Barley grain weight related gene HvTGW1s and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a barley grain weight related gene HvTGW1s and application thereof. The invention discloses a barley grain weight related gene HvTGW1s, the base sequence of the barley grain weight related gene HvTGW1s is shown as SEQ ID NO: 1, and the barley grain weight related gene HvTGW1s has the function of increasing the thousand grain weight, grain width and grain length of barley. The invention further discloses the KASP marker associated with the barley yield character and application of the KASP marker in barley yield assisted selective breeding. The invention further discloses a kit for assisting in identifying the barley yield. The method is applied to barley yield assisted selection breeding, the breeding efficiency can be effectively improved, and the breeding process is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to genes related to barley grain weight. HkDJ s and its applications. Background Art

[0002] The yield per unit area of ​​barley is composed of the number of ears per unit area, the number of grains per ear and the grain weight. Studies have shown that grain weight has the greatest impact on genetic characteristics among the yield components, and the broad heritability is generally high (>80%) (Hu Yanji et al., 1994). Therefore, grain weight is one of the key traits for high-yield breeding of barley, and the cloning and identification of key genes is the basis for molecular genetic improvement.

[0003] Barley grain weight is a qualitative-quantitative trait locus, which is controlled by major and minor genes. Although a large number of QTL loci related to barley grain weight have been reported, the fine positioning of major QTL loci related to grain weight is relatively lagging. Watt et al. finely located the major QTL for grain length on chromosomes 2H and 5H, respectively. qG The interval was narrowed to 140.9 Kb (Watt et al., 2020); q5H It has shrunk from a 106.8 Mb interval to a 1.7 Mb interval (Watt et al., 2019).

[0004] In rice, which is also a grass crop, a large number of grain weight-related genes have been identified using the map-based cloning strategy, such as GS5 , OsSPL16 , GW2 and GW5 , regulating granule length GS3 , regulating grain size ANT , GIF1 and TGW6 (Mao et al., 2010; Takano-Kai et al., 2009; Weng et al., 2008; Li et al., 2011; Wang et al., 2008; Ishimaru et al., 2013; Wang et al., 2012; Mizukami et al., 2000). In barley, some grain weight-related genes were also cloned using homologous cloning strategies. For example, the negatively regulated grain length gene GW2 Homologous clones of HvG2 Two transposon insertions in the sixth intron of the gene may lead to reduced gene transcriptional activity, thereby affecting grain weight. So far, no map-based cloning study of barley grain weight QTL has been reported.

[0005] With the completion of genome sequencing of multiple barley varieties (Mascher et al. 2017, Mayer et al. 2012, Russell et al. 2016, Zeng et al. 2015), the gene cloning research of barley has been greatly promoted. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a barley grain weight-related gene having the function of increasing barley thousand-grain weight, grain width and grain length. HkDJ s , and KASP markers associated with barley yield traits and their application in assisted selection breeding of barley yield. The specific technical solutions provided by the present invention are: A gene related to barley grain weight HkDJ s , which has the function of increasing the thousand-grain weight, grain width and grain length of barley, and its nucleotide sequence is shown in SEQ ID NO: 1. Also provided is the barley grain weight-related gene HkDJ s Application in assisted selection breeding of barley yield and in breeding transgenic barley varieties with improved barley yield.

[0007] Also provided is a barley grain weight-related protein, which is obtained by the barley grain weight-related gene according to claim 1 HkDJ s Place Express, Its amino acid sequence is shown in SEQ ID NO:2.

[0008] Also provided is a KASP marker SNP53 associated with barley yield traits, wherein the molecular marker is located on a barley kernel weight-related gene on barley chromosome 6HL. HkDJ At the locus, there is an allele C / A, and when the allele is A, the thousand-grain weight, grain length and grain width of barley are significantly increased. And the application of the KASP marker SNP53 in assisted selection breeding of barley yield.

