A molecular marker for identifying tiller number QTL LNT2 and its application

Abstract

The present invention discloses a molecular marker for identifying tiller number QTL LNT2 and its application, and relates to the technical field of molecular markers. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is a SNP site at the 26bp base of the nucleotide sequence, which is an A / G mutation. The present invention discloses for the first time a molecular marker KASP1765 for accurately detecting tiller number QTL LNT2 of barley CIHO 11530 based on a fluorescent quantitative PCR platform, with accurate and efficient detection, convenient and stable amplification. The molecular marker KASP1765 disclosed by the present invention is significantly correlated with tiller number QTL LNT2, has high accuracy in molecular marker-assisted selection, improves the selection and identification efficiency of tiller number varieties adapted to different environments, and has a high success rate.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and in particular to a molecular marker for identifying tiller number QTL LNT2 and an application thereof. Background Art

[0002] Barley (Hordeum vulgare L.) is an annual or biennial herb belonging to the Gramineae family and the Triticum tribe. Barley is one of the world's important cereal crops, planted all over the world, ranking fifth in global crop production. It is widely used in feed and food industries, and is also the main raw material for brewing beer. At the same time, research has shown that the β-glucan contained in barley grains is the most valuable health functional component of highland barley, and it has been confirmed that β-glucan has the effects of lowering blood lipids, lowering cholesterol and preventing cardiovascular diseases.

[0003] Tillering is one of the decisive factors affecting crop yield. The occurrence of tillering is a very complex process, which is regulated by internal genetic factors and external environmental conditions. The growth and development of tillering buds will directly affect the number of effective ears of crops and thus affect their unit yield. The location of genes related to tillering number can provide a theoretical basis for breeding high-yield barley varieties.

[0004] Tiller number is a quantitative trait (QTL), which is controlled by multiple genes and greatly affected by environmental effects. In recent years, the reasonable tiller number has gradually become a research hotspot. There are reports on the QTL study of tiller number in rice, corn and wheat, but there are few reports on the flag leaf of barley. Babb studied the uniculm2 (cul2.b) mutant and found that the cul2.b gene plays a role in the growth of axillary bud meristems to the formation of tillers, and located cul2.b between RFLP molecular markers ABG458 and KFP128 on chromosome 6H of barley, which is closely linked to the gene rob1. lnt1.a is a recessive single gene, which may regulate the initiation of axillary bud meristems like the rice gene MONOCULM1. lnt1.a was located 5.8cM away from the SSR molecular marker GBM1043, and sequence analysis proved that JuBel2 is the candidate gene of lnt1.a.

[0005] Molecular marker-assisted selection does not rely on phenotypic selection, that is, it is not affected by environmental conditions, gene interactions, genotype-environment interactions and other factors, but directly selects genotypes, thus greatly improving breeding efficiency. High Resolution Melting (HRM) technology is a SNP and mutation research tool that has emerged internationally in recent years. This detection method has the advantages of simple operation, good specificity, high throughput, rapidity, low detection cost, accurate results, and has achieved true closed-tube operation and has received widespread attention. Therefore, screening out molecular markers that are closely linked to the tiller number QTL and are suitable for the fluorescent quantitative PCR platform KASP technology can not only select barley tiller number genes, effectively regulate barley plant type, and shape a reasonable tiller number population, but also improve the selection flux, speed and accuracy, which is conducive to solving the technical bottleneck of large-scale promotion and application, and is of great significance to the large-scale improvement of barley breeding population quality and yield. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker for identifying the tiller number QTL LNT2 and its application, so as to solve the problems existing in the above-mentioned prior art. The molecular marker is significantly correlated with the barley tiller number QTL LNT2, has high accuracy in molecular marker-assisted selection, improves the selection and identification efficiency of barley tiller number varieties adapted to different environments, and has a high success rate.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a molecular marker KASP1765 for identifying tiller number QTL LNT2. The nucleotide sequence of the molecular marker KASP1765 is shown in SEQ ID NO.1. There is a SNP site at the 26 bp base of the nucleotide sequence, which is an A / G mutation.

[0009] The present invention also provides a KASP primer combination for identifying tiller number traits, comprising two forward primers and a universal reverse primer; the nucleotide sequences of the two forward primers are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively, and the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.4.

[0010] The present invention also provides the use of the above-mentioned KASP primer combination in preparing a product for identifying the trait of barley tiller number.

[0011] Furthermore, the product is a kit.

