CN111979345A - Development and application of KASP (Kaempferi protein) marker related to wheat biomass and yield under salt stress condition - Google Patents
Development and application of KASP (Kaempferi protein) marker related to wheat biomass and yield under salt stress condition Download PDFInfo
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Abstract
The invention discloses development and application of KASP markers related to wheat biomass and yield under a salt stress condition. The invention firstly discloses the application of a substance for detecting the polymorphism or genotype of an SNP marker AX-108839508 in identification or auxiliary identification of wheat biomass and/or yield; the SNP marker AX-108839508 is the 692158124 th nucleotide located on the wheat 5A chromosome, which is A or G. The invention further discloses a method for identifying or assisting in identifying wheat biomass and/or yield under salt stress conditions. The KASP marker related to the SNP marker AX-108839508 developed by the invention is obviously related to the yield and biomass traits under the salt stress condition, lays a theoretical foundation for breeding salt-tolerant wheat varieties with high and stable yield and excellent quality, and provides a molecular auxiliary selection means.
Description
Technical Field
The invention relates to the technical field of biology, in particular to development and application of KASP markers related to wheat biomass and yield under a salt stress condition.
Background
Soil salinization is an important limiting factor affecting grain yield. How to utilize middle and low yield fields such as saline-alkali soil and the like and effectively improve the total yield of grains is an important problem to be solved urgently. Wheat is one of the most important grain crops in the world and is also one of the main cultivation crops of salinized soil. The salinization of soil seriously affects the wheat yield, and the cultivation and planting of salt-tolerant wheat varieties is an important way for effectively utilizing the salinization soil resources and increasing the wheat yield.
Molecular marker assisted selection is an aid to selection using molecular markers in crop improvement processes. The genotype of the target gene can be known by detecting the genotype of a molecular marker by means of a molecular marker closely linked to the target gene. Wheat yield and yield-related traits are controlled by polygenes and are susceptible to environmental factors. The molecular marker closely linked with the wheat yield and the yield-related characters is used for screening the target region and the whole genome of the individual, so that the breeding process can be accelerated, the breeding workload can be reduced, and the aim of improving the breeding efficiency can be achieved.
Single Nucleotide Polymorphisms (SNPs) refer to DNA sequence Polymorphisms resulting from variation of a Single Nucleotide at the genomic level. SNP can be inherited stably, is widely distributed and rich in polymorphism, and is easy to realize automatic analysis. The current methods for SNP detection mainly comprise two methods: genome sequencing and SNP chips are simplified. However, when the SNP marker linked to the target trait is obtained by linkage analysis or association analysis and then whether other materials contain the excellent site or not is detected, the technical cost of simplified genome sequencing or high-density SNP chip is obviously too high and is not necessary. Competitive Allele-Specific PCR (KASP) technology can achieve SNP typing and detection of Insertions and Deletions based on Specific matching of the terminal bases of primers (Insertions and Deletions, InDels). The KASP technique was developed by LGC (Laboratory of the Goverment Chemist Government Chemist Laboratory) Inc. in the United kingdom and has now become one of the mainstream methods of SNP analysis internationally. It only needs to synthesize two general fluorescent probes and two general quenching probes, and then synthesizes a plurality of SNP PCR primers aiming at specific sites, and can detect a plurality of sites. The fluorescent probe and the quenching probe are expensive, so compared with the Taqman probe, the KASP method can replace the fluorescent probe aiming at the site by the universal fluorescent probe, thereby greatly saving the cost. The micro-fluidic chip detection system is an SNP detection platform independently developed by Beijing Boao classical biotechnology limited, and has the advantages of simple operation, quick reaction and high cost performance. The method can complete typing detection of 96 reactions within 2.5 hours by only one-step sample adding and four-step simple taking and placing work, and realizes low-cost, simple and quick SNP/InDel typing.
Disclosure of Invention
The invention aims to solve the technical problem of developing KASP markers related to the biomass and yield of wheat, particularly the biomass and yield of wheat under the condition of salt stress so as to breed high-yield salt-tolerant wheat varieties.
