CN109468406B - KASP marker related to wheat seedling stage root system configuration and application thereof - Google Patents
KASP marker related to wheat seedling stage root system configuration and application thereof Download PDFInfo
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Abstract
本发明涉及一个与小麦苗期根系构型相关的SNP位点，该位点位于小麦4D染色体16.64Mb位置处的基因的第36位。本发明还基于该SNP位点开发了KASP标记专用引物、包含该KASP标记引物的试剂盒以及利用该KASP标记引物鉴定或辅助鉴定小麦根系性状的方法。本发明的KASP标记专用引物可用于鉴定待测小麦的基因型，根据待测小麦的基因型可确定AA基因型的待测小麦的根系性状优于AG或GG基因型的待测小麦，进而用于筛选苗期根系性状优良的小麦品种。The present invention relates to a SNP site related to the root system configuration of wheat seedling stage, which is located at the 36th position of the gene at the 16.64Mb position of wheat 4D chromosome. Based on the SNP site, the present invention also develops a KASP marker-specific primer, a kit comprising the KASP marker primer, and a method for identifying or assisting in identifying wheat root traits using the KASP marker primer. The KASP marker special primer of the present invention can be used to identify the genotype of the wheat to be tested. According to the genotype of the wheat to be tested, it can be determined that the root character of the wheat to be tested of the AA genotype is better than that of the wheat to be tested of the AG or GG genotype. To screen wheat varieties with excellent root traits at seedling stage.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a KASP (KaSP) labeled primer related to wheat seedling stage root system configuration, a kit containing the KASP labeled primer and a method for identifying or assisting in identifying wheat root system characters by using the KASP labeled primer.
Background
The root system is used for supporting and fixing important organs of the overground part of the plant and is also a main place for synthesizing various hormones and amino acid substances, the excellent root system configuration and the rhizosphere process provide important guarantee for the efficient utilization of plant nutrients, and play a key supporting role in promoting the growth of crops and the formation of yield (Kaushik et al, 2017). The development of the root system in the early growth stage of the plant is extremely sensitive to the application of nutrients, and the utilization of a genetic breeding method to master the root system which is adaptive to the growth and development of the overground part and has lasting, stable and active activity becomes an important link of the modern molecular breeding technology. The wheat root system configuration not only lays the foundation of strong and weak plant growth potential, but also is closely related to the drought resistance and dry hot wind resistance of the wheat root system configuration. In combination with frequent extreme climate and scarce resources in the world, the cultivation of wheat varieties with high, stable and stress-resistant yield becomes a research hotspot of breeders. Aiming at the difference of the root system characteristics of wheat with different genotypes, a genetic regulation and control mechanism for root system growth and development in a seedling stage is excavated, a scientific basis can be provided for correctly selecting root system indexes in the breeding process of drought-resistant water-saving wheat varieties, and the method has important significance for high-yield and high-efficiency cultivation of wheat in dry land and full utilization of soil resources (Baker and the like, 2015). Because the roots grow underground, the sampling research on the roots has a larger bottleneck, and the labor intensity can be reduced, the breeding efficiency can be improved and the practical significance is provided for the high yield and variety improvement of the wheat by digging related molecular markers of the root system structure and combining the breeding work (Zhang Weijun et al, 2018).
The molecular marker assisted selection technology can track the genes of the transferred or polymerized root system, and provides an effective technical means for cultivating the wheat variety with excellent root system and high yield and wide adaptability. The development of genes for controlling root system characters and gene markers thereof is a precondition for developing molecular marker-assisted breeding, and the research on rice and corn is relatively extensive at present. Yusaku et al (2013) proved that DRO1 is located on chromosome 9 through research on a rice deep root gene DRO1(DEEPER ROOTING 1), and the enhanced expression of the DRO1 can reduce the included angle of a root system and increase the downward growth tendency of the root system. Research shows that DRO1 is an early auxin response gene, plays a negative regulation role through auxin, participates in the elongation of root tip cells, and is probably directly influenced by ARFs (auxin signaling pathway). QTL carried on chromosome 9 of rice for controlling root depth correlation is beneficial to improving drought resistance of plants and enhancing the absorption of the plants to root system nutrient elements, and the corresponding markers of the genetic sections RM242-RM201 of the gene are applied to molecular assisted breeding, and strains carrying the gene and having excellent root system characters are obtained (Steele et al, 2006). In addition, a major QTL associated with corn yield and root system has been explored and applied (Landi et al, 2010). The wheat genome structure is more complex than that of rice and corn, root system characters such as root length, surface area, volume, dry matter weight and the like are controlled by multiple genes and are also influenced by a plant height gene Rht to a certain extent, and the root length, the root surface area and the root volume of the wheat in a seedling stage are obviously reduced by dwarf genes Rht-B1c, Rht-D1c and Rht12 related to plant height; part of the root trait QTL and the thousand seed weight QTL in the seedling stage are in the same marking interval, which shows that the thousand seed weight is closely related to the root system in the seedling stage (Bai et al, 2013; Lopes et al, 2010). Most root trait genes are related to genes related to fertilizer water utilization, agronomic traits and yield traits (Bai et al, 2013), but there are few reports of closely linked markers. CAO and the like (2014) find that qTaLRO-B1 is a Quantitative Trait Locus (QTL) influencing the root length and related traits of common wheat by researching root systems of filial generations F1 and F2 of wheat varieties with two different root systems of 178A (short root system) and 178B (long root system).