[0009] The present invention also provides primers for detecting the KASP marker SNP53 associated with the barley yield trait, wherein the amplification primers are nucleotide sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The present invention also provides a primer for assisting in identifying a gene with high yield in barley. HkDJ s The kit comprises amplification primers for the KASP marker SNP53.

[0010] Finally, the present invention provides a method for improving barley yield traits, comprising the following steps: The barley plant containing the barley grain weight-related gene is used as a donor parent and hybridized with a barley recipient parent to be improved to obtain F1 seeds. The F1 seeds are hybridized with the recipient parent to be improved to obtain BC1F1 seeds. BC1F1 seeds were planted and selected to contain QUR S A single plant with the same genotype and phenotype as the recipient parent to be improved is hybridized with the recipient parent to be improved to obtain BC2F1 generation seeds. The BC2F1 generation seeds are then backcrossed for two generations to finally obtain the BC4F1 generation seeds. The BC4F1 generation seeds are self-pollinated for two generations to obtain the BC4F3 pure line containing the barley grain weight-related gene, which is the high-yield barley cultivated.

[0011] In the above method for improving barley yield traits, it is preferred that the barley recipient parent to be improved is Barley Xila 22, and the donor parent is QUR S Allele of Australia's choice 3.

[0012] The present invention has at least the following beneficial effects: The present invention uses barley RIL population and adopts the strategy of map-based cloning to identify a grain weight gene on 6HL, which is named HkDJ At the same time, the present invention has been identified QUR The molecular marker SNP53 (C / A) is designed at the key gene variation site, and the marker is significantly correlated with grain weight. The present invention is applied to auxiliary selection breeding of barley yield, which can effectively improve breeding efficiency and accelerate the breeding process.

[0013] Other advantages of the present invention will be apparent in part from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the genetic linkage map and positioning map for the identification of the major QTL locus (QTgw.ZiSc-6H) in the example of the present invention.

[0015] Figure 2 These are the plant and grain phenotypes of the near-isogenic pure lines (NIL-Z and NIL-S) in the embodiments of the present invention. NIL-Z represents a subpopulation whose target interval is a homozygous purple-light naked two-row genotype, and NIL-S represents a subpopulation whose target interval is a homozygous Australian-selected 3 genotype.

[0016] Figure 3 In the embodiment of the present invention QTgw.ZiSc-6HFine positioning and candidate gene cloning diagram, where A is the BC3F3 exchange single plant genotype and derived population progeny test diagram; B is the BC3F4 exchange single plant genotype and derived population progeny test diagram; AA represents the target interval is the purple light naked two-row genotype, BB represents the target interval is the Australian selection 3 genotype; TGW, thousand-grain weight; * indicates significant level P ≤ 0.05; ** indicates significant level P ≤ 0.01, where C is the gene QUR Location map.

[0017] Figure 4 This is a comparison chart of the grain weight performance of different alleles in the examples of the present invention.

[0018] Figure 5 In the embodiment of the present invention, the BC4F3 and the BC4F3 QUR S )Photographs of field performance.

[0019] Figure 6 In the embodiment of the present invention, the BC4F3 and the BC4F3 QUR S ) Characteristic investigation diagram, * indicates significant level P≤0.05; NS indicates no significant difference.

[0020] Figure 7 This is a comparison chart between BC4F3 and Xila 22 in agronomic traits such as plant height, ear length and number of grains per ear. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0022] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0024] The present invention identifies the gene through a positional cloning strategy, identifies key mutation sites of the gene, and develops applications of molecular markers targeting the mutation sites.

[0025] The present invention uses a RIL population containing 190 lines provided by Zeng Yawen's research group of Yunnan Academy of Agricultural Sciences (Fan et al; 2017) to perform QTL positioning analysis on barley yield-related traits such as 1000-grain weight, grain width and grain length. The population parents include a local variety Ziguangmang naked two-row and a beer barley variety Aoxuan No. 3; a near-isogenic line population is used for fine positioning, with Aoxuan No. 3 as the recurrent parent and Ziguangmang naked two-row as the donor parent. After three and four generations of backcrossing, BC3F1 populations and BC4F1 populations were obtained, respectively. Heterozygous plants containing two alleles in the target interval were further selected for self-pollination to obtain BC3F2 and BC4F2 populations, respectively; 454 core barley germplasm resources at home and abroad are used for correlation analysis of key variation sites.

