A barley grain weight-related gene HvTGW1 s and its applications

Abstract

The present invention belongs to the field of biotechnology and particularly relates to a barley grain weight-related gene. HvTGW1 s The present invention discloses a gene related to barley grain weight. HvTGW1 s The base sequence is shown in SEQ ID NO: 1. The grain weight-related gene has the function of increasing barley thousand-grain weight, grain width, and grain length. The present invention also discloses a KASP marker associated with barley yield traits and its application in assisted selection breeding for barley yield. The present invention also discloses a kit for assisted identification of barley yield. Applying the present invention to assisted selection breeding for barley yield can effectively improve breeding efficiency and accelerate the breeding process.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to barley grain weight-related genes. HvTGW1 s and its applications. Background Art

[0002] Barley yield per unit area is composed of the number of ears per unit area, the number of grains per ear, and grain weight. Research has shown that grain weight has the greatest genetic influence among yield components, with a generally high broad-sense heritability (>80%) (Hu Yanji et al., 1994). Therefore, grain weight is a key trait in high-yield barley breeding, and the cloning and identification of key genes is the foundation of molecular genetic improvement.

[0003] Barley grain weight is a qualitative-quantitative trait locus controlled by major and minor genes. Although a large number of QTL loci related to barley grain weight have been reported, the fine mapping of major QTL loci related to grain weight is relatively lagging behind. Watt et al. finely mapped the major QTLs 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 narrowed 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 map-based cloning strategies, such as those that regulate grain width. GS5 、 OsSPL16 、 GW2 and GW5 , regulating granule length GS3 , regulating grain size ANT 、 GIF1 and TGW6 etc. (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 homology cloning strategies. For example, the negatively regulated grain length gene GW2 Homologous clones were found in barley HvGW2 Two transposon insertions in the sixth intron of the gene may reduce the gene's transcriptional activity, thereby affecting grain weight. To date, no map-based cloning studies of QTLs for grain weight in barley have 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), gene cloning research in 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. HvTGW1 s , and KASP markers associated with barley yield traits and their application in assisted selection breeding for barley yield. The specific technical solutions provided by the present invention are:

[0007] A barley grain weight-related gene HvTGW1 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 HvTGW1 s Application in assisted selection breeding of barley yield and in breeding transgenic barley varieties with improved barley yield.

[0008] Also provided is a barley grain weight-related protein, which is obtained from the barley grain weight-related gene according to claim 1. HvTGW1 s Place Express, Its amino acid sequence is shown in SEQ ID NO: 2.

[0009] 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 chromosome 6HL of barley. HvTGW1 The allele C / A exists at the locus. When the allele is A, the thousand-grain weight, grain length, and grain width of barley are significantly increased. And the use of the KASP marker SNP53 in assisted selection breeding for barley yield.

[0010] The present invention also provides primers for detecting the KASP marker SNP53 associated with barley yield traits, 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 method for assisting in identifying high-yield barley genes. HvTGW1 s The kit comprises amplification primers for the KASP marker SNP53.

[0011] Finally, the present invention provides a method for improving barley yield traits, comprising the following steps:

[0012] A 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.

[0013] The F1 seeds are hybridized with the recipient parent to be improved to obtain BC1F1 seeds.

[0014] Plant the BC1F1 seeds and select the ones containing HvQTGW1 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.

[0015] 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.

[0016] In the above method for improving barley yield traits, it is preferred that the barley recipient parent to be improved is Damaixila 22, and the donor parent is a barley containing HvQTGW1 S Allele of Australia's choice 3.

[0017] The present invention has at least the following beneficial effects:

[0018] 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 HvTGW1 At the same time, the present invention has been identified HvQTGW1 A molecular marker, SNP53 (C / A), was designed for the key gene mutation site, which is significantly correlated with grain weight. Applying this method to assisted selection breeding for barley yield can effectively improve breeding efficiency and accelerate the breeding process.

[0019] Other advantages of the present invention will be apparent in part from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the genetic linkage map and positioning map for the identification of the major effect QTL locus (QTgw.ZiSc-6H) in the examples of the present invention.

[0021] Figure 2 These are the plant and grain phenotypes of the near-isogenic pure lines (NIL-Z and NIL-S) in the examples of the present invention. NIL-Z represents a subpopulation whose target interval is the homozygous purple-light naked two-angle genotype, and NIL-S represents a subpopulation whose target interval is the homozygous Australian selection 3 genotype.

