A gene and molecular marker related to plant height traits in oats

Abstract

The present invention belongs to the technical field of molecular genetics, and specifically relates to a gene and a molecular marker related to the plant height trait of oats. The gene related to the plant height trait of oats is characterized in that the gene is <h2 style=";text-align:left;direction:ltr">AsbHLH19, Its CDS nucleotide sequence is shown in SEQ ID NO.3, and its encoded amino acid sequence is shown in SEQ ID NO.4. <h2 style=";text-align:left;direction:ltr"> AsbHLH19 The linked KASP molecular marker is a SNP located at <h2 style=";text-align:left;direction:ltr"> AsbHLH19 The 115 bp of the first exon of the gene, the nucleotide at the SNP site is T / G. <h2 style=";text-align:left;direction:ltr"> AsbHLH19 It can be used as a positive regulatory factor for oat plant height to accelerate the growth rate of oats, increase the length of oat stem nodes, and thus increase the plant height of oats; the KASP molecular marker linked to the gene provided can effectively and quickly screen out germplasm resources or hybrid offspring materials with <h2 style=";text-align:left;direction:ltr"> AsbHLH19 Individuals with excellent allelic variations can significantly improve the breeding efficiency of oats with plant height.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics, and in particular relates to a gene and a molecular marker related to oat plant height traits. Background Art

[0002] Oats (Avena sativa L.) is a dual-purpose crop of the Poaceae family. It has the characteristics of high yield, high quality and strong stress resistance. It is one of the key forages to solve the shortage of forage supply in winter and spring pastures in northern my country, and occupies an important position in the development of the domestic forage industry. In recent years, with the rapid development of my country's animal husbandry, the domestic demand for the production of forage oats hay has become increasingly urgent, which in turn puts higher requirements on the breeding of high-yield forage oat varieties. Plant height is one of the most important yield components of oat biomass. There is a significant positive correlation between plant height and biomass, and the correlation coefficient can reach 0.84; it is closely related to multiple yield traits, affecting the productivity of individual plants, and acting together with planting density to affect the yield of oats. Increasing the plant height of oats is an effective way to increase its biomass.

[0003] However, oat breeding in my country is still in the traditional breeding stage, and there is a serious lack of genes and molecular markers related to oat plant height, which seriously limits the development of oat molecular breeding. Therefore, it is necessary to identify genes related to oat plant height, discover the allelic variation of key genes in the oat population, and develop them into molecular markers that can be used for breeding. On the one hand, it provides key candidate genes for oat high plant height breeding, and on the other hand, the developed molecular markers can be used to select target genes in low-generation materials after hybridization, combined with rapid generation technology, to promote the rapid stabilization of target sites and speed up the selection of high plant height varieties.

[0004] Based on the above needs, a gene and molecular marker related to the plant height trait of oats were invented to provide candidate genes and molecular markers for the field of oat molecular breeding, which is of great significance in the field of molecular genetics technology. Summary of the invention

[0005] The present invention first provides a gene related to the plant height trait of oats, wherein the gene is AsbHLH19, its CDS Nucleotide sequence such as The amino acid sequence encoded by the gene is shown in SEQ ID NO.3, and the amino acid sequence encoded by the gene is shown in SEQ ID NO.4.

[0006] The present invention also provides a gene related to oat plant height AsbHLH19 Linked KASP molecular marker, the KASP molecular marker is a SNP, which is located AsbHLH19 The nucleotide at the SNP site is T / G at the 115 bp of the first exon of the gene.

[0007] The present invention also provides the use of the above-mentioned gene related to the oat plant height trait, or the above-mentioned gene AsbHLH19 linked KASP molecular marker related to the oat plant height in identifying or assisting in identifying the oat plant height trait.

[0008] The present invention also provides a method for detecting the above-mentioned gene related to oat plant height trait, or a method for detecting the above-mentioned gene AsbHLH19 linked to KASP molecular marker related to oat plant height in identifying or assisting in identifying the oat plant height trait.

[0009] In certain embodiments, the substance is a primer or a kit.

[0010] In certain embodiments, the primer sequences are shown in SEQ ID NO.9-SEQ ID NO.13.