Therefore, the field needs to develop a linkage marker related to the root system configuration of wheat in the seedling stage, so that the marker is used for screening wheat varieties with excellent root system characters in the seedling stage, theoretical basis is laid for breeding wheat varieties with high and stable yield and excellent quality, and a molecular auxiliary selection means is provided.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an SNP locus related to the wheat seedling stage root system configuration, and develops a KASP marker special primer based on the SNP locus, wherein the KASP marker primer can be used for identification or auxiliary identification of the wheat root system character, so that a wheat variety with excellent seedling stage root system character can be screened.
In a first aspect, the present invention provides a KASP marker primer set (hereinafter sometimes simply referred to as "KASP marker primer set of the present invention") which can be used for detecting the genotype of the 36 th deoxyribonucleotide of a gene at the 16.64Mb position of wheat 4D chromosome (the nucleotide sequence of which is shown as sequence 4 in the sequence listing). The KASP labeled primer group comprises at least two primers of an upstream primer F1, an upstream primer F2 and a downstream primer R, wherein the sequence of the upstream primer F1 is shown as a sequence 1; the upstream primer F2 is shown as a sequence 2; the sequence of the downstream primer R is shown as the sequence 3.
In one embodiment, the upstream primer F1 comprises a FAM fluorescent sequence and a single-stranded DNA at positions 22-39 which are arranged in sequence from 5 'end to 3' end, wherein the FAM fluorescent sequence is a single-stranded DNA shown at positions 1-21 in a sequence 1, and the nucleotide sequence of the FAM fluorescent sequence is shown as a sequence 9; the upstream primer F2 comprises a HEX fluorescent sequence and single-stranded DNA of 22 th to 39 th positions which are sequentially arranged from 5 'end to 3' end, the HEX fluorescent sequence is the single-stranded DNA shown in 1 st to 21 th positions in the sequence 2, and the nucleotide sequence is shown as the sequence 10.
In one embodiment, the KASP marker primer set of the present invention may comprise only two primers, primer F1 and primer R, which are used for amplifying or detecting the AA genotype of the 36 th DNA of the gene shown in sequence No. 4.
In another embodiment, the primer set of the present invention may comprise only two primers, primer F2 and primer R, which are used for amplifying or detecting a fragment of DNA at position 36 of a gene represented by sequence 4, the genotype of which is GG.
In a second aspect, the present invention provides a kit (hereinafter sometimes simply referred to as "kit of the present invention") comprising the KASP-labeled primer set of the present invention. The kit can be used for identifying or assisting in identifying the root system character of wheat to be detected.
In a preferred embodiment, the kit of the present invention further comprises necessary reagents required for completing PCR amplification, such as a fluorescent probe, a quenching probe, Taq enzyme and dNTP.
In a third aspect, the invention provides a DNA fragment, which is located at the 16.64Mb position of wheat 4D chromosome and has the nucleotide sequence shown as sequence 4.
In a fourth aspect, the present invention provides a SNP site (hereinafter sometimes simply referred to as "SNP site of the present invention") associated with the root architecture of wheat at the seedling stage, at position 36 of the gene at position 16.64Mb on the 4D chromosome (the nucleotide sequence of which is shown as sequence 4) (the relative positions of the gene at position 16.64Mb on the 4D chromosome and the SNP site of the present invention can also be referred to in FIG. 1). In the present invention, this SNP site is designated AX-109816583.
In one embodiment, the genotype of the SNP site is AA, AG, or GG, wherein the AA genotype is a homozygote of a at position 36 of the gene at position 16.64Mb of the 4D chromosome; the AG genotype is a hybrid of A and G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the GG genotype is a homozygote of G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome.
In a fifth aspect, the present invention provides a method for identifying or assisting in identifying a wheat root trait (hereinafter sometimes simply referred to as "the identifying method of the present invention"), the method comprising the steps of:
1) detecting which of AA, GG and AG the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome of the wheat to be detected is; and
2) determining the root system character of the wheat to be detected with the AA genotype better than that of the wheat to be detected with the GG or AG genotype according to the genotype of the wheat to be detected obtained in the step 1),
wherein the AA genotype is a homozygote of A at position 36 of the gene at position 16.64Mb of the 4D chromosome; the AG genotype is a hybrid of A and G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the GG genotype is a homozygote of G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
In a preferred embodiment, the wheat root trait is a wheat seedling root trait. In another preferred embodiment, the wheat root system trait is expressed as root-shoot ratio (RRS), i.e., the root-shoot ratio of the test wheat determined to have the AA genotype in step 2) is greater than that of the test wheat having the GG or AG genotype. Preferably, the wheat root system trait is the wheat seedling root-seedling ratio.