[0022] Figure 3 In the embodiment of the present invention QTgw.ZiSc-6H Fine positioning and candidate gene cloning diagram, where A is the BC3F3 exchange single plant genotype and derived population progeny test diagram; where 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 the significance level P ≤ 0.05; ** indicates significant level P ≤ 0.01, where C is the gene HvQTGW1 Location map.

[0023] Figure 4 This is a comparison chart of grain weight performance of different alleles in the examples of the present invention.

[0024] Figure 5 For the embodiment of the present invention, Hi-22 and BC4F3 ( HvQTGW1 S )Photos of field performance.

[0025] Figure 6 For the embodiment of the present invention, Hi-22 and BC4F3 ( HvQTGW1 S ) Characteristic investigation diagram, * indicates significant level P≤0.05; NS indicates no significant difference.

[0026] 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

[0027] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be understood that terms such as “having,” “including,” and “comprising” used herein do not prescribe the existence or addition of one or more other elements or combinations thereof.

[0029] 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.

[0030] The present invention identifies the gene through a map-based cloning strategy, identifies key mutation sites of the gene, and develops molecular markers for the mutation sites.

[0031] The present invention used a 190-line RIL population provided by Zeng Yawen's research group at the Yunnan Academy of Agricultural Sciences (Fan et al; 2017) to conduct QTL mapping analysis on barley yield-related traits such as 1000-grain weight, grain width, and grain length. The population parents included a local variety Ziguangmang Nuerlong and a beer barley variety Aoxuan No. 3. Fine mapping was performed using a near-isogenic line population, with Aoxuan No. 3 as the recurrent parent and Ziguangmang Nuerlong as the donor parent. After three and four generations of backcrossing, BC3F1 and BC4F1 populations were obtained, respectively. Heterozygous plants containing two allelic variants in the target interval were further selected for self-pollination to obtain BC3F2 and BC4F2 populations, respectively. 454 core barley germplasm resources from China and abroad were used for correlation analysis of key variant sites.

[0032] 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.

[0033] After the barley matures, the SC-G batch seed analysis module system of Hangzhou Wanshen Company is used to assist in inspecting the thousand-grain weight, grain width and grain length.

[0034] The present invention conducted preliminary QTL mapping analysis based on an existing population genetic linkage map (Fan et al.; 2017) combined with data from five environmental sites. WinQTLcart mapping software used the composite interval mapping method to detect QTLs. Default parameters were selected: the step speed was set to 1 cM and the LOD threshold was less than or equal to 0.05 after 1000 calculations.

[0035] Based on the initial positioning, the present invention uses boundary markers of the major effect QTL interval to screen a BC3F2 population to obtain recombinant exchange plants, and conducts progeny tests on these plants, that is, obtains a secondary F2 population through self-pollination of the plants, uses boundary markers to screen plants homozygous for the genotype of the major effect QTL interval for trait investigation, obtains the phenotypic average value of two pure lines with different alleles, uses the statistical software IBM SPSS 20 software to perform a Student's t-test, compares the average values ​​of the two pure lines, and narrows the target interval carrying the major effect QTL segment; on this basis, uses a BC4F2 population to screen recombinant exchange plants to further narrow the QTL interval.

[0036] To identify candidate genes for the target QTL interval, the present invention used the latest barley reference genome, Morex v3.0 (https: / / galaxy-web.ipk-gatersleben.de / ), to perform BLASTN alignment analysis on the QTL boundary marker sequences, anchor the QTL interval, search for annotated genes within it, and obtain the gene sequences of both parents through sequence amplification. The amplification system was as follows: parental whole genomic DNA was used as a template, and the gene PCR product was amplified using the Tks Gflex DNA Polymerase kit produced by TaKaRa. The 25µl system: 1.0µl genomic DNA (40ngμL -1 ); 0.5µl (10µmolL) of each of the left and right primers (NC1) -1 ); 12.5µl 2× Gflex PCR Buffer; 10µl ddH2O; 0.5µl Tks Gflex DNA Polymerase (1.25U / μl). The amplification program was as follows: denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 68°C for 30 seconds. The PCR products were stored at 12°C and sent to Qingke Biotech Co., Ltd. for sequencing.