[0011] The present invention also provides a kit for identifying or assisting in identifying the oat plant height trait, wherein the kit contains primer sequences as shown in SEQ ID NO.9-SEQ ID NO.13.

[0012] The present invention also provides the use of the above-mentioned kit in any of the following:

[0013] (A1) Identify or assist in identifying oat plant height traits;

[0014] (A2) Identify or assist in identifying oat germplasm resources;

[0015] (A3) Breeding of oats.

[0016] The present invention also provides a method for identifying or assisting in identifying oat plant height traits, comprising the step of detecting the above-mentioned gene related to oat plant height traits, or the above-mentioned oat plant height related gene AsbHLH19 linked to KASP molecular markers.

[0017] The present invention finally provides the application of the above method in any of the following:

[0018] (A1) Identify or assist in identifying oat plant height traits;

[0019] (A2) Identify or assist in identifying oat germplasm resources;

[0020] (A3) Breeding of oats.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects:

[0022] The AsbHLH19 provided by the present invention can serve as a positive regulatory factor for oat plant height to accelerate the growth rate of oats, increase the length of oat stem nodes, and thus improve the plant height of oats; the KASP molecular marker linked to the gene provided can effectively and quickly screen out individuals with excellent allele variation of AsbHLH19 in germplasm resources or hybrid offspring materials, and can significantly improve the breeding efficiency of plant height oats. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Phenotypes of oat germplasm resources with extreme plant height (a) and Venn diagram of differentially expressed genes and differentially expressed proteins (b);

[0024] Figure 2 DNA level identification of transgenic oats (a) and the expression level of AsbHLH19 in transgenic oats (b);

[0025] Figure 3 Growth rate of transgenic oats (a) and phenotype of transgenic oats (b);

[0026] Figure 4 Phenotypic comparison of different genotypes of 80 oat germplasm resources (a) and KASP molecular marker typing diagram;

[0027] Figure 5 Phenotypic comparison of different genotypes in 700 oat germplasm resources. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0029] Embodiment 1, oat AsbHLH19 Gene screening and cloning

[0030] (1) Screening of germplasm resources with extreme plant height

[0031] 700 forage oat germplasm resources from all over the world were collected to form a natural population, which was sown in Changping District, Beijing in 2021 and 2022. The planting method was single seed sowing, with a row spacing of 30 cm and conventional field management. During the milky stage of oats, the plant height traits of the above oat germplasm resources were investigated to obtain the phenotypic data of the natural population. Based on the phenotypic traits, 172 germplasm resources with extremely high plant height were selected, with an average plant height of 154 cm, and 589 germplasm resources with extremely low plant height, with an average plant height of 46 cm ( Figure 1 a).

[0032] (2) Transcriptome and proteome sequencing of extreme plant height materials

[0033] We extracted RNA and total protein from the stem nodes of the above extreme oat germplasms, commissioned Wuhan Bena Technology Co., Ltd. to perform RNA-seq and TMT protein sequencing, and obtained 1147 common differentially expressed genes and proteins in the above two extreme plant height germplasms through library construction, sequencing and analysis. Figure 1 b).

[0034] (3) Candidate genes related to plant height AsbHLH19 Clone

[0035] Through functional annotation, GO and KEGG analysis of the above 1147 differentially expressed genes (proteins), AsbHLH19 As a candidate gene for plant height trait. Based on the sequence information of AsbHLH19, amplification primers CDS-F: ATGGACGCGGACATG (SEQ ID NO.1) and CDS-R: TTATAGCTCATGGAGC (SEQ ID NO.2) were designed, and PCR amplification was performed using mixed cDNA from different oat tissues as templates. The amplification system was as follows: 50 μL reaction system, including 20 ng DNA, Mg 2+ 2μL, PCR buffer 5μL, dNTP 5μL, KOD enzyme 1.5μL, upstream and downstream primers 1.5μL respectively. The PCR amplification procedure is as follows: 34 PCR cycles after 95℃ pre-denaturation for 3 min, 95℃ denaturation for 35s, 58℃ annealing for 35s, 72℃ extension for 2min, and 72℃ extension for 10min after the cycle ends. After amplification, agarose gel electrophoresis was performed, and the amplified products were recovered and sequenced to obtain AsbHLH19 CDS sequence.