In the identification method of the present invention, the detection step of step 1) comprises:
a) using the genomic DNA of wheat to be detected as a template, performing PCR amplification by using the KASP marker primer group of the invention, performing fluorescence signal scanning on the obtained amplification product, and
b) and judging the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the wheat 4D chromosome to be detected is AA, GG and AG according to the fluorescence signal.
The KASP primer group adopted in the step a) consists of an upstream primer F1, an upstream primer F2 and a downstream primer R; wherein the sequence of the upstream primer F1 is shown as the sequence 1; the sequence of the upstream primer F2 is shown as the sequence 2; and the sequence of the downstream primer R is shown as a sequence 3.
In one embodiment, the upstream primer F1 comprises a FAM fluorescent sequence and a single-stranded DNA at positions 22-39 which are arranged in sequence from 5 'end to 3' end, wherein the FAM fluorescent sequence is the single-stranded DNA shown as positions 1-21 in the sequence 1, and the nucleotide sequence of the FAM fluorescent sequence is shown as the sequence 9; the upstream primer F2 comprises a HEX fluorescent sequence and single-stranded DNA of 22 th to 39 th positions which are sequentially arranged from 5 'end to 3' end, the HEX fluorescent sequence is the single-stranded DNA shown in 1 st to 21 st positions in the sequence 2, and the nucleotide sequence is shown as the sequence 10.
In the identification method of the present invention, the step of judging from the fluorescent signal may comprise using KlustercallerTMThe software judges the genotype according to the fluorescence signal: if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMThe genotype of the wheat to be detected is AA if the blue color is obtained by software analysis; if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMThe genotype of the wheat to be detected is GG if the red color is analyzed by software; if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMAnd if the software analysis shows green, the genotype of the wheat to be detected is AG.
In a specific embodiment of the identification method of the present invention, a PCR amplification system for PCR amplification may employ the following system (total volume of 5.2. mu.l): 20 ng/. mu.l template DNA 3.0. mu.l, 2 XKASP reaction mix 2.0. mu.l, primer mix reagent 0.1. mu.l, ddH2O0.1 μ l, wherein the 2 xKASP reaction mix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B, high fidelity Taq enzyme, dNTP and the like. The sequence of the fluorescent probe A is 5'-GAAGGTGACCAAGTTCATGCT-3' (sequence 5), and 1 fluorophore FAM is connected to the 5 ' end; the sequence of the fluorescent probe B is 5' -GAAGGTCGGAGTCAACGGATT-3 '(SEQ ID NO: 6), wherein 1 fluorescent group HEX is connected to the 5' terminal; the sequence of the quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3' (sequence 7), and the 3 ' terminal is connected with a quenching group BHQ; the sequence of the quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3' (sequence 8), and a quenching group BHQ is connected to the 3 ' terminal. The primer mixture reagent comprises a primer F1, a primer F2 and a primer R. In a preferred embodiment, the volume ratio of the primer F1 to the primer F2 in the PCR amplification system is 1:1, for example, the final concentration of both in the PCR amplification system is 0.134. mu.M, and the final concentration of the primer R in the PCR amplification system is 0.336. mu.M.
In a preferred embodiment, the PCR amplification may employ the Touch down PCR amplification procedure, as follows: pre-denaturation at 94 ℃ for 15 min; (Touch down program) denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 60s, extension at 72 ℃ for 30s, 11 cycles, and annealing temperature reduction of 0.6 ℃ per cycle; (amplification procedure) denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 30s, 26 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
In a preferred embodiment, the PCR amplification reaction can be performed on a PCR amplification apparatus commonly used in the art (e.g., a PTC-200PCR amplification apparatus) to obtain PCR amplification products, and then the PCR amplification products are subjected to genotyping by fluorescent irradiation on a fluorescence microplate reader commonly used in the art (e.g., a PHERAStarplus fluorescence microplate reader), and then fluorescence genotyping software commonly used in the art (e.g., Klustercalenler)TMSoftware) reads the typed data.
In a sixth aspect, the present invention provides use of the SNP site of the invention in any one of (a1) - (a6) below:
(a1) identifying or assisting in identifying the root system characters of the wheat to be detected;
(a2) preparing a product for identifying or assisting in identifying the root system character of the wheat to be detected;
(a3) breeding a wheat variety with excellent root system character;
(a4) preparing a product for breeding a wheat variety with excellent root system characters;
(a5) breeding wheat; and
(a6) preparing a wheat breeding product.
In a seventh aspect, the invention provides a method for breeding wheat with excellent root system characters, which comprises the steps of selecting a wheat variety with an AA genotype for breeding to obtain target wheat; the AA genotype is a homozygote of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the wheat 4D chromosome, namely A; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
In the present invention, the root system characteristics of wheat are expressed in terms of a root-shoot ratio, and therefore, wheat having excellent root system characteristics refers to a wheat variety having a relatively large root-shoot ratio. Preferably, the root trait is a seedling stage root trait. Therefore, preferably, the wheat root system character refers to the wheat seedling stage root-seedling ratio.
In the present invention, wheat includes, but is not limited to, any one or any several of the following varieties: afu, CA1055, CA1133, alternate 987, jindong No. 8, qinong 731, wan wheat 52, jining 16, jimai 21, lumai 15, lumai 21, chuan nong 52, mianyang 26, ningmai No. 9, zhenmi No. 6, and the like.