[0012] The invention also provides a product for identifying the trait of tiller number of barley, comprising the above-mentioned KASP primer combination.

[0013] Furthermore, the product is a kit.

[0014] The present invention also provides the use of the molecular marker KASP1765, KASP primer combination or product in identifying the tiller number trait of barley.

[0015] The present invention also provides a method for identifying the trait of barley tiller number, comprising the following steps:

[0016] Extracting the DNA of the barley to be tested;

[0017] The DNA is used as a template and the KASP primer combination is used to perform fluorescence quantitative PCR amplification to obtain the genotype of the barley to be tested. The tiller number trait of the barley to be tested is determined according to the genotype: the tiller number of the GG genotype barley is lower than that of the AA genotype barley.

[0018] Furthermore, the reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Master mix 5 μL, KASPAssay Mix 0.14 μL, template DNA 50 ng, and DNase / RNase-free deionized water supplemented to 10 μL.

[0019] Furthermore, the reaction procedure of the fluorescent quantitative PCR amplification is: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

[0020] The present invention discloses the following technical effects:

[0021] The present invention discloses for the first time the tiller number QTL LNT2 from barley CIHO 11530, which is located on the barley chromosome 6H and is significantly correlated with the barley tiller number trait. The QTL has a high utilization value in barley yield (regulating the tiller number trait) breeding. The present invention discloses for the first time the molecular marker KASP1765 for accurately detecting the tiller number QTL LNT2 of barley CIHO 11530 based on a fluorescent quantitative PCR platform, with accurate and efficient detection, convenient and stable amplification. The molecular marker KASP1765 disclosed by the present invention is significantly correlated with the tiller number QTL LNT2, has high accuracy in molecular marker-assisted selection, improves the selection and identification efficiency of barley tiller number varieties that adapt to different environments, and has a high success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The present invention shows the location of the tiller number QTL LNT2 on chromosome 6H and the linkage genetic map between the QTL and the molecular marker CIHO 11530;

[0024] Figure 2 The results of genotyping the leaf DNA of CIHO 11530, CIHO 1789 and Bowman at the three-leaf stage using fluorescent quantitative PCR primers in Example 2 of the present invention;

[0025] Figure 3 This is the result of genotyping the F2 population progeny of CIHO 11530×Bowman in Example 3 of the present invention using fluorescent quantitative PCR primers. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Obtaining the tiller number QTL LNT2 and molecular marker KASP1765

[0032] In the present invention, the tiller number QTL LNT2 and the molecular marker KASP1765 were obtained by the following method:

[0033] (1) Barley CIHO 11530 with few tillers was used as the female parent and barley CIHO 1789 as the male parent to obtain hybrid F1. The F1 generation plants were self-pollinated to obtain an F2 population containing 91 lines to form the genetic population.

[0034] (2) Identify the tiller number of the F2 population plants in the field during the barley maturity period.

[0035] (3) The DNA of each strain of the genetic population was extracted by the CTAB method, and genotyping was performed using the DArT chip technology with the DNA of the parents CIHO11530 and CIHO 1789 as templates to obtain the genotype data of the F2 population. The band type of the parent CIHO11530 was recorded as a (i.e., GG genotype), and the band type of the parent CIHO 1789 was recorded as b (i.e., AA genotype). The band type of the F2 population strains derived from the band type of CIHO 11530 was recorded as a, and the band type derived from CIHO 1789 was recorded as b.

[0036] (4) The obtained F2 population genotype data were used to construct a barley molecular linkage map using JoinMap4.0 mapping software to find the optimal number of markers and marker sequence and determine the linkage group to be used subsequently. The tiller number QTL LNT2 was located in the KASP1765-SNP526 segment on chromosome 6H using the interval mapping method of Map QTL6.0 software and combined with the tiller number phenotypic data of the F2 population. The KASP1765 marker was closely linked to the tiller number QTL LNT2.

[0037] (5) Convert the SNP sites in the target segment into fluorescent quantitative PCR primers for subsequent screening. Poly Marker website designed 4 pairs of fluorescent quantitative PCR primers (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-30 bp, amplification product length 73-105 bp, annealing temperature 57-62°C, GC content between 40% and 60%.

[0038] The synthetic primer sequences are:

[0039] Forward primer 1: F probe + amplification primer sequence;

[0040] Forward primer 2: H probe + amplification primer sequence;

[0041] Reverse primer: amplification primer sequence;

[0042] F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO. 14, with FAM fluorescent group bound to the 5' end);

[0043] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 15, with a HEX fluorescent group bound to the 5' end).