[0036] AsbHLH19 CDS Sequence:

[0037] ATGGACGCGGACATGGGCGAGAACTTCGCGTACTACTGGGAGACGCAGCGCTACCTCGAGTCGGAGGAGCTGGTGGACAGCATCTTCGTCGGGGCGACCGAGGACGCCGTTTCGTACTACGACTCGAGCTCGCCGGACGGGTCCCACTCGTCCTCGACGCCGACCGGGGCTGCCATGCCCGGTGCTGCGATGGGGATGGGGATGGGGATGGGCGAGACGGGGGCAAACAAGAACATCCTGATGGAGCGCGACCGCCGCCGCAAGCTGAACGAGAAGCTCTACGCGCTCCGCAGCGTCGTGCCCAACATCACCAAGATGGACAAGGCGTCGATCATCAAAGACGCGATCGAGTACATCCAGGTGCTGCAGGCGGAGGAGCGTCAGATATCGGCGGAGGTGTCCGCGCTCGAGTCCGCCGCGGGTGCGGAGGAGGTGGACAACGAGGACGGCCTCTTCGCGGAGCAGGTCTCCTCGGGGCAGAGGAAGAAGGTGAAGCGCGCGCTTTCGGTCTCGTCGATGAACGACGCGCTGCTCGCCGCGGCGGCCGAGGCGGCCTCGCCGCCCGTGGAGGTCCTGGAGCTGCGCGTGTCGGAGGTGAGCGAGAAGGTGCTGGTGGTGAGCGTGACGTGCACCAAGCAGCGCGACGCCATGGCCAAGGTGTGCCGCGTGCTGGAGGAGCTCCGCCTCAGGGTCATCACCGCCAACATCACCTCCGTGTCGGGCTTCCTCATGCACACCCTCTTCGTCGAGGCAGATGACATGGACCGTTTCCAGATGAAGGAGATCGTGGAGGCTGCTCTCTCGCAGCTTGATGCTCCAGGAAGCCCGCCACTTAGCTCCATGAGCTATAA(SEQ ID NO.3);

[0038] AsbHLH19 Coding protein sequence:

[0039] MDADMGENFAYYWETQRYLESEELVDSIFVGATEDAVSYYDSSSPDGSHSSSSTPTGAAMPGAAMGMGMGMGETGANKNILMERDRRRKLNEKLYALRSVVPNITKMDKASIIKDAIEYIQVLQAEERQISAEVSALESAAGAE EVDNEDGLFAEQVSSGQRKKVKRALSVSSMNDALLAAAAEAASPPVEVLELRVSEVSEKVLVVSVTCTKQRDAMAKVCRVLEELRLRVITANITSVSGFLMHTLFVEADDMDRFQMKEIVEAALSQLDAPGSPPLSSSMY (SEQ ID NO.4).

[0040] Example 2: Overexpression AsbHLH19 Acquisition and phenotypic observation of transgenic oats

[0041] (1) Overexpression AsbHLH19 Transgenic vector construction

[0042] The plant overexpression vector uflag was double-digested with restriction endonucleases HindIII and BanmHI, and primers uflag-F: tacttctgcagaagcttATGGACGCGGACATG (SEQ ID NO.5) and uflag-R: ttgtagtccatggatccTAGCTCATG

[0043] GAGC (SEQ ID NO.6) amplification AsbHLH19 CDS region to obtain vector arms containing AsbHLH19 The CDS region of the gene was recombined using the LanGene™ Seamless Cloning & Assembly Kit. The recombinant product was transformed into DH5a E. coli competent cells, and overexpression was obtained by sequencing verification. AsbHLH19 The transgenic vector uflag:: AsbHLH19 .