In order to solve the problems, the invention provides an application of a substance for detecting polymorphism or genotype of SNP marker AX-108839508 in identification or auxiliary identification of wheat biomass and/or yield;
the SNP marker AX-108839508 is the 692158124 th nucleotide located on the wheat 5A chromosome, which is A or G.
In the above application, the application of identification or auxiliary identification of wheat biomass and/or yield is application of identification or auxiliary identification of wheat biomass and/or yield under salt stress conditions.
The application of the substance for detecting the polymorphism or genotype of the SNP marker AX-108839508 in wheat breeding, particularly the application in salt-tolerant wheat breeding, is also within the protection scope of the invention.
In the application, the wheat breeding is to cultivate salt-tolerant wheat varieties with high biomass and/or yield.
In the above application, the SNP marker AX-108839508 corresponds to the 106 th nucleotide of SEQ ID NO.1, i.e., r represents A or G.
In the application, the polymorphism or the genotype for detecting the SNP marker AX-108839508 can be specifically used for detecting the nucleotide type of the SNP marker AX-108839508 in the wheat genome. The genotype of the SNP marker AX-108839508 is AA (AA genotype for short), GG (GG genotype for short) or AG (AG genotype for short). Wherein the AA genotype is homozygous for the SNP marker AX-108839508A in the wheat genome, the GG genotype is homozygous for the SNP marker AX-108839508G in the wheat genome, and the AG genotype is heterozygous for the SNP marker AX-108839508A and G in the wheat genome.
In the above application, the substance for detecting the polymorphism or genotype of the SNP marker AX-108839508 is A1) or A2) or A3):
A1) the primer set comprises a single-stranded DNA molecule shown in SEQ ID NO.2 or a derivative thereof, a single-stranded DNA molecule shown in SEQ ID NO.3 or a derivative thereof and a single-stranded DNA molecule shown in SEQ ID NO. 4;
A2) PCR reagents containing the primer sets;
A3) a kit comprising a1) or a 2);
the derivative of the single-stranded DNA molecule shown in SEQ ID NO.2 is obtained by connecting the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.2 with a fluorescent label;
the derivative of the single-stranded DNA molecule shown in SEQ ID NO.3 is obtained by connecting the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.3 with another fluorescent label.
Wherein, the fluorescent label in the derivative of the single-stranded DNA molecule shown in SEQ ID NO.2 can be FAM or HEX;
in a specific embodiment of the invention, the derivative of the single-stranded DNA molecule represented by SEQ ID NO.2 is specifically FAM-KASP-F1, the nucleotide sequence of which is 5-GAAGGTGACCAAGTTCATGCTCCGAAGTCCGCTCTATGTTGCAA-3' (underlined sequence is fluorescent tag FAM).
The fluorescent label in the derivative of the single-stranded DNA molecule shown in SEQ ID NO.3 can be HEX or FAM.
In the present inventionIn a specific embodiment, the derivative of the single-stranded DNA molecule represented by SEQ ID NO.3 is HEX-KASP-F2, the nucleotide sequence of which is 5-GAAGGTCGGAGTCAACGGATTCGAAGTCCGCTCTATGTTGCAG-3' (underlined sequence is the fluorescent tag HEX).
The invention also provides a product.
The product is a substance for detecting the polymorphism or genotype of the SNP marker AX-108839508;
it has at least one function of 1) or 2) or 3) or 4) or 5) or 6) as follows:
1) identifying or aiding in identifying biomass and/or yield of wheat;
2) identifying or assisting in identifying biomass and/or yield of wheat under salt stress conditions;
3) breeding of high biomass and/or yield wheat;
4) breeding high biomass and/or yield salt-tolerant wheat;
5) breeding wheat with high biomass and/or yield;
6) and (3) breeding salt-tolerant wheat with high biomass and/or yield.
The invention further provides a method for identifying or assisting in identifying wheat biomass and/or yield under salt stress conditions.
