until an amplification strip is clearly visible, and washed by water for 2 times.
The developed gel block was placed on a film viewer, the tape pattern was recorded, and a picture of the gel was taken, as shown in fig. 1.
Band pattern read results show: the amplification product size of the wheat variety Kenong 9204 is 312bp, the amplification product size of the wheat variety Jing 411 is 344bp, and 94 banding patterns of 188 groups of KJ-RIL are identical to those of Kenong 9204, and 93 banding patterns are identical to those of Jing 411.
And step 3: construction of wheat molecular marker genetic linkage map
The obtained genetic linkage map of the RIL population genotype is constructed by using JoinMap 2.0 mapping software, wherein the genetic linkage map of the wheat molecular marker with 119566 marker loci is constructed by using the LOD (Long-Strand-Diels distance Detector) which is more than or equal to 2.5 as a standard.
As a result: the 119566 markers included 119001 SNP markers, 287 DArTs markers, 153 g-SSR markers, 50 e-SSR markers, 17 STS markers, 45 SRAPs markers, 5 ISSRs markers, 7 functional PCR markers, and 1 morphological marker.
And 4, step 4: examine the grain length and thousand grain weight of 188 families of the KJ-RIL group
188 families of the KJ-RIL group are planted in 10 different environments for field phenotype identification, and the grain length and thousand grain weight of each strain under different nitrogen level treatment conditions in different years and different places are respectively considered.
The different environments include: 2011-2012, 2012-2013 and 2013-2014 are planted in agriculture ecosystem test stations (T1, T2 and T3: 37 ° 53 'N and 114 ° 41' E) of Koelreuterian of China academy of sciences, 2012-2013 and Beijing Pingxi fu farms (T4: 40 ° 06 'N and 116 ° 24' E) of China academy of genetics and developmental biology and test bases (T5: 35 ° 27 'N and 113 ° 48' E) of Xinxiang city science research institute of Henan province, wherein each environment is provided with two nitrogen levels of low nitrogen and high nitrogen, and 10 environments are provided, wherein the low nitrogen treatment is not applied with fertilizer all the year round, and the high nitrogen treatment is applied with 300kg hm before each year-2Phosphoric diamine and 225 kg hm-2Urea as base fertilizer and applying 150 kg.hm in jointing stage-2And (5) urea topdressing.
The wheat planting method under 10 different environments comprises the following steps: each strain is planted in 2 rows and 40 grains/row; the row length is 3m, the plant spacing is 7.5cm, and the row spacing is 25 cm.
The grain length measuring method comprises the following steps: after harvesting, randomly selecting 5 plants from each plant line, separating main stem ears, threshing, selecting 10 grains, and measuring by using a vernier caliper, wherein the average value of the 5 plants is taken as the final grain length of the plant line to be measured.
And 5: performing additive QTL localization analysis
The 188 KJ-RIL high-density genetic linkage maps, the genotypes and the phenotypic values are utilized, MapQTL6.0 and IcMapping 4.1 are adopted to carry out additive QTL positioning analysis, and the analysis results are shown in table 1, figure 2 and figure 3.
TABLE 1 major QTL-qKl-1BLResult of positioning
| trait-Environment | Straight bit (MB) | Left side mark | Right marker | LOD value | PVE(%) | Additive effect |
| Grain length-El | 698.71 | AX-109370525 | AX-110979003 | 2.27 | 5.54 | -0.05 |
| Grain Length-E2 | 703.31 | AX-111496745 | AX-110579468 | 5.86 | 13.47 | -0.10 |
| Grain Length-E3 | 695.01 | AX-86178495 | AX-109370525 | 3.18 | 7.19 | -0.10 |
| Grain Length-E4 | 698.31 | AX-86178495 | AX-109370525 | 5.95 | 14.05 | -0.12 |
| Grain Length-E5 | 696.61 | AX-86178495 | AX-109370525 | 2.24 | 5.16 | -0.08 |
| Grain Length-E6 | 698.81 | AX-109370525 | AX-110979003 | 4.34 | 10.45 | -0.09 |
| Grain Length-E7 | 704.61 | AX-111496745 | AX-110579468 | 2.15 | 5.23 | -0.07 |
| Grain Length-E9 | 700.41 | AX-110979003 | AX-111496745 | 2.41 | 5.78 | -0.06 |
| Grain Length-E10 | 699.21 | AX-109370525 | AX-110979003 | 4.24 | 9.89 | -0.09 |
QTL analysis results show that: the major QTL for controlling grain length can be stably detected in 10 environmentsqKl-1BL Wherein 9 of the environmental LOD values are 2 or more, 1 of the environmental LOD values is < 2 (grain length-E8, not shown in Table 1), and the interval is located from AX-86178495 to AX-110579468 on the long arm of chromosome 1B (see Table 1, below)Physical location: KN1B: 687.193291-710.238150) 23MB segment, and the LOD peak occurs in the segment with a physical location of KN1B: 695.01-704.61 MB. The located grain length QTL can account for 5.16-14.05% of the phenotypic variation of grain length, with alleles from Jing 411 increasing the grain length by 0.5-1.3 mm.