[0044] (2) Overexpression AsbHLH19 Genetically modified oats

[0045] The constructed overexpression vector uflag:: AsbHLH19Transform the competent cells of Agrobacterium tumefaciens EHA105, pick the positive clones and culture them to 50 ml, use the Agrobacterium-mediated transformation method to infect oat callus tissue, and obtain regenerated plants through dark culture, induction and regeneration. Extract the genomic DNA of the regenerated plants, and use the primer pair uflagseq-F: cttggatgatggcatatgc (SEQ ID NO.7); uflagseq-R: ttcgagctggtcagagctc (SEQ ID NO.8) to identify the regenerated plants at the DNA level. The positive plants are obtained. AsbHLH19 Overexpression of transgenic oats ( Figure 2 a).

[0046] (3) In genetically modified oats AsbHLH19 Expression analysis

[0047] extract AsbHLH19 RNA from overexpressed oats was converted to cDNA and diluted 2-fold as a template for expression analysis. AsbHLH19 -RT-F: CGAGAAGGTGCTGGTGGTGAG (SEQ ID NO.9); AsbHLH19-RT-R: AGGTGATGTTGGCGGTGATGAC (SEQ ID NO.10), qRT-PCR amplification was performed using ABI Q7 fluorescent quantitative PCR instrument to detect the positive plants. AsbHLH19 The expression level of -ΔΔCt The relative expression level of the target gene was calculated by the method. Figure 2 b shows that the overexpression plants numbered OE3, OE5 and OE11 AsbHLH19 The expression levels were higher, 483, 327 and 307 times that of the control plants, respectively.

[0048] (4) Overexpression AsbHLH19 Observation of transgenic oats phenotype

[0049] The T0 seeds of OE25, OE7 and OE13 were harvested respectively, and T1 plants were screened in 1 / 2 MS medium (30 mg / ml hygromycin was added). The T1 plants obtained by screening were planted and seeds were harvested. Screening was continued in 1 / 2 MS medium (30 mg / ml hygromycin was added). Homozygous T2 plants were obtained based on the screening performance of T1 plants. Seeds of T2 homozygous plants were harvested to obtain overexpression AsbHLH19 Homozygous transgenic oats. AsbHLH19 The plant height phenotypes of the homozygous transgenic oats and controls were observed and recorded during the entire growth period. AsbHLH19 The transgenic oats grew significantly faster than the control plants ( Figure 3 a); Finally, at the milk stage of oats, the height of the transgenic plants was about 1.7 times that of the control plants ( Figure 3 b). The significant differences in the above phenotypic traits indicate that the overexpression of AsbHLH19 It can speed up the growth of oats, increase the length of oat stem nodes, and thus increase the height of oats.

[0050] Example 3: Genes related to oat plant height AsbHLH19 Development and validation of molecular markers linked to KASP

[0051] (1) AsbHLH19 Gene sequence polymorphism discovery

[0052] From 700 oat germplasm resources, 40 germplasms with high plant height and 40 germplasms with low plant height were selected. DNA of the above 80 germplasm resources was extracted and amplified by PCR and sequenced to obtain the DNA of the above 80 oat germplasm resources. Asb Gene sequence. Multiple sequence alignment was performed on 80 sequences, and local manual adjustment was performed, and finally 8 SNPs were obtained. In 80 germplasms, the genotype and phenotype of each site were tested to screen polymorphic sites significantly associated with phenotypic traits. The results showed that there was a SNP site (115bp, T / G) in the first exon of the gene that was extremely significantly correlated with the plant height trait, with a detection P value of 0.00014, among which the TT type was associated with high plant height, and the GG type was associated with low plant height ( Figure 4 a).