The method for identifying or assisting in identifying the biomass and/or the yield of the wheat under the condition of salt stress comprises the steps of detecting the genotype of an SNP marker AX-108839508 of the wheat to be detected, and determining the biomass and/or the yield of the wheat to be detected according to the genotype;
the genotype of the SNP marker AX-108839508 is GG, the biomass of the wheat to be detected and/or the yield of the wheat to be detected are higher than those of the SNP marker AX-108839508, and the genotype of the wheat to be detected is AA;
wherein the AA genotype is homozygous for the SNP marker AX-108839508A in the wheat genome, and the GG genotype is homozygous for the SNP marker AX-108839508G in the wheat genome.
The invention also provides a breeding method of salt-tolerant wheat.
The breeding method of salt-tolerant wheat comprises the following steps: detecting the genotype of the SNP marker AX-108839508 in the genome of the wheat to be detected, and selecting the wheat with the genotype of the SNP marker AX-108839508 in the genome of the wheat to be detected as GG for breeding.
In the method, the method for detecting whether the genotype of the wheat SNP marker AX-108839508 to be detected is GG or AA is A) or B) as follows:
A) detecting the genotype of a wheat SNP marker AX-108839508 through an SNP chip;
B) and (3) carrying out PCR reaction on the wheat genome DNA to be detected by using the substance for detecting the genotype of the SNP marker AX-108839508 to obtain a product, and carrying out genotyping on the product.
In the above-mentioned method, the first step of the method,
the genotyping method comprises detecting the fluorescent signal of the product, determining the genotype according to the fluorescent signal: if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in SEQ ID NO.2, the genotype of the wheat SNP marker AX-108839508 to be detected is AA; if the product only shows the color of the fluorescent label connected to the 5' end of the DNA molecule shown in SEQ ID NO.3, the genotype of the wheat SNP marker AX-108839508 to be detected is GG.
The salt stress condition in the invention is that the mass percentage of salt in soil is 0.3%.
The invention develops the KASP marker associated with the SNP marker AX-108839508, and proves that the KASP marker is obviously related to the yield and biomass traits under the salt stress condition in a DH group (No. 10 XLUMAI 14 for dry selection) consisting of 150 strains, which shows that the KASP marker developed in the invention can realize the auxiliary selection effect on the yield and biomass under the salt stress condition, lays the theoretical foundation for breeding salt-tolerant wheat varieties with high and stable yield and excellent quality, and provides a molecular auxiliary selection means.
Drawings
FIG. 1 is a statistical analysis of yield and biomass at site Q-5A.
FIG. 2 shows the result of genotyping 24 randomly selected wheat samples with SNP marker AX-108839508.
FIG. 3 shows the result of genotyping 150 strains of DH population (Dry selection No. 10 XRomai 14) with SNP marker AX-108839508.
FIG. 4 is a graph showing the statistical analysis of the yield and biomass of the result of typing of SNP marker AX-108839508.
In the figure, "×" has a very significant difference (p < 0.01).
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the 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.
The following examples, dry election No. 10, are described in the following documents: the Shanxi agricultural science, 1980,4:5-7, is created by the institute of crop science of the Chinese institute of agricultural science, and the public can obtain the same from the institute of crop science of the Chinese institute of agricultural science, so as to repeat the experiment of the application, and can not be used for other purposes.
Examples of the following examples, ruma 14 is described in the following documents: the method is just, a wheat germplasm resource improvement strategy from the breeding theory of Lumai No. 14, a wheat crop academic newspaper, 2005,25(6): 121-.
In the following examples, the DH population (dry selection No. 10 × lumai 14) was created by the crop science institute of the chinese agricultural science institute, using dry selection No. 10 as the female parent and lumai 14 as the male parent, and the public can obtain it from the crop science institute of the chinese agricultural science institute, repeat the experiment of the present application, and cannot be used for other purposes.