[0026] The RIL population was planted in multiple environments for many years, namely: Beijing in 2016, Guyuan, Hebei in 2016, Datong, Shanxi in 2016, Beijing in 2017 and Guyuan, Hebei in 2017, obtaining phenotypic data from a total of 5 environmental points; the BC3F2 population and domestic and foreign core germplasm materials were planted in Beijing in 2018; and the BC4F2 population was planted in Beijing in 2019.

[0027] After the barley matures, the SC-G batch seed analysis module system of Hangzhou Wanshen Company is used to assist in the inspection of thousand-grain weight, grain width and grain length.

[0028] The present invention is based on the existing population genetic linkage map (Fan et al; 2017) combined with the data of five environmental points for preliminary QTL positioning analysis. WinQTLcart mapping software uses the composite interval mapping method to detect QTL, and selects the default parameters: the step speed is set to 1 cM; the LOD threshold of 1000 operations is less than or equal to 0.05.

[0029] The invention, based on the initial positioning, uses boundary markers of the main effect QTL interval to screen the BC3F2 population, obtains recombinant exchange plants, performs progeny test on the plants, i.e. obtains a secondary F2 population by self-pollination of the plants, uses boundary markers to screen plants with homozygous genotypes in the main effect QTL interval for trait investigation, obtains the phenotypic average values ​​of two pure lines with different alleles, uses statistical software IBM SPSS 20 to perform Student's t-test, compares the average values ​​of the two pure lines, and narrows the target interval carrying the main effect QTL segment; on this basis, uses the BC4F2 population to screen the recombinant exchange plants to further narrow the QTL interval.

[0030] In order to determine the candidate genes of the target QTL interval, the present invention performed BLASTN comparison analysis on the QTL boundary marker sequences based on the latest barley reference genome Morex v3.0 (https: / / galaxy-web.ipk-gatersleben.de / ), anchored the QTL interval and searched for annotated genes in it, and obtained the gene sequences of the two parents by sequence amplification. The amplification system is: the whole genome DNA of the parents is used as a template, and the gene PCR product is amplified using the Tks Gflex DNA Polymerase kit produced by TaKaRa, 25µl system: 1.0µl genomic DNA (40ngμL -1 ) ; 0.5µl (10µmolL) of each left and right primer (NC1) -1 ); 12.5µl 2×Gflex PCR Buffer; 10µl ddH2O; 0.5µl Tks Gflex DNA Polymerase (1.25U / μl). The amplification program was: 98℃ denaturation for 1min; 30 cycles of 98℃ denaturation for 10s, 60℃ annealing for 15s, and 68℃ extension for 30s; stored at 12℃, and the PCR product was sent to Qingke Biotech Co., Ltd. for sequencing.

[0031] Candidate genes HkDJ The cDNA sequence was obtained: using early development seeds (two days after pollination) as templates, the seed RNA was extracted using the Trizol method, and reverse transcription was performed using the Promega reverse transcription kit to synthesize single-stranded c DNA. RCR amplification was performed using primer NC2, and amplification was performed using the Tks Gflex DNA Polymerase kit. The amplification template was 2 µl single-stranded cDNA. The rest of the system was consistent with the above, and the amplification procedure was consistent with the above. The nucleotide sequence containing the open reading frame was obtained by PCR amplification, and the sequence was ligated to T sequencing. The 5 µl system: 1 µl T vector, 4 µl amplification product, gently mixed on ice, and ligated at 25°C for 15 minutes. The ligation product was added to the competent cells that had melted on ice, heat-shocked for 40 seconds, ice-bathed for 5 minutes, transformed into Escherichia coli DH5α, and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0032] Candidate genes HkDJ Obtaining protein sequence and analyzing its conservative structure: The nucleotide sequence obtained by cDNA sequencing was translated to obtain its protein sequence. On this basis, Smart was used to analyze the conservative functional domains of the HvQTGW1 protein sequence. At the same time, BLASTP comparison was performed using the plant protein database to find QUR Compare and analyze the conserved functional domains of proteins from Arabidopsis thaliana, rice and other Poaceae crops with homologous genes.