[0037] candidate genes HvTGW1 To obtain the cDNA sequence: RNA was extracted from early-stage seeds (two days after pollination) using the Trizol method. Single-stranded cDNA was synthesized by reverse transcription using a Promega reverse transcription kit. PCR amplification was performed using primer NC2 and the Tks Gflex DNA Polymerase Kit. 2 µl of single-stranded cDNA was used as template. The remaining system and amplification procedures were identical to those described above. PCR amplified the nucleotide sequence containing the open reading frame. This sequence was subjected to T-ligation sequencing. A 5 µl aliquot consisted of 1 µl of T-vector and 4 µl of amplified product. The amplified product was gently mixed on ice and ligated at 25°C for 15 minutes. The ligation product was added to thawed competent cells on ice, heat-shocked for 40 seconds, then incubated on ice for 5 minutes. The product was then transformed into Escherichia coli DH5α and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0038] candidate genes HvTGW1 The protein sequence of HvQTGW1 was obtained and its conserved structure was analyzed: the nucleotide sequence obtained by cDNA sequencing was translated to obtain its protein sequence. On this basis, the conserved functional domain of HvQTGW1 protein sequence was analyzed by Smart. At the same time, BLASTP comparison was performed using the plant protein database to find HvQTGW1 Compare and analyze the conserved functional domains of proteins from gramineous crops such as Arabidopsis and rice that have similar genes.

[0039] The simple sequence repeat (SSR) markers used for QTL interval encryption in the present invention were developed as follows: Based on the Morex v3.0 reference genome information, the QTL target interval was defined by flanking markers, the nucleotide sequence within the target interval was obtained, and SSR markers were designed.

[0040] Diagnostic markers: based on HvTGW1 Methods: We designed competitive allele-specific PCR-single nucleotide polymorphism (KASPSNP) markers to identify differentially expressed SNPs. The genotypes of 454 core barley germplasm accessions from China and abroad were analyzed. Fluorescence signals were detected by an Applied Biosystems 7900 HT, and Kluster caller software was used to analyze the population typing results.

[0041] The recipient parent Xila 22 to be improved is one of the highland barley varieties widely planted in Tibet. HvQTGW1 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 further generations and hybridized with Xila 22 to obtain BC1F1 seeds. In October 2019, the BC1F1 seeds were planted in a greenhouse for further generations. Samples were taken at the seedling stage, and the genotypes of all BC1F1 generations were analyzed using the SNP53 marker to screen out those containing HvQTGW1 S The genotype of the single 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 HvQTGW1 S BC4F3 pure lines of alleles.

[0042] 1) KASP primer mix system:

[0043] FAM-Primer 12 μL

[0044] HEX-Primer 12 μL

[0045] Common Reverse-Primer 30 μL

[0046] ddH2O 46 μL

[0047] Total 100 μL

[0048] 2) KASP genotyping PCR reaction system:

[0049] 2x KASP Master Mix 2.50 μL

[0050] Primer Mix 0.07 μL

[0051] ddH2O 1.25 μL

[0052] DNA 1.25 μL

[0053] Total 5.07 μL

[0054] The reaction process is divided into four stages:

[0055] Stage 1: DNA denaturation, pre-denaturation at 94°C for 15 minutes;

[0056] Stage 2: denaturation at 94°C for 20 seconds, annealing and extension at 61°C for 1 minute, with the temperature decreasing by 0.6°C each cycle, for 10 cycles;

[0057] Stage 3: denaturation at 94°C for 20 s, annealing and extension at 55°C for 1 min, for 34 cycles;

[0058] Stage 4: Store at 16°C.

[0059] KASP amplification reaction: performed in an Applied Biosystems® Veriti® 384-Well Thermal Cycler gradient gene amplification instrument.

[0060] 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.

[0061] 2. Experimental Results

[0062] 2. Experimental Results

[0063] (1) QTL location analysis

[0064] Combining the existing genetic linkage map of the RIL population (Fan et al; 2017) and data from five environmental sites, 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 1The agronomic and yield traits of two near-isogenic pure lines, NIL-S and NIL-Z, were investigated after four generations of backcrossing. 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 ).