Example 1 discovery of SNP markers related to wheat biomass and yield under salt stress conditions and establishment of KASP marker development and detection method
Identification of wheat yield under salt stress condition
191 varieties (lines) screened from the collected wheat germplasm resources are research materials, and the geographical sources comprise 14 provinces, wherein 17 Beijing is selected, 40 Hebei is selected, 30 Henan is selected, 34 Shandong is selected, 8 Shanxi is selected, 39 Shanxi is selected, 6 Qinghai is selected, 3 Gansu is selected, 1 Jilin is selected, 1 inner Mongolia is selected, 1 Xizang is selected, 1 Tianjin is selected, 1 Anhui is selected, 3 Sichuan is selected, and 5 foreign introduced species and 1 unknown wheat germplasm are selected.
2014-2015, 2015-2016 and 2016-2017, 191 varieties (lines) of yield traits were identified in Nanpi ecological agriculture test station (116 DEG 40'E, 38 DEG 00' N, altitude 11m) of China academy of sciences. 1 salinity gradient is set, and the salt content is 0.3 percent (m/m). The experiment adopts a completely random block design, and each variety (line) is planted in a hole sowing mode, namely 1 hole is sowed in each variety, 3 exposed white seeds are sowed in each hole, the row spacing is 23cm, and the hole spacing is 15 cm. 5 times of repetition are set, and the seeds in each repetition are randomly arranged for sowing. After seedling emergence, irrigation is not needed in the growth period, the field is managed conventionally, and serious plant diseases and insect pests and lodging are not caused in the growth period. After the plant is mature, harvesting 5 times according to the holes, weighing the overground part of each hole plant to obtain biomass, and after threshing, weighing the seed weight of each hole plant to obtain the yield.
Second, genotype scanning and whole genome correlation analysis
A wheat 660K SNP chip is used for scanning the whole genome of a 191 wheat variety (line) to obtain the genotype of the wheat variety, and software TASSEL5.0 is used for carrying out whole genome association analysis and positioning the sites associated with the wheat yield and biomass traits under the salt stress condition.
The DNA of the 191 triticale cultivar (line) was extracted by a slightly modified SLS method. The method comprises the following specific steps:
(1) taking 0.2g of fresh wheat leaves into a 2mL centrifuge tube (2 steel balls with the diameter of 4mm are put in the tube in advance), precooling by liquid nitrogen, grinding into powder in a tissue grinder (tissue lyser II, QIAGEN, Germany), adding 0.8mL of 1% SLS (sodium lauryl sarcosinate) lysate, violently shaking to fully mix the sample uniformly, and standing for 10min at room temperature to fully crack the cells.
(2) Adding an extracting solution (phenol: chloroform: isoamyl alcohol: 25:24:1) with the same volume as the lysate into a centrifuge tube, slowly shaking the mixture until the mixture is emulsion, standing the mixture for 10min at room temperature, centrifuging the mixture for 10min at 12000rpm, and transferring the supernatant into a new centrifuge tube. This operation was repeated 1 more time.
(3) Sucking the supernatant into another new 1.5mL centrifuge tube, adding 0.6 times volume of pre-cooled isopropanol into the supernatant, mixing well, precipitating at-20 deg.C for 30min, if there is much precipitate, centrifuging instantaneously, removing supernatant, and washing.
(4) Centrifuging at 4 deg.C and 10000rpm for 10min, discarding supernatant, adding 1mL 75% ethanol, washing precipitate, centrifuging at 4 deg.C and 10000rpm for 5min, discarding supernatant, and washing again. Finally, washing with absolute ethyl alcohol, centrifuging, then removing supernatant, inverting, and drying DNA at room temperature;
(5) to the dried DNA, 100. mu.L of 1 XTE buffer (containing RNase A) was added to dissolve the DNA.