Step 6: single-label 1 BL-16-based grain length difference significance analysis
1BL-16 is adopted to carry out genotyping on 188 KJ-RIL families, and the difference significance analysis is carried out on different genotype grain lengths.
The results of single-marker analysis of 188 KJ-RIL families based on grain length of 1BL-16 in 9 environments are shown in FIG. 4.
The results show that the allele from kyoton 411 can both increase grain length compared to the effect of the allele from corm 9204 on decreasing grain length.
This indicates that: the molecular weight of an amplification product which is obtained from DNA of a wheat variety Jing 411 by an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2 is 344bp, namely a molecular marker (namely, the molecular marker 1 BL-16) which is closely linked with the wheat grain length QTL.
Application of molecular marker 1BL-16
Based on the previous experiments and analyses:
(1) the primer pair consisting of the upstream primer shown in SEQ ID NO. 1 and the downstream primer shown in SEQ ID NO. 2 can be applied to identification or auxiliary identification of wheat grain length traits and can also be applied to acquisition of molecular markers related to wheat grain length;
(2) the molecular marker 1BL-16 can be applied to identification or auxiliary identification of wheat grain length traits and can also be applied to breeding wheat with grain length genotypes.
In conclusion, the primer pair and the molecular marker 1BL-16 provided by the invention can greatly accelerate the molecular marker-assisted selective breeding process of the wheat grain length, and have important application values in the genetic improvement of wheat grain types and the cultivation of high-yield and high-quality wheat varieties.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Sequence listing
<110> university of Ludong
<120> molecular marker closely linked with major QTL of wheat grain length and application thereof
<141>2019-12-25
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<170>SIPOSequenceListing 1.0
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<213>Artificial Sequence
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<213>Artificial Sequence
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<213>Artificial Sequence
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gagccatctg tgactccgtc agccattggg tgtcaaccta tgtatataaa gggacgaccc 60
ggtggcggtt cggggcaaga aacaagggat caaaagctag gttaagcgta ttcgctccct 120
ggtaatcgag acataagcaa taccacctca aactagatta ggcctttacc ttcactgcaa 180
ggggctgaac tagtataaac tcctgtgtcc tttgtcccgt ttaacccctt taagctaacc 240
tagttgcgat ggctcgacga ctaagccctc acactaggac atctgccgtg actattccac 300
gatagttggc gcccaccgtg gggccagcgc acaatggttt agag 344
Claims (2)
1. The molecular marker 1BL-16 related to the wheat grain length property is characterized in that the molecular marker 1BL-16 is a DNA fragment obtained by carrying out PCR amplification by using genome DNA of wheat Jing 411 as a template and using an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and the nucleotide sequence of the molecular marker 1BL-16 is shown in SEQ ID NO. 3.
2. The use of the molecular marker 1BL-16 of claim 1 in any of:
(1) the application in identification or auxiliary identification of wheat grain length traits;
(2) application in breeding wheat with grain length genotype.
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| CN112593007B (en) * | 2021-01-11 | 2022-07-01 | 四川农业大学 | SNP molecular marker linked with wheat grain length QTL and application thereof |
| CN114107537B (en) * | 2021-09-22 | 2023-06-13 | 青岛农业大学 | Molecular marker closely linked with main effect QTL of lateral root number in wheat seedling stage, detection primer and application thereof |
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| CN102690822B (en) * | 2012-06-01 | 2014-11-05 | 山东农业大学 | Molecular marker closely linked with major quantitative trait locus (QTL) of thousand-grain weight and grain length of wheat and obtaining method and application of molecular marker |
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