[0033] The simple sequence repeat (SSR) marker used for QTL interval encryption in the present invention is developed as follows: based on the Morex v3.0 reference genome information, the QTL target interval is defined by flanking markers, the nucleotide sequence in the target interval is obtained, and the SSR marker is designed.

[0034] Diagnostic markers: based on HkDJ Differential SNP sites were identified and competitive allele specific PCR-single nucleotide polymorphism (KASPSNP) markers were designed to analyze the genotypes of 454 core barley germplasm resources from home and abroad. The fluorescence signals were detected by Applied Biosystems7900 HT, and the Kluster caller software was used to perform population typing statistics.

[0035] The recipient parent Xila 22 to be improved is one of the highland barley varieties widely planted in Tibet. QUR S In March 2019, the recipient parents Xila 22 and Aoxuan 3 were sown, and the two parents were hybridized to obtain F1 seeds; in July 2019, the F1 seeds were planted in a greenhouse for generation and hybridized with Xila 22 to obtain BC1F1 seeds; in October 2019, the BC1F1 seeds were planted in a greenhouse for generation, and samples were taken at the seedling stage. The genotypes of all BC1F1 generations were analyzed using the SNP53 marker, and the genotypes containing QUR S The genotype of the individual plant and the phenotype of Xila 22 were hybridized with Xila 22 to obtain BC2F1 generation seeds. After two generations of backcrossing, BC4F1 generation seeds were finally obtained. After two generations of self-pollination, the seeds containing QUR S BC4F3 is a pure line of alleles.

[0036] 1) KASP primer mixture system: FAM-Primer 12 μL HEX-Primer 12 μL Common Reverse-Primer 30 μL ddH2O 46 μL Total 100 μL 2) KASP genotyping PCR reaction system: 2x KASP Master Mix 2.50 μL Primer Mix 0.07 μL ddH2O 1.25 μL DNA 1.25 μL Total 5.07 μL The reaction process is divided into four stages: Stage 1: DNA denaturation, pre-denaturation at 94°C for 15 minutes; Stage 2: denaturation at 94°C for 20 s, annealing and extension at 61°C for 1 min, with the temperature decreasing by 0.6°C for each cycle, for 10 cycles; Stage 3: denaturation at 94°C for 20 s, annealing and extension at 55°C for 1 min, for 34 cycles; Stage 4: Store at 16°C.

[0037] KASP amplification reaction: performed in an Applied Biosystems® Veriti® 384-Well Thermal Cycler gradient gene amplification instrument.

[0038] KASP amplification reaction products: Fluorescence signal detection was performed using the real-time fluorescence quantitative PCR instrument Fluostar Omege SNP, and then genotyping results were statistically analyzed using Kluster caller software.

[0039] 2. Experimental results 2. Experimental results (1) QTL location analysis Combining the existing genetic linkage map of the RIL population (Fan et al; 2017) and data from five environmental points, the present invention detected a major QTL locus on the long arm of chromosome 6H that can simultaneously regulate 1000-grain weight, grain width and grain length ( QTgw.ZiSc- 6H )( Figure 1 ). The agronomic and yield traits of two near-isogenic pure lines NIL-S and NIL-Z of four generations of backcrossing were investigated. The results showed that the thousand-grain weight, grain length and grain width of NIL-S were significantly higher than those of NIL-Z, while there were no significant differences in agronomic traits such as plant height, ear length and number of grains per ear (Table 1; Figure 3 ).