Integrity of DNA samples was checked using agarose gel and DNA concentration and quality was checked using NanoDrop (A)260/280And A260/230). After the detection is qualified (the total amount of DNA is more than 1 mu g, the DNA is complete, no RNA pollution is caused, A260/280And A260/230Meeting the requirements), hybridizing a sample DNA with a wheat 660K SNP chip according to the requirements of the American African company operation manual AXIOM Array 2.0, scanning the hybridization signals of the SNP chip, reading the data of the original scanning result by using Affymetrix software APT1.17.0 and SNPolisher1.5.0, and performing SNP marking quality control on the obtained original typing data to obtain the genotype of 191 triticale varieties (lines). The quality control standards are as follows:
a) removing SNPs with a typing success rate (call rate) of less than 80%;
b) eliminating SNPs with a Minor Allele Frequency (MAF) of less than 0.05;
c) removing SNPs which do not accord with Hardy-Weinberg balance test;
d) samples with a typing success rate (call rate) of less than 90% were rejected.
Third, correlation analysis of yield traits
The software TASSEL5.0 was used to perform genome-wide association analysis using the mixed linear model MLM, mining the genetic variation associated with wheat yield and biomass traits under salt stress conditions across the genome. Based on the SNPs typing results after quality control and 191 triticale varieties, group stratification evaluation is performed by using a PCA function under Analysis option in software TASSEL5.0, a Q matrix is constructed, and default values (number of components 5, minimum eigenvalue 0.0, and total variance 0.5) are selected for parameter setting. Population genetic relationship Analysis was performed using the Kinship function under Analysis option in TASSEL5.0 to construct K matrices with parameter settings chosen as default (Kinship method center IBS, Max Alleles 6). Integrating SNPs typing results of 191 wheat varieties with phenotypic data of yield and biomass characters, and performing yield and biomass character correlation analysis under a mixed linear model MLM by combining a population hierarchical Q matrix and a genetic relationship K matrix. And drawing a Manhattan graph and a QQ graph, and displaying analysis results of the 191 wheat variety yield and biomass character MLM model.
The results showed that a QTL (Q-5A) associated with yield and biomass under salt stress conditions, comprising 3 SNPs, was detected at 692158124-692387337 bp 5A chromosome, with an interpretation of phenotypic variation for yield and biomass of 12.15-12.70% and 12.15-12.74%, respectively. As shown in figure 1, the excellent allelic variation type (GCC type) of the site Q-5A can improve the yield of a wheat single plant by 1.53g and increase the biomass of the single plant by 2.73g under the salt stress condition. The SNP marker AX-108839508 in the site was selected, developed as KASP marker, and applied to other materials.
Four, KASP mark development and verification
KASP tag development
After the DNA sequence of the SNP marker AX-108839508 is compared with a wheat genome reference sequence (iwgsc _ refseq 1.0), 211bp sequences are obtained after extending two ends, the sequences correspond to SEQ ID NO.1, 5 '→ 3' is arranged from left to right of SEQ ID NO.1, and the SNP marker AX-108839508 is positioned at the 106 th position of SEQ ID NO.1, namely r represents A or G.
The SNP marker AX-108839508 is a SNP site of the wheat genome, and the SNP marker AX-108839508 is located at position 692158124 (corresponding to position 106 of SEQ ID NO. 1) on the wheat 5A chromosome, and the nucleotide is A or G.
The genotype of the SNP marker AX-108839508 is AA (abbreviated as AA genotype), GG (abbreviated as GG genotype) or AG (abbreviated as AG genotype). The AA genotype is homozygous for the SNP marker AX-108839508A in the wheat genome, the GG genotype is homozygous for the SNP marker AX-108839508G in the wheat genome, and the AG genotype is heterozygous for the SNP marker AX-108839508A and G in the wheat genome.
(II) design of specific primer and establishment of method
Aiming at two alleles of a target SNP marker AX-108839508, designing a set of KASP primers, wherein the KASP primers comprise 2 upstream primers and 1 universal downstream primer, the upstream primers are DNA molecules with fluorescent labels FAM added at the 5 'ends, DNA molecules with fluorescent labels HEX added at the 5' ends, and the universal downstream primer is a single-stranded DNA molecule (KASP-R) shown in SEQ ID NO. 4;
the DNA molecule with the fluorescence label FAM added at the 5 'end is obtained by adding the fluorescence label FAM at the 5' end of a single-stranded DNA molecule (KASP-F1) shown in SEQ ID NO.2, namely FAM-KASP-F1;
the DNA molecule added with the fluorescent label HEX at the 5 'end is obtained by adding the fluorescent label HEX at the 5' end of a single-stranded DNA molecule (KASP-F2) shown in SEQ ID NO.3, namely HEX-KASP-F2.