[0044] Table 14 Pairs of KASP primer sequences and amplified fragment lengths

[0045]

[0046]

[0047] Note: Primers with "-1" in their names are forward primers 1; primers with "-2" in their names are forward primers 2; primers with "-3" in their names are reverse primers.

[0048] (6) Competitive allele-specific PCR (KASP) analysis

[0049] a) Screening of polymorphic molecular markers between parents: The four pairs of primers designed above were selected, and DNA of parents CIHO 11530 and CIHO 1789 was used as template for PCR amplification, and a pair of molecular marker primers with good effect was obtained, named KASP1765-1 / 2 / 3 (nucleotide sequences are shown in SEQ ID NO.2-4 respectively). The amplified product is the polymorphic molecular marker KASP1765, and the nucleotide sequence is shown in SEQ ID NO.1.

[0050] SEQ ID NO.1:

[0051] 5'-TGCAGGAGGACAAGCAAAAGAGAAGRGAGGGCGACGCGTGTCCTGCATGCACGCCACGGCCGAGATCGG-3'; where R is A or G.

[0052] b) KASP analysis of F2 population: PCR primers of molecular marker KASP1765 with polymorphism obtained in the above steps were used to amplify DNA of parent CIHO 11530, CIHO 1789 and F2 population plants, and genotype identification was performed to obtain molecular marker data. The type of parent CIHO 11530 was recorded as a (i.e., GG genotype), the amplified fragment size was 95 bp, and the single base difference site was G. The type of parent CIHO 1789 was recorded as b (i.e., AA genotype), the amplified fragment length was 95 bp, and the single base difference site was A. The type of F2 population strains derived from CIHO 11530 was recorded as a, and those derived from CIHO 1789 were recorded as b.

[0053] c) Using the interval mapping method of MapQTL6.0 software and combining the tiller number phenotypic data of the F2 population, it was found that the molecular marker KASP1765 was closely linked to the tiller number QTL LNT2. The results are as follows Figure 1 As shown. Figure 1 It can be seen that the tiller number QTL LNT2 is located in the 3.39 cM segment between markers KASP1765 and KASP526, and the molecular marker KASP1765 is closely linked to the tiller number QTL LNT2.

[0054] The location of tiller number QTL LNT2 on chromosome 6H and its linkage genetic map with molecular marker KASP1765 are shown in Figure 1 .

[0055] Example 2 Application of molecular marker KASP1765 tightly linked to tiller number QTL LNT2

[0056] 1. DNA extraction

[0057] The experimental materials selected were CIHO 11530, CIHO 1789 and Bowman, among which CIHO 1789 and Bowman were multi-tillering varieties, and CIHO 11530 was a oligo-tillering variety. The leaf DNA of barley samples at the three-leaf stage was extracted using the CTAB method.

[0058] 2. Screening of primers for detecting tiller number QTL LNT2

[0059] 2.1 Fluorescence quantitative PCR primers

[0060] The primers KASP1765-1 / 2 / 3 of Example 1 were used to detect the tiller number QTL LNT2, wherein the 5' ends of KASP1765-1 and KASP1765-2 were connected to a FAM fluorescent group and a HEX fluorescent group, respectively.

[0061] 2.2 Fluorescence quantitative PCR platform to test the differences between primers and their parents

[0062] (1) Extract DNA from leaves of CIHO 11530, CIHO 1789 and Bowman at the three-leaf stage.

[0063] (2) Using the genomic DNA of the barley to be tested as a template, primers were designed based on the KASP detection platform technology to perform fluorescence quantitative PCR amplification;

[0064] Fluorescence quantitative PCR amplification reaction system: 2×KASP Master mix 5μL, KASPAssay Mix 0.14μL, template DNA 50ng, DNase / RNase-free deionized water to 10μL; KASP Assay Mix contains primers KASP1765-1, KASP1765-2 and KASP1765-3 in a volume ratio of 2:2:5. That is, in KASPAssay Mix, primers KASP1765-1, KASP1765-2 and KASP1765-3 with a concentration of 100μM are mixed in a volume ratio of 2:2:5.

[0065] Fluorescence quantitative PCR program: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

[0066] (3) The specific method for analyzing PCR products is as follows: barley samples containing the tiller number QTL LNT2 all showed the same genotype as barley CIHO 11530, recorded as type a (i.e., GG genotype), while barley samples without the tiller number QTL LNT2 all showed fluorescent signals that were significantly different from barley CIHO 11530, such as CIHO 1789 and Bowman, recorded as type b (i.e., AA genotype). The results of genotyping CIHO 11530, CIHO 1789, and Bowman using KASP primers are shown in Figure 2 .