The invention has the advantages of
The invention provides an SNP locus related to the wheat seedling root system configuration, and develops a special primer for KASP marker based on the SNP locus. The KASP special-purpose primer can be used for identifying whether the genotype of the wheat to be detected is AA, GG or AG, can determine the root system character (root-seedling ratio) of the wheat to be detected with the AA genotype larger than the genotype of AG or GG according to the genotype of the wheat to be detected, and is further used for screening the wheat variety with excellent root system character (root-seedling ratio) in the seedling stage, thereby laying a theoretical foundation for breeding the wheat variety with high yield, stable yield and excellent quality and providing a molecular auxiliary selection means.
Drawings
FIG. 1 shows the nucleotide sequence of the gene at position 16.64Mb of wheat 4D chromosome and the position of the SNP site of the present invention, shown in FIG. 1 as being located at position 36 deoxyribonucleotide (see underlining, A/G in parentheses indicates that A may be mutated to G at this site).
Fig. 2 illustrates the seedling root phenotype of yangmai 16 and midgmai 895 at high nitrogen concentration.
Fig. 3 illustrates the linkage diagram of the SNP markers of the invention with the qrrs.
FIG. 4 is a graph showing the results of KASP marker detection of the root shoot ratio gene of the wheat variety tested.
Detailed Description
Defining:
KASP：KASPTMthe genotyping technology is a unique competitive allele-specific PCR, and can perform high-precision double-allele genotyping on various genome DNA samples including complex genomes aiming at SNP and InDel of re-sequencing data or other data, or KASP can perform large-population verification work on candidate markers obtained through trait localization. The KASP technique is based on specific matching of the terminal bases of primers to type SNPs and detect InDels. Due to the high flexibility of SNpline, the application of SNpline is very wide, and the SNpline is suitable for various genotyping researches, and is applicable to low-throughput research and development projects, SNP verification after NGS, agricultural population research and the like. The conventional method has high cost for identifying the variety or evaluating the seed quality and long identification period, while the KASP technology can effectively avoid the two defects, so that the breeding cost is greatly reduced, and the identification period is obviously shortened, therefore, the KASP technology is a preferred technology for identifying the variety and controlling the seed quality in molecular breeding work.
The following examples are intended to illustrate the invention, but are not intended to limit the scope of 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. In the quantitative tests in the following examples, three replicates were set up and the results averaged. All primers in the following examples were synthesized by Olympic Biotechnology, Inc. of Beijing. All wheat material in the examples below was from the national center for crop germplasm preservation, academy of agricultural sciences, china.
Example 1 obtaining of primers specific for KASP marker associated with root architecture of wheat at seedling stage
Root system character investigation and SNP marker analysis
1. Root system character investigation
Yangmai 16 (China agriculture development group, Inc., variety number: CNA 20030436.4)/Zhongmai 895 (Henan New big agriculture development Co., Ltd., national scrutiny wheat 2012010) DH groups including 200 families in total are selected as experimental materials to carry out root tests, and root characters are investigated. (Note: Zhongmai 895 is a semiwinterness multi-spike middle and late maturing variety bred by hybridization of Zhongmai 16 as a female parent and litchi No. 4 as a male parent, which is a crop scientific institute of Chinese academy of agricultural sciences, and cotton research of Chinese academy of agricultural sciences, and has the characteristics of long leaf function period, strong tillering capability, high grouting speed and the like.9 th of 2009 is approved by a southern leaf of the Huang-Huai wheat area.in a variety comparison test and a field demonstration in 2013 and 2015 three years, Zhongmai 895 has the characteristics of high yield, wide adaptability, strong root activity, high temperature resistance in the late grouting period and the like.Yangma 16 is a variety with the largest planting area in the middle and downstream wheat areas of Yangtze river and has the characteristics of high grouting speed, high grain weight and the like.)
The root test adopts a water culture method, namely 30 seeds are selected from each experimental material. The method comprises the following specific steps: with 10% H2O2Soaking the seeds for 15-20 minutes, and washing with sterile water for 5-6 times; then, selecting plump seeds with consistent size from the treated seeds, placing the seeds in a culture dish paved with filter paper, and accelerating germination in a dark room of an incubator; seedlings with consistent germination were selected for each experimental material and cultured, the plates were placed in nutrient solution for culturing in a controlled greenhouse, and the nutrient solution was prepared according to the method described in the reference (Ren et al, 2012). The root traits at seedling stage were investigated after 10 days of continuous culture (FIG. 2 shows the root phenotype at seedling stage of Yangmai 16 and Miao 895 at high nitrogen concentration). Survey traits included total root length, total root tip number, and dry root weight.