[0053] (2) AsbHLH19 Gene-linked KASP marker development

[0054] According to the design principles of KASP molecular marker primers, primers for this site (T / G) were designed, and "FAM" and "HEX" fluorescent sequence labels were added to the front ends of the primers, respectively. The primers were sent to Shanghai Biotechnology Co., Ltd. for primer synthesis. The primer sequences were F1: GAAGGTGACCAAGTTCATGCTGGCGACCGAGGACGCCGTTTCGT (SEQ ID NO.11), F2: GAAGGTCGGAGTCAACGGATTGGCGACCGAGGACGCCGTTTCGG (SEQ ID NO.12), and R: GAAGGTGACCAAGTTCATGCTGCGGTCGCGCTCCATCAGGATGT (SEQ ID NO.13). Using the DNA of the 80 germplasms in step (1) as templates, PCR amplification was performed separately. The amplification system was as follows: 10 μL reaction system, including 5 μL 2×KASP master mix, 20 ng DNA, and 1 μL primer mixture (the primer mixture was 6 μL upstream primer F1, 6 μL upstream primer F2, 15 μL downstream primer R, and 23 μL ddH2O). The PCR amplification program was as follows: 95°C pre-denaturation for 15 min, 95°C denaturation for 20 s, 61°C annealing and extension for 40 s, 95°C denaturation for 20 s, and the above program was repeated for 10 cycles, in which the annealing temperature was reduced by 0.6°C each cycle; followed by 55°C annealing and extension for 40 s, for 32 cycles. After obtaining the PCR product, the fluorescence was collected at 37 degrees by ABI Q7 real-time fluorescence quantitative PCR, and the PCR product was typed. The typing results were compared with the true variant types of the site, and it was found that the consistency rate between the site variant types detected by the molecular marker and the true variant types was 100%, indicating that the KASP marker for this site was successfully developed ( Figure 4 b).

[0055] (3) AsbHLH19 Validation of gene-linked KASP molecular markers

[0056] To further verify the effectiveness of the developed KASP molecular marker, the marker was used to perform genotyping analysis on 700 oat germplasm resources. The detection method was the same as step (2) in Example 3. The t-test method was used to detect whether oats with different genotypes had statistical differences in plant height traits. The results showed that among the 800 oat germplasm resources, 250 germplasms were TT type at this site, and 426 germplasms were GG type at this site. There was a very significant difference in plant height traits between the two types of oat germplasms ( P =2.11 E-11 ); the average plant height of oat germplasm resources with TT type is 108.35cm, and the average plant height of oat germplasm resources with GG type is 95.28cm ( Figure 5 ). The above results further confirmed the accuracy of the KASP molecular marker, indicating that it is a molecular marker significantly associated with the plant height trait of oats.

[0057] It can be seen from this that the present invention provides AsbHLH19 It can be used as a positive regulatory factor for oat plant height to accelerate the growth rate of oats, increase the length of oat stem nodes, and thus increase the plant height of oats; the KASP molecular marker linked to the gene provided can effectively and quickly screen out germplasm resources or hybrid offspring materials with AsbHLH19 Individuals with excellent allelic variations can significantly improve the breeding efficiency of oats with plant height.

[0058] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene related to oat plant height trait, characterized in that: The gene is AsbHLH19, the CDS nucleotide sequence of which is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.

4.

2. A KASP molecular marker linked to the oat plant height-related gene AsbHLH19, characterized in that: The KASP molecular marker is a SNP, which is located at the 115bp of the first exon of the AsbHLH19 gene, and the nucleotide at the SNP site is T / G; the CDS nucleotide sequence of the AsbHLH19 is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.

4. When the nucleotide at the SNP site is G, the oat has a low plant height, and when the nucleotide at the SNP site is T, the oat has a high plant height.

3. Use of primers for detecting the KASP molecular marker linked to the gene AsbHLH19 related to oat plant height as claimed in claim 2 in identifying or assisting in identifying the plant height trait of oat; The primer sequences are shown in SEQ ID NO.11-SEQ ID NO.

13.

4. A kit for identifying or assisting in identifying oat plant height traits, characterized in that: The kit contains a primer set, and the sequence of the primer set is shown as SEQ ID NO.11-SEQ ID NO.

13.

5. Use of the kit according to claim 4 in any of the following: (A1) Identify or assist in identifying oat plant height traits; (A2) Identify or assist in identifying oat germplasm resources; (A3) Breeding of oats.

6. A method for identifying or assisting in identifying oat plant height traits, characterized in that: The method comprises the step of detecting the KASP molecular marker linked to the gene AsbHLH19 related to oat plant height as described in claim 2.

7. Use of the method according to claim 6 in any of the following: (A1) Identify or assist in identifying oat plant height traits; (A2) Identify or assist in identifying oat germplasm resources; (A3) Breeding of oats.

Images