The primer sequence of the SNP marker AX-108839508 is as follows:
KASP-F1:5′-CCGAAGTCCGCTCTATGTTGCAA-3′(SEQ ID NO.2)
KASP-F2:5′-CGAAGTCCGCTCTATGTTGCAG-3′(SEQ ID NO.3)
KASP-R:5′-GCGAGCTTGAGTGTCCACCGT-3′(SEQ ID NO.4)
fluorescent label FAM 5'-GAAGGTGACCAAGTTCATGCT-3';
fluorescent label HEX is 5'-GAAGGTCGGAGTCAACGGATT-3';
FAM-KASP-F1：
5′-GAAGGTGACCAAGTTCATGCTCCGAAGTCCGCTCTATGTTGCAA-3' (underlined sequence is fluorescent tag FAM);
HEX-KASP-F2：
5′-GAAGGTCGGAGTCAACGGATTCGAAGTCCGCTCTATGTTGCAG-3' (underlined sequence is the fluorescent tag HEX).
The single-stranded DNA molecule (FAM-KASP-F1) obtained by adding fluorescent label FAM at the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.2 and the single-stranded DNA molecule (KASP-R) shown in SEQ ID NO.4 amplify the fragment with the SNP locus genotype of AA (i.e. the SNP label AX-108839508 is homozygous for A), and the product obtained after PCR amplification of the FAM-carrying sequence shows blue color by fluorescent irradiation;
the single-stranded DNA molecule (HEX-KASP-F2) obtained by adding fluorescent label HEX to the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.3 and the single-stranded DNA molecule (KASP-R) shown in SEQ ID NO.4 amplify the fragment with SNP locus genotype GG (i.e. the SNP marker AX-108839508 is homozygous for G), and the product obtained after PCR amplification of the HEX-carrying sequence shows red color by fluorescent irradiation.
(III) SNP typing
SNP typing is carried out by utilizing a micro-fluidic chip detection system (a product of Beijing Boo classical biotechnology limited). The method comprises the following specific steps:
(1) the DNA of 24 randomly selected wheat samples (Table 3) including 10 lines of the RIL population (Zhongmai 175. times. Xiaoyan 60) and two parents thereof (Zhongmai 175. times. Xiaoyan 60), 10 lines of the RIL population (Xiaoyan 54. times. Jing 411) and two parents thereof (Xiaoyan 54. times. Jing 411) were extracted according to the SLS method slightly modified in the second step.
(2) And (3) configuring a PCR amplification reaction system, injecting the configured reaction system into the microfluidic chip from the chip inlet by using a liquid transfer device, and sealing the inlet and the outlet.
The PCR reaction system is shown in Table 1 below:
TABLE 1 PCR reaction System
Wherein the primer Mix consists of FAM-KASP-F1, HEX-KASP-F2 and KASP-R, and the final concentration of FAM-KASP-F1 is 0.6 mu M, HEX-KASP-F2 and the final concentration of 0.6 mu M, KASP-R is 1.5 mu M.
The chip was placed in a centrifuge at 4000rpm and centrifuged for 1 min. And placing the wafer into a chip heat sealing instrument for heat sealing for 1 sec. The plate was placed on a PCR plate for amplification reaction, and the PCR amplification program is shown in Table 2:
TABLE 2 PCR amplification procedure
(3) Scanning the reaction product by a LuxScan-10K/D scanner to generate a tif file, converting the tif file into a data signal value through software, and typing through a typing software SNPTyper. The genotype showing blue fluorescence was AA, and the genotype showing red fluorescence was GG.