2. SNP marker analysis
SNP (Single nucleotide polymorphism) markers in Boo biological Limited (CapitalBio Corporation, Beijing, China; http:// bioservices. CapitalBio. com) Using Illumina SNP genotyping assays, the major steps were as follows: 1) carrying out whole genome amplification on the genome DNA of the experimental wheat material to be detected; 2) cutting the amplified product by using random endonuclease; 3) hybridizing the DNA fragment with a chip, connecting a 50-mers length specific capture probe on a microbead of the chip, and combining a product obtained after digestion of gDNA with a probe complementary sequence; 4) washing to remove DNA fragments which are not hybridized or mismatched and hybridized; 5) carrying out single-base extension on dinitrophenol (dinitrophenol) and biotin (biotin) -labeled nucleotide substrates (A/T and C/G) on a capture probe, wherein only a probe which is complementarily combined with gDNA can be extended; by staining, A/T and C/G will mark different fluorochromes respectively; 6) and scanning the chip, interpreting and outputting a typing result according to the two types of fluorescence by using software.
Carrying out 660k SNP chip typing on Yangmai 16/Migmai 895DH groups by using an Illumina SNP genotyping research platform, wherein the 660k SNP chip typing comprises series markers such as BS, Bobwhite, CAP, D _ contig and the like, and the total number of the markers is 630518, wherein 626276 SNP markers have differences in the Yangmai 16/Migmai 895DH groups.
Second, the discovery of the associated Gene mapping and the linkage marker AX-109816583
Basic statistics and multiple comparison analysis are carried out by using SAS9.2 software (SAS institute.2000), stepwise regression is carried out on SNP data and root system characters by combining with a Glmselect program of SAS, and the associated sites are judged according to a P value (P < 0.01). AX-109816583 was located in association with the site qrrs. caas-4DS (P < 0.001).
Identification of allele-specific marker at position III, AX-109816583
The whole genome DNA of 12 Yangmai 16/Zhongmai 895DH groups was extracted. Allele-specific marker KASP of SNP marker AX-109816583 site using each genomic DNA as templateTMGenotyping assays revealed AA-typed fragments (fig. 4). The SNP marker AX-109816583 is shown to be capable of effectively identifying the root-seedling ratio of the wheat variety.
TABLE 1 isolation of AX-109816583 marker allelic variants in the Yangmai 16/Miyami 895DH population
The results show that: when the genotype of the AX-109816583 locus is only AA, the phenotype of the locus is consistent with that of the wheat variety with a larger root-shoot ratio, which shows that the AX-109816583 locus can effectively identify the root-shoot ratio gene and the genotype of the wheat variety.
The marker AX-109816583 was integrated into the Wheat genetic map according to the Wheat molecular marker map published by Wheat DArT maps Version 1.2(http:// www.triticarte.com.au) and Allen et al (2011), and the results are shown in FIG. 3. The 16.64Mb position of qrrs. caas-4DS on chromosome 4D (also known as the root-shoot ratio gene) was determined.
Development of primers special for KASP marker
The primer special for KASP marker developed by the invention aiming at SNP site (36 th site in sequence 4) consists of 2 upstream primers, namely primer F1, primer F2 and 1 downstream primer, namely primer R. The sequence of the primer special for KASP marking is as follows:
primer F1:GAAGGTGACCAAGTTCATGCTGCAGGAATTCCATCTACTCCATGAA (SEQ ID NO: 1);
primer F2:GAAGGTCGGAGTCAACGGATTGCAGGAATTCCATCTACTCCATGAG (SEQ ID NO: 2);
and (3) primer R: TGGCGAGCCTTTTACTAAGCA (SEQ ID NO: 3).
Among them, underlined sequences in the primer F1 shown in the sequence 1 are FAM fluorescent sequences (5'-GAAGGTGACCAAGTTCATGCT-3', sequence 9), and underlined sequences in the primer F2 shown in the sequence 2 are HEX fluorescent sequences (5'-GAAGGTCGGAGTCAACGGATT-3', sequence 10). The combination of the primer F1 and the primer R can amplify a fragment with the SNP locus genotype of AA, and the combination of the primer F2 and the primer R can amplify a fragment with the SNP locus genotype of GG.
Example 2 application of SNP
Offspring varieties of 15 non-Yangmai 16/Zhongmai 895DH populations, parent Zhongmai 895 and Yangmai 16 are selected. Wherein 15 offspring varieties of non-Yangmai 16/Zhongmai 895DH populations are used as a test group, and Zhongmai 895 and Yangmai 16 are used as a control group; comprises Aff, CA1055, CA1133, alternate 987, Jingdong No. 8, Qinong 731, Wanmai 52, Jining 16, Jimai 21, Lumai 15, Lumai 21, Chuannong 52, Mianyang 26, Ningmai No. 9 and Zhenmai No. 6.