[0067] Example 3

[0068] Applicability of primer sequence KASP1765-1 / 2 / 3 in population detection:

[0069] (1) CIHO 11530 was used as the female parent and Bowman as the male parent to obtain F1, which was then self-fertilized to obtain the F2 validation population. Leaf DNA of each strain in the population was extracted at the three-leaf stage.

[0070] (2) Using the DNA obtained in step (1) as a template, fluorescent quantitative PCR amplification was performed using the primers KASP1765-1 / 2 / 3 of Example 1. The reaction system and procedure of fluorescent quantitative PCR amplification were the same as those of Example 2.

[0071] (3) The specific method for analyzing PCR products is as follows: barley samples containing the tiller number QTL LNT2 all showed the same genotype as barley CIHO 11530, recorded as type a (i.e., GG genotype), while barley samples without the tiller number QTL LNT2 all showed fluorescence signals that were significantly different from barley CIHO 11530, recorded as type b (i.e., AA genotype). The results are shown in Figure 3 136 lines were tested and the homozygotes in the lines were analyzed. Among them, 33 lines could amplify the same type of fragment as CIHO 11530, which were homozygous plants containing the tiller number QTL LNT2, and it was predicted that the plants in the line would have few tillers at maturity. 35 lines could amplify the same b-type fragment as CIHO 1789 and Bowman, which were homozygous plants without the tiller number QTL LNT2, and it was predicted that these plants would have many tillers at maturity.

[0072] (4) The tiller number of the 75 F2 homozygous plants was identified in the field during the barley maturity period. The results are shown in Table 2. The results show that the molecular marker KASP1765 of the tiller number QTL LNT2 can be used to predict the tiller number of the genetic population. The barley varieties containing the tiller number QTL LNT2 all showed the same fluorescence signal as the fluorescent probe connected to the primer shown in SEQ ID NO.2, while the barley varieties without the tiller number QTL LNT2 all showed a fluorescent signal significantly different from the fluorescent probe connected to the primer shown in SEQ ID NO.2. The average tiller number of plants with the same type as CIHO 11530 was 7.29, which was significantly lower than the average tiller number of plants with the same type as CIHO 1789 and Bowman, which was 53.73. The actual results were consistent with the expected results, indicating that the tiller number QTL LNT2 of the present invention does have a significant effect on improving the tiller number, and the molecular marker KASP1765 can be used to track and identify the tiller number QTL LNT2.

[0073] Table 2 Statistics of genotype and tiller number of 75 homozygous plants in F2

[0074]

[0075]

[0076]

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A KASP primer combination for identifying tiller number traits, characterized in that: It comprises two forward primers and a universal reverse primer; the nucleotide sequences of the two forward primers are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively, and the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.4; The 5' end of the forward primer with a nucleotide sequence as shown in SEQ ID NO.2 is bound to a FAM fluorescent group; The 5' end of the forward primer having a nucleotide sequence as shown in SEQ ID NO.3 is bound to a HEX fluorescent group.

2. Use of the KASP primer combination according to claim 1 in preparing a product for identifying the trait of tiller number of barley.

3. The use according to claim 2, characterized in that: The product is a test kit.

4. A product for identifying the trait of tiller number of barley, characterized in that: Comprising the KASP primer combination according to claim 1.

5. The product according to claim 4, characterized in that The product is a test kit.

6. Use of the KASP primer combination according to claim 1 or the product according to claim 4 or 5 in identifying the trait of tiller number in barley.

7. A method for identifying the trait of tiller number of barley, characterized in that: The following steps are involved: Extracting the DNA of the barley to be tested; The DNA is used as a template and the KASP primer combination according to claim 1 is used to perform fluorescent quantitative PCR amplification to obtain the genotype of the barley to be tested, and the tiller number trait of the barley to be tested is determined according to the genotype: the tiller number of the GG genotype barley is lower than that of the AA genotype barley.

8. The method according to claim 7, characterized in that The reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Master mix 5 μL, KASP Assay Mix 0.14 μL, template DNA 50 ng, and DNase / RNase-free deionized water to 10 μL.

9. The method according to claim 7, characterized in that: The reaction procedure of the fluorescent quantitative PCR amplification is as follows: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

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