[0040] Table 1 The expression of traits such as thousand-grain weight in near-isogenic pure lines

[0041] Note: NIL-S represents the subpopulation with homozygous Aoxuan 3 genotype in the target interval, and NIL-Z represents the subpopulation with homozygous purple light naked two-leng genotype in the target interval. * indicates a significant level of P ≤ 0.05; ** indicates a significant level of P ≤ 0.01 rightA major QTL locus on the long arm of chromosome 6H that simultaneously regulates 1000-grain weight, grain width and grain length ( QTgw.ZiSc-6H) Fine positioning A total of 10 SSR markers were developed for the encrypted genetic linkage map of the QTL target interval, as shown in Table 2: 6L155, 6BC7, 6C19, 6C1, 6C7, 6C52, 6C54, B1, 6C29 and 6B2 ( Figure 3 A), using the two markers 6L155 and 6B to screen the BC3F2 population, and obtain individuals that have undergone recombination between these two markers. A total of 10 types of recombination were obtained, and one plant of each exchange type was selected for self-pollination to obtain secondary F2 populations, namely H3, H12, H8, H52, H7, H29, H26, H28, H30 and H2 populations ( Figure 3 A). Comparing the average grain weight of the pure lines of the offspring of these F2 populations, the grain weight of the offspring of the exchanged individual plants containing the heterozygous fragment between markers 6C52 and B1 was significantly different ( Figure 3 A), therefore, the target QTL interval was located within an interval of approximately 114 Kb between markers 6C52 and B1.

[0042] Table 2 Genetic molecular marker sequences used for QTL map encryption and gene amplification

[0043] 6C52 and B1 were further used to screen the recombinant strains in the BC4F2 population. At the same time, the target intervals were encrypted, such as 6L15, M1, and M2 (Table 2; Figure 3 B), a total of 5 exchange types were screened, and one plant of each exchange type was selected for self-pollination to obtain a secondary F2 population, namely 29-16, 10-21, 7-93, 25-171 and 25-83 ( Figure 3 B). Combined with the pure line phenotype of its offspring, it was found that the grain weight of the offspring of the two pure lines of the recombinant single plant derived population heterozygous between marker 6L15 and marker M2 was significantly different ( Figure 3 B). The present invention finally narrowed the QTL target interval to a physical interval of about 31.7 Kb between markers 6L15 and M2.

[0044] (3) Candidate genes HkDJ Clone carry QTgw.ZiSc-6H Only one open reading frame is annotated within the target interval of the site, which is named HkDJ ( Figure 3 C indicates the gene QUR), the gene is 1938 bp in length. Through its cDNA sequencing, it was found that the gene encodes a protein sequence containing 310 amino acids, which contains 3 exons in total. Parental sequence analysis found that compared with the purple light naked two-leng, the SNP (C→A) located in the first exon of Australia No. 3 led to an amino acid variation (proline P→glutamine Q), containing 2 SNP variation sites. The other variation is located in the third exon and is a synonymous mutation. Multi-species protein homology comparison analysis found that the amino acid variation site in HvTGW1 is located in the N-terminal conserved functional domain, and proline is a conserved amino acid. Therefore, it is speculated that the SNP site on the first exon is the determining factor. HkDJ Key variation sites of gene biological function.

[0045] against QUR A KASP marker SNP53 was designed for the variant site in the gene coding region, and its amplification primers were the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. This marker can effectively distinguish the two parents. The marker was used to genotype 454 barley core germplasm resources, and the genetic effect of the variant site was analyzed in combination with the grain weight phenotypic data. The results showed that QUR S The grain weight was significantly higher than QUR Z ( P <0.001) ( Figure 4 ), indicating that the variant site is significantly correlated with grain weight and can be further used in molecular marker-assisted selection breeding of grain weight and yield.