Detecting root system characters of wheat of different varieties in seedling stage
The root system character of the wheat variety in the seedling stage is identified in 2016 in the greenhouse of the institute of crop science of the academy of agricultural sciences of China. Selecting 30 full seeds with the same size from each variety, and using 10% of H2O2Treating for 20-30 min, and washing with sterile water for 5-6 times; then placing the seeds in a culture dish paved with filter paper, and accelerating germination in a dark room in an incubator for 18-24 hours; then 25 seeds with consistent germination are selected and placed on a seedling growing net, 10 wheat seedlings with consistent size are selected and transferred to culture plates after 6 days of growth, and each culture plate is repeated for 3 times; then the culture dish is placed in nutrient solution to be cultured in a controllable greenhouse. Nutrient solution preparation reference literature (Ren Y, He X, Liu D, Li J, ZHao X, Li B, Tong Y, Zhang A, Li Z. major qualitative trail location for a minor root morphology of wheat seeds, 2012,30: 139) -148). The seedling culture conditions are 22 +/-1 ℃ and 50-60% of relative humidity. The nutrient solution is changed every 3 days, and the roots in the seedling stage are harvested after continuous culture for 10 days. The root systems of the harvested varieties were scanned by a scanner, and then analyzed by image analysis software Win RHIZO (Regent Instruments, canada) for the following root traits: total root length, root surface area, total root tip number, dry root weight, and the like. Secondly, detecting the genotype of SNP loci of different varieties of wheat
Respectively extracting the genome DNA of each variety of wheat, and carrying out PCR amplification by using the genome DNA as a template and adopting a KASP labeled special primer to obtain a PCR amplification product. Wherein, the PCR amplification product carrying the fluorescence sequence FAM shows blue after fluorescent irradiation, and the PCR amplification product carrying the fluorescence sequence HEX shows red after fluorescent irradiation.
The PCR amplification system was as follows (total volume 5.2. mu.l): 20 ng/. mu.l template DNA 3.0. mu.l, 2 XKASP reaction mix 2.0. mu.l, primer mix reagent (Assay mix) 0.1. mu.l, ddH2O0.1. mu.l. The 2 xKASP reaction mix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B, as well as high-fidelity Taq enzyme, dNTP and the like. The sequence of the fluorescent probe A is5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO: 5), wherein 1 fluorophore FAM is linked to the 5 ' end; the sequence of the fluorescent probe B is 5'-GAAGGTCGGAGTCAACGGATT-3' (sequence 6), and 1 fluorophore HEX is connected to the 5 ' end; the sequence of the quenching probe A is 5'-AGCATGAACTTGGTCACCTTC-3' (sequence 7), and the 3 ' terminal is connected with a quenching group BHQ; the sequence of the quenching probe B is 5'-AATCCGTTGACTCCGACCTTC-3' (sequence 8), and a quenching group BHQ is connected to the 3 ' terminal. The primer mixed reagent comprises a primer F1, a primer F2 and a primer R, wherein the final concentration of the primer F1 and the final concentration of the primer F2 in a PCR amplification system are both 0.134 mu M, and the final concentration of the primer R in the PCR amplification system is 0.336 mu M.
The PCR amplification reaction is carried out on a PTC-200PCR amplification instrument, and the Touch down PCR amplification program is as follows: pre-denaturation at 94 ℃ for 15 min; (Touch down program) denaturation at 94 ℃ for 30s, annealing at 61 ℃ for 60s, extension at 72 ℃ for 30s, 11 cycles, and annealing temperature reduction of 0.6 ℃ per cycle; (amplification procedure) denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 60s, extension at 72 ℃ for 30s, 26 cycles; extending for 5min at 72 ℃; storing at 10 deg.C.
Placing the PCR amplification product in PHERAStarplusGenotyping with fluorescent irradiation on a fluorescent microplate reader, followed by KlustercallerTMReading the data after typing by software, and only displaying a blue image to show that the 36 th SNP site base of the gene (sequence 4) at the 16.64Mb position of the 4D chromosome of the wheat to be tested is A, and the genotype of the wheat is AA; only red images are displayed, which indicates that the 36 th SNP site base of the gene (sequence 4) at the 16.64Mb position of the 4D chromosome of the wheat to be detected is G, and the genotype of the wheat is GG; and displaying a green image, which indicates that the 36 th SNP site base of the gene (sequence 4) at the 16.64Mb position of the 4D chromosome of the wheat to be detected is A and G, and the genotype of the wheat is AG.
And comparing the PCR amplification products of the wheat varieties pairwise, wherein if the PCR amplification products of the wheat A only show blue color (the 36 th deoxyribonucleotide of the sequence 4 is a homozygote of A, and the genotype is AA), and the PCR amplification products of the wheat B only show red color (the 36 th deoxyribonucleotide of the sequence 4 is a homozygote of G, and the genotype is GG) or green color (the 36 th deoxyribonucleotide of the sequence 4 is a heterozygote of A and G, and the genotype is AG), the root system character (root-seedling ratio) of the wheat A is superior to that of the wheat B.
The genotype and the root system characteristics (root-seedling ratio) of each wheat at the seedling stage are shown in Table 2.
TABLE 2, 15 results of genotype and root-shoot ratio of SNP sites of wheat varieties
Note: chinese rye 895 is AA; yangmai 16 is GG.
The above results show that: the genotypes of the Anhui wheat 52, the Ji wheat 21, the Lu wheat 15, the Luo wheat 21, the Chuannong 52 and the Mianyang 26 at the SNP sites are all AA, and the wheat has a larger root-seedling ratio. From the above results, it can also be seen that the root-shoot ratio of wheat with the genotype of the SNP site AA is greater than that of wheat with the genotype of GG.