[0065] Table 1. Performance of traits such as thousand-grain weight in near-isogenic pure lines

[0066]

[0067] Note: NIL-S represents the subpopulation with the homozygous Aoxuan 3 genotype, and NIL-Z represents the subpopulation with the homozygous Ziguangmang naked Erling genotype. * indicates a significant level of P ≤ 0.05; ** indicates a significant level of P ≤ 0.01

[0068] right A 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 of

[0069] 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, we screened the BC3F2 population and obtained individuals that underwent recombination between these two markers. A total of 10 recombinant types were obtained, and one plant from 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 individual pure lines of these F2 population offspring, the grain weight difference between the pure lines of the exchanged individual plants containing the heterozygous fragment between markers 6C52 and B1 was significant ( Figure 3 A), therefore, the target QTL interval was located within an interval of approximately 114 Kb between markers 6C52 and B1.

[0070] Table 2 Genetic molecular marker sequences used for QTL map encryption and gene amplification

[0071]

[0072] 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 3B), 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 phenotypes of its offspring, it was found that the grain weight difference between the two pure lines of the offspring of the recombinant single plant derived from the heterozygous marker 6L15 and marker M2 was significant ( Figure 3 B). The present invention ultimately narrowed the QTL target interval to a physical interval of approximately 31.7 Kb between markers 6L15 and M2.

[0073] (3) Candidate genes HvTGW1 clone

[0074] carry QTgw.ZiSc-6H Only one open reading frame is annotated within the target interval of the site, which is named HvTGW1 ( Figure 3 C indicates the gene HvQTGW1 ), the gene is 1938 bp long, and its cDNA sequencing revealed that the gene encodes a protein sequence containing 310 amino acids, which contains 3 exons. Parental sequence analysis found that compared with Purple Light Naked Two-Legged, the SNP (C→A) located in the first exon of Australia No. 3 led to an amino acid mutation (proline P→glutamine Q), containing 2 SNP mutation sites. The other mutation is located in the third exon and is a synonymous mutation. Multi-species protein homology comparison analysis found that the amino acid mutation 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. HvTGW1 Key mutation sites in gene biological functions.

[0075] against HvQTGW1 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. 454 core barley germplasm resources were genotyped using this marker, and the genetic effect of the variant site was analyzed in combination with the grain weight phenotypic data. HvQTGW1 S The grain weight was significantly higher than HvQTGW1 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.

[0076] In order to verify the authenticity and validity of the variant site, the present invention will HvQTGW1S The allele was introduced into the Xila 22 variety ( HvQTGW1 Z ). Backcross assisted selection was performed using the SNP53 marker ( Figure 5 ), and finally screened out HvQTGW1 S High-generation pure lines 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. 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 Xila 22 parent, and about half of the remaining plants are HkDJ S genotype; the other half are heterozygous genotype, i.e. HkDJ S / 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 ( HvQTGW1 S ) has similar field performance to that of Figure 5 ), further investigation of the yield and agronomic traits of the recipient parent Xila 22 and the backcross lines revealed 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 ); however, there were no significant differences in agronomic traits such as plant height, ear length and number of grains per ear ( Figure 7 (Figure 2 compares agronomic traits such as plant height, ear length, and grain number 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 variant locus can be effectively used for genetic improvement of barley yield traits.

[0077] SNP53 marker sequence

[0078] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. 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 illustrations shown and described herein.

Claims

1. A gene related to barley grain weight HvTGW1 s Application of the barley grain weight-related gene in assisted selection breeding of barley yield HvTGW1 s The nucleotide sequence is shown in SEQ ID NO:

1.

2. A gene related to barley grain weight HvTGW1 s Application of the barley kernel weight-related gene in cultivating transgenic barley varieties with improved barley yield HvTGW1 s The nucleotide sequence is shown in SEQ ID NO:

1.

3. Used to detect genes related to barley grain weight HvTGW1 s The primer is characterized in that The nucleotide sequences of the primers are shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5. HvTGW1 s Located on the barley chromosome 6HL, 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 the barley are significantly increased. HvTGW1 s The nucleotide sequence is shown in SEQ ID NO:

1.

4. Used to assist in identifying the barley grain weight-related genes HvTGW1 s The kit is characterized in that The kit comprises the primers according to claim 3, the barley grain weight-related gene HvTGW1 s The nucleotide sequence is shown in SEQ ID NO:

1.

5. Use of the kit according to claim 4 in assisted selection breeding of barley yield.

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