As a result, as shown in FIG. 2, the cultivar with the SNP marker AX-108839508 genotype of GG was located at the upper left, and the cultivar with the SNP marker AX-108839508 genotype of AA was located at the lower right; the marker can distinguish different genotypes of the SNP marker AX-108839508. Through detection, the KASP marker typing result (Table 3) is consistent with the genotype result obtained by the chip, and the developed KASP marker can realize successful typing of the SNP marker AX-108839508.
TABLE 3 typing results of KASP marker amplified in 24 wheat samples
Note: the document 1 is: molecular detection of trait genes related to quality of Guilingi et al, 2012, Elytrigia elongata No. 6 and its derived progeny;
the document 2 is: chinese wheat, namely Sunzouzhong, 2010, identification of germination resistance of wheat main-pushed varieties and evaluation of related molecular markers;
the document 3 is: engineering peak, etc., 2009, little elytrigia 54 and Jing 411 and their filial generation stabilize and optimize the dynamic change of the photosynthetic property of the strain;
The above materials can be obtained by the public from the institute of genetics and developmental biology of the Chinese academy of sciences to repeat the experiment of the present application, and cannot be used for other purposes.
Example 2 validation of the SNP marker AX-108839508 in the doubled haploid population
In 2017 and 2018, under the salt stress condition, the yield and biomass traits of 150 strains of a DH population (dry selection No. 10 multiplied by Lumai 14) and two parents (namely, dry selection No. 10 and Lumai 14) thereof are identified. The field test was carried out at southern skin ecological agriculture testing station (116 ° 40'E, 38 ° 00' N, elevation 11m) of the chinese academy of sciences, setting 1 salinity gradient with a soil salinity of 0.3% (m/m). The experiment adopts a random block design, the row spacing is 20cm, and the plant spacing is 10 cm. There were 10 replicates, with only 1 strain per material in each replicate. After seedling emergence, irrigation is avoided in the growth period, field conventional management is realized, and serious plant diseases and insect pests and lodging are avoided in the growth period. The entire harvest was repeated 10 times. Weighing the overground part of each individual plant to obtain the biomass of the individual plant, and weighing the total grain weight of each individual plant after threshing the individual plant to obtain the yield of the individual plant.
DNA of 150 strains and their parents were extracted according to the SLS method slightly modified in example 1, and 152 DNA samples were SNP-typed according to the method in example 1 using the KASP marker which was successfully developed.
The KASP marker typing results are shown in fig. 3, fig. 4 and table 4: the genotypes of 81 strains (54.00%) were AA, the same as ruma 14; the genotypes of 65 strains (43.33%) are GG, which is the same as No. 10 dry election; 4 lines (2.67%) were deleted. According to the typing results, the yield (5.50 + -1.68 g (mean + -SD)) of the wheat strain with genotype GG of SNP marker AX-108839508 was found to be significantly greater than that of the strain with genotype AA (4.80 + -1.40 g (mean + -SD)) under the salt stress condition and the biomass (13.16 + -3.44 g (mean + -SD)) of the wheat strain with genotype GG was also significantly greater than that of the strain with genotype AA (11.80 + -2.85 g (mean + -SD)) under the salt stress condition in combination with the yield and biomass phenotype data analysis of the DH population (Dry election No. 10 × Roumai 14).
TABLE 4 genotype of SNP marker AX-108839508 with wheat Biomass and/or yield profiles
Note: "- -" indicates a deletion.
Combining the above results, the KASP marker successfully developed by the present invention was also significantly related to yield and biomass traits under salt stress conditions in DH population consisting of 150 lines (drought selection No. 10 × lugmai 14). This indicates that the excellent allelic sites identified in the natural population consisting of 191 triticum aestivum varieties for yield and biomass under salt stress conditions were equally applicable in the DH population (drought selection No. 10 × lugmai 14), and the experimental results were consistent with expectations. The KASP marker successfully developed in the invention can realize the auxiliary selection effect on the yield of wheat in saline-alkali soil.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120> KASP marker associated with wheat biomass and yield under salt stress conditions and use thereof
<130> GNCFY200073
<160> 4
<170> PatentIn version 3.5
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. The application of the substance for detecting the polymorphism or genotype of the SNP marker AX-108839508 in identification or auxiliary identification of wheat biomass and/or yield;
the SNP marker AX-108839508 is the 692158124 th nucleotide located on the wheat 5A chromosome, which is A or G.