The results show that the SNP locus can rapidly and accurately identify whether the wheat variety has a larger root-seedling ratio.
The present invention includes the following embodiments:
1. a KASP marker primer set, wherein the KASP marker primer set comprises at least two primers of an upstream primer F1, an upstream primer F2 and a downstream primer R, wherein the sequence of the upstream primer F1 is shown as sequence 1; the sequence of the upstream primer F2 is shown as the sequence 2; and the sequence of the downstream primer R is shown as a sequence 3.
2. The KASP-labeled primer set according to embodiment 1, wherein the forward primer F1 comprises a FAM fluorescent sequence and a single-stranded DNA at positions 22 to 39 arranged in this order from the 5 'end to the 3' end, the FAM fluorescent sequence being a single-stranded DNA represented by positions 1 to 21 in sequence 1, and a nucleotide sequence thereof being represented by sequence 9; the upstream primer F2 comprises a HEX fluorescent sequence and single-stranded DNA of 22 th to 39 th positions which are sequentially arranged from 5 'end to 3' end, the HEX fluorescent sequence is the single-stranded DNA shown in 1 st to 21 th positions in the sequence 2, and the nucleotide sequence is shown as the sequence 10.
3. The KASP labeled primer set of embodiment 1 or 2, wherein the KASP labeled primer set comprises only two primers, primer F1 and primer R.
4. The KASP labeled primer set of embodiment 1 or 2, wherein the KASP labeled primer set comprises only two primers, primer F2 and primer R.
5. A kit for identifying or assisting in identifying a wheat root trait to be tested, wherein the kit comprises a KASP marker primer set of any one of embodiments 1-4.
6. The kit of embodiment 5, wherein the kit further comprises a fluorescent probe, a quencher probe, Taq enzyme, and dntps.
7. A DNA fragment, wherein the DNA fragment is located at the 16.64Mb position of the wheat 4D chromosome, and the nucleotide sequence of the DNA fragment is shown as a sequence 4.
8. An SNP locus related to the wheat seedling root system configuration, wherein the SNP locus is positioned at the 36 th position of a gene at the 16.64Mb position of a wheat 4D chromosome, and the nucleotide sequence of the gene is shown as a sequence 4.
9. The SNP site of embodiment 8, wherein the genotype of the SNP site is AA, AG, or GG, wherein the AA genotype is a homozygote of A for the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the AG genotype is a hybrid of A and G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the GG genotype is a homozygote of G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome.
10. A method for identifying or assisting in identifying a wheat root trait, wherein the method comprises the steps of:
1) detecting which of AA, GG and AG the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome of the wheat to be detected is; and
2) determining the root system character of the wheat to be detected with the AA genotype better than that of the wheat to be detected with the GG or AG genotype according to the genotype of the wheat to be detected obtained in the step 1),
wherein the AA genotype is a homozygote of A at position 36 of the gene at position 16.64Mb of the 4D chromosome; the AG genotype is a hybrid of A and G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the GG genotype is a homozygote of G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
11. The method of embodiment 10, wherein the root system trait is root-to-shoot ratio.
12. The method of embodiment 10, wherein the detecting step of step 1) comprises:
a) using the genomic DNA of wheat to be detected as a template, performing PCR amplification by using the KASP labeled primer group described in any one of embodiments 1 to 4, scanning the obtained amplification product with a fluorescent signal, and
b) judging which of AA, GG and AG the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome of the wheat to be detected is according to the fluorescence signal;
wherein the KASP marker primer group consists of an upstream primer F1, an upstream primer F2 and a downstream primer R; wherein the sequence of the upstream primer F1 is shown as the sequence 1; the sequence of the upstream primer F2 is shown as the sequence 2; and the sequence of the downstream primer R is shown as a sequence 3.
13. The method of embodiment 12, wherein the step of determining from the fluorescent signal comprises using KlustercallerTMThe software judges the genotype according to the fluorescence signal: if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMThe genotype of the wheat to be detected is AA if the blue color is analyzed by the software; if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMIf the software analyzes that the color is red, the genotype of the wheat to be detected is GG; if the fluorescence signal data of the amplification product of the wheat to be detected is processed by KlustercallerTMAnd if the software analysis shows green, the genotype of the wheat to be detected is AG.
14. The use of the SNP site of embodiment 8 or embodiment 9 in any one of (a1) - (a6) as follows:
(a1) identifying or assisting in identifying the root system characters of the wheat to be detected;
(a2) preparing a product for identifying or assisting in identifying the root system character of the wheat to be detected;
(a3) breeding a wheat variety with excellent root system character;
(a4) preparing a product for breeding a wheat variety with excellent root system characters;
(a5) breeding wheat; and
(a6) preparing a wheat breeding product.
15. A method for breeding wheat with excellent root system character includes selecting wheat variety with AA gene type to obtain target wheat; the AA genotype is a homozygote of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the wheat 4D chromosome, namely A; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
16. The kit of embodiment 5 or 6, the method of any one of embodiments 10-13, the use of embodiment 14, or the method of embodiment 15, wherein the root trait is a seedling stage root trait.