[0046] In order to verify the authenticity and validity of the variant site, the present invention will QUR S The allele was introduced into the Xila 22 variety ( QUR Z ). Backcross assisted selection using SNP53 marker ( Figure 5 ), and finally screened out QUR S A high-generation pure line of alleles, such as BC4F1, Figure 5 The middle is the genotype detection of BC4F1 generation single plants using SNP53 marker, among which the parent Aoxuan No. 3 is distributed in the upper left corner, that is, HkDJ S Genotype, the lower right corner is the genotype of the parent Xila 22, that is HkDJ Z Genotype; 2 of them have the same genotype as the Xila 22 parent, and about half of the remaining plants are HkDJ S genotype; the other half is heterozygous genotype, that is HkDJS / HvTGW Z The genotype is HkDJ S The base was selected for self-pollination, and two generations of self-pollination were combined with phenotypic selection to obtain BC4F3 pure line single plants. Compared with the parent Xila 22, BC4F3 ( QUR S ) has similar field performance to that of Figure 5 ), further investigated the yield and agronomic traits of the recipient parent Xila 22 and the backcross lines, and found that the thousand-grain weight, grain length and grain width of BC4F3 were significantly higher than those of Xila 22, and the thousand-grain weight could be increased by about 4.5 g ( Figure 6 ); while there were no significant differences in agronomic traits such as plant height, ear length and number of grains per ear ( Figure 7 (Figure 2) Comparison of agronomic traits such as plant height, ear length and number of grains per ear between BC4F3 and Xila 22. This result is consistent with the comparison between the near-isogenic lines NIL-S and NIL-Z. Therefore, this mutation site can effectively improve the yield traits of barley.

[0047] SNP53 marker sequence Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A barley grain weight-related gene HvTGW1s, which has the function of increasing barley thousand-grain weight, grain width and grain length, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A barley grain weight-related protein, characterized in that The barley grain weight-related gene according to claim 1 HkDJ s Expressed, Its amino acid sequence is shown in SEQ ID NO:

2.

3. The barley grain weight-related gene according to claim 1 HkDJ s Application in assisted selection breeding for barley yield.

4. The barley grain weight-related gene according to claim 1 HkDJ s Application in breeding transgenic barley varieties with improved barley yield.

5. KASP marker SNP53 associated with barley yield traits, characterized in that The molecular marker is located on the barley kernel weight-related gene on barley chromosome 6HL HkDJ The 53rd nucleotide of the gene has a C / A mutation. ， When the nucleotide is A, the thousand-grain weight, grain length and grain width of barley are significantly increased.

6. A primer for detecting the KASP marker SNP53 associated with barley yield traits according to claim 5, characterized in that: The amplification primers are nucleotide sequences shown as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:

5.

7. Used to assist in identifying genes with high yield in barley HkDJ s The kit is characterized in that The kit comprises the amplification primers for the KASP marker SNP53 as claimed in claim 6.

8. Use of the KASP marker SNP53 according to claim 5 in assisted selection breeding of barley yield.

9. A method for improving barley yield traits, characterized in that: The steps include: A barley plant containing the barley grain weight-related gene as claimed in claim 1 is used as a donor parent and hybridized with a barley recipient parent to be improved to obtain F1 seeds, The F1 seeds are hybridized with the recipient parent to be improved to obtain BC1F1 seeds. Plant the BC1F1 seeds, and select the individual plants with the HvQTGW1S genotype and phenotype close to the recipient parent to be improved, and hybridize them with the recipient parent to be improved to obtain the BC2F1 generation seeds. The BC2F1 generation seeds are then backcrossed for two generations to finally obtain the BC4F1 generation seeds, and the BC4F1 generation seeds are self-pollinated for two generations to obtain the BC4F3 pure line containing the barley grain weight-related gene as described in claim 1, which is the high-yield barley cultivated.

10. The method for improving barley yield traits according to claim 9, characterized in that: The recipient parent of the barley to be improved is Barley Xila 22, and the donor parent is Aoxuan 3 containing the HvQTGW1S allele.

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