2. The application of the substance for detecting the polymorphism or genotype of the SNP marker AX-108839508 in wheat breeding;
the SNP marker AX-108839508 is the 692158124 th nucleotide located on the wheat 5A chromosome, which is A or G.
3. The use according to claim 1 or 2, wherein the identification or assisted identification of wheat biomass and/or yield is identification or assisted identification of wheat biomass and/or yield under salt stress conditions;
the wheat breeding is to cultivate salt-tolerant wheat varieties with high biomass and/or yield.
4. Use according to any one of claims 1 to 3, characterized in that:
the substance for detecting the polymorphism or genotype of the SNP marker AX-108839508 is A1) or A2) or A3):
A1) the primer set comprises a single-stranded DNA molecule shown in SEQ ID NO.2 or a derivative thereof, a single-stranded DNA molecule shown in SEQ ID NO.3 or a derivative thereof and a single-stranded DNA molecule shown in SEQ ID NO. 4;
A2) PCR reagents containing the primer sets;
A3) a kit comprising a1) or a 2).
5. Use according to claim 4, characterized in that:
the derivative of the single-stranded DNA molecule shown in SEQ ID NO.2 is obtained by connecting the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.2 with a fluorescent label;
the derivative of the single-stranded DNA molecule shown in SEQ ID NO.3 is obtained by connecting the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.3 with another fluorescent label.
6. A product for detecting the polymorphism or genotype of SNP marker AX-108839508 for use according to claims 1 to 5;
the product has at least one function of 1) or 2) or 3) or 4) or 5) or 6) as follows:
1) identifying or aiding in identifying biomass and/or yield of wheat;
2) identifying or assisting in identifying biomass and/or yield of wheat under salt stress conditions;
3) breeding of high biomass and/or yield wheat;
4) breeding high biomass and/or yield salt-tolerant wheat;
5) breeding wheat with high biomass and/or yield;
6) and (3) breeding salt-tolerant wheat with high biomass and/or yield.
7. A method for identifying or aiding in identifying wheat biomass and/or yield under salt stress conditions, comprising: the method comprises the steps of detecting the genotype of the SNP marker AX-108839508 of wheat to be detected, and determining the biomass and/or the yield of the wheat to be detected according to the genotype.
8. A breeding method of salt-tolerant wheat comprises the following steps: detecting the genotype of the SNP marker AX-108839508 according to any one of claims 1 to 4 in a genome of a test wheat, and selecting the wheat with the genotype of GG as the genotype of the SNP marker AX-108839508 in the genome of the test wheat for breeding.
9. The method of claim 8, wherein:
the method for detecting whether the genotype of the wheat SNP marker AX-108839508 to be detected is GG or AA is A) or B) as follows:
A) detecting the genotype of a wheat SNP marker AX-108839508 through an SNP chip;
B) carrying out PCR reaction on the wheat genome DNA to be tested by using the substance for detecting the genotype of the SNP marker AX-108839508 according to any one of claims 1 to 4 to obtain a product, and carrying out genotyping on the product.
10. The method of claim 9, wherein:
the genotyping method comprises detecting the fluorescent signal of the product, determining the genotype according to the fluorescent signal: if the product only shows the color of a fluorescent label connected with the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.2, the genotype of the wheat SNP marker AX-108839508 to be detected is AA; and if the product only shows the color of the fluorescent label connected with the 5' end of the single-stranded DNA molecule shown in SEQ ID NO.3, the genotype of the wheat SNP marker AX-108839508 to be detected is GG.
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