17. The kit of embodiment 5 or 6, the SNP site of embodiment 8 or 9, the method of any one of embodiments 10-13, the use of embodiment 14, the method of embodiment 15 or 16, wherein the wheat is selected from any one or any of the following varieties: afu, CA1055, CA1133, alternate 987, kyowan No. 8, qinong 731, wan wheat 52, jining 16, jimai 21, lumai 15, lomai 21, chuan nong 52, mianyang 26, ningmai No. 9 and zhenmi No. 6.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one 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.
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Claims (13)
1. A KASP marker primer group, which is characterized in that the KASP marker primer group comprises an upstream primer F1, an upstream primer F2 and a downstream primer R, wherein the sequence of the upstream primer F1 is shown as a sequence 1; the sequence of the upstream primer F2 is shown as the sequence 2; the sequence of the downstream primer R is shown as the sequence 3.
2. The KASP labeled primer set as defined in claim 1, wherein said forward primer F1 comprises a FAM fluorescent sequence and a single-stranded DNA at positions 22-39 arranged in sequence from 5 'end to 3' end, said FAM fluorescent sequence being a single-stranded DNA represented by positions 1-21 in SEQ ID NO. 1, the nucleotide sequence of which is represented by SEQ ID NO. 9; the upstream primer F2 comprises a HEX fluorescent sequence and single-stranded DNA of 22 th to 39 th positions which are sequentially arranged from 5 'end to 3' end, the HEX fluorescent sequence is the single-stranded DNA shown in 1 st to 21 th positions in the sequence 2, and the nucleotide sequence is shown as the sequence 10.
3. A kit for identifying or assisting in identifying a root trait of wheat to be detected, characterized in that the kit comprises the KASP marker primer set of claim 1 or 2.
4. A DNA fragment, which is characterized in that the DNA fragment is positioned at the 16.64Mb position of the wheat 4D chromosome, and the nucleotide sequence of the DNA fragment is shown as a sequence 4.
5. An SNP molecular marker related to the wheat seedling root system configuration, which is characterized in that the SNP molecular marker is positioned at the 36 th site of a gene at the 16.64Mb position of a wheat 4D chromosome, the nucleotide sequence of the gene is shown as a sequence 4, and the genotype of the SNP is AA, GG or AG.
6. A method for identifying or assisting in identifying wheat root system characters is characterized by comprising the following steps:
1) detecting which of AA, GG and AG the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome of the wheat to be detected is; and
2) determining the root system character of the wheat to be detected with the AA genotype better than that of the wheat to be detected with the GG or AG genotype according to the genotype of the wheat to be detected obtained in the step 1),
wherein the AA genotype is a homozygote of A at position 36 of the gene at position 16.64Mb of the 4D chromosome; the AG genotype is a hybrid of A and G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the GG genotype is a homozygote of G at the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
7. The method of claim 6, wherein the detecting step of step 1) comprises:
a) performing PCR amplification using the genomic DNA of wheat to be tested as a template and the KASP-labeled primer set of claim 3, scanning the obtained amplification product for a fluorescent signal, and
b) judging which of AA, GG and AG the genotype of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the 4D chromosome of the wheat to be detected is according to the fluorescence signal;
wherein the KASP marker primer group consists of an upstream primer F1, an upstream primer F2 and a downstream primer R; wherein the sequence of the upstream primer F1 is shown as the sequence 1; the upstream primer F2 is shown as a sequence 2; and the sequence of the downstream primer R is shown as a sequence 3.
8. The use of a KASP marker primer set according to claim 1 in any one of the following (a1) - (a 6):
(a1) identifying or assisting in identifying the root system characters of the wheat to be detected;
(a2) preparing a product for identifying or assisting in identifying the root system character of the wheat to be detected;
(a3) breeding a wheat variety with excellent root system character;
(a4) preparing a product for breeding a wheat variety with excellent root system characters;
(a5) breeding wheat; and
(a6) preparing a wheat breeding product.
9. A method for breeding wheat with excellent root system character is characterized in that the method comprises the steps of selecting wheat varieties with AA genotype for breeding to obtain target wheat; the AA genotype is a homozygote of the 36 th deoxyribonucleotide of the gene at the 16.64Mb position of the wheat 4D chromosome, namely A; the nucleotide sequence of the gene at the 16.64Mb position on the 4D chromosome is shown as sequence 4.
10. The kit of claim 3, wherein the root system trait is a seedling stage root system trait.
11. The method according to claim 6 or 7, characterized in that the root system trait is a seedling stage root system trait.
12. Use according to claim 8, characterized in that the root system trait is a seedling stage root system trait.
13. The method of claim 9, wherein the root system trait is a seedling stage root system trait.
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| CN107794308A (en) * | 2016-08-29 | 2018-03-13 | 中国农业科学院作物科学研究所 | Identify special SNP and its application of wheat seed character |
| CN108060262A (en) * | 2018-02-08 | 2018-05-22 | 中国农业科学院作物科学研究所 | KASP marks relevant with wheat root character and its application |
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| CN107619874A (en) * | 2017-09-06 | 2018-01-23 | 山东农业大学 | The anti-wheat ear germinating molecular labeling of one high flux and its application in breeding |
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