CN111593060B

CN111593060B - Rice grain length gene and application of molecular marker thereof

Info

Publication number: CN111593060B
Application number: CN202010505614.0A
Authority: CN
Inventors: 李一博; 李翠翠; 樊亚伟
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2022-04-19
Anticipated expiration: 2040-06-05
Also published as: CN111593060A

Abstract

The invention discloses a rice grain length gene and application of a molecular marker thereof, belonging to the technical field of plant functional genomes, wherein a regulatory gene is GS3-5, a nucleotide sequence of a GS3-5 coding region is shown as a sequence table SEQ ID NO.1, and an amino acid sequence is shown as a sequence table SEQ ID NO. 2. GS3-5 is the GS3 allele with the strongest function for negatively regulating the grain length of rice, and lays a foundation for fully utilizing the GS3 excellent allele in rice yield breeding and quality breeding. The invention also provides a functional marker GS3-CDS which can distinguish multiple GS3 alleles, and can simply, quickly and inexpensively identify different allelic forms of GS3 so as to predict rice grain length variation; meanwhile, molecular markers for finely positioning the GS3-5 gene are provided, so that the ultrashort-grained genotype can be screened. The functional marker and the molecular marker can assist breeding to improve breeding efficiency and provide more gene resources and reference values for realizing rice molecular design breeding.

Description

Rice grain length gene and application of molecular marker thereof

Technical Field

The invention belongs to the technical field of plant functional genomes, and particularly relates to a rice grain length gene and application of a molecular marker thereof.

Background

Rice is one of the most important crops in China and is staple food for more than half of the world population. How to increase the rice yield as soon as possible, and improving the rice quality becomes an important target of rice genetic breeding. The rice grain shape (grain length, grain width, grain thickness and length-width ratio) is a complex quantitative character, is regulated and controlled by multiple genes, and is also a character with higher heritability. The grain shape not only directly affects the yield of a single rice plant, but also affects the appearance quality and processing quality of rice. Meanwhile, the rice grain shape character is easy to select and fix in the evolution process, and can be used for researching the rice evolution process. Therefore, cloning of functional genes related to grain shape and elucidating the mechanism of formation thereof are the basis for further improving rice yield and rice quality. Meanwhile, functional genetic variation of the grain shape gene is excavated, and corresponding functional markers are developed, so that more gene resources and reference values are provided for realizing rice molecular design and breeding.

The grain shape of rice is a typical quantitative trait and is regulated by a plurality of major genes and a plurality of minor genes. Carrying out primary positioning and effect value analysis on QTL (quantitative Trait Locus) for controlling the grain shape traits by utilizing molecular markers in a primary mapping population, and decomposing complex quantitative traits into simple Mendelian factors for research; and constructing a secondary mapping population, screening more recombinant single plants for a progeny test, so as to narrow the interval, finish fine positioning and finally clone the target gene. The traditional map-based cloning method mainly comprises the steps of constructing a genetic linkage map, positioning and cloning corresponding target genes, constructing a mapping population and screening molecular markers, and is time-consuming and labor-consuming. With the rapid development of high throughput sequencing technologies, a variety of simple methods for locating genes by sequencing means have been developed. Two methods for rapidly mapping QTLs, MutMap and QTL-seq, were developed by combining whole genome sequencing with BSA analysis (Michelmore et al, 1991, Proc Nat Acad Sci USA,88: 9828-. By combining the advantages of a BSA Analysis method (bulk Segregant Analysis) and a chip technology/second-generation sequencing technology, a new rapid map-based cloning method is developed by introducing a cosegregation marker, and the possibility is provided for rapid large-scale cloning of natural variation regulatory genes.

Based on the map-based cloning method, a series of QTL sites for controlling grain shape on a rice genome are identified, and mainly comprise rice grain length controlling genes GS3, qGL3, TGW6, GW6a, GS2, GLW7, TGW3 and the like, and rice grain width controlling genes GW2, GW5, GS5, GW8, GW7 and the like (Fan et al, 2006, Theor Appl Genet,112: 1164-.

GS3 is the first major gene for controlling rice grain shape cloned by Fan et al using map cloning method, and the gene codes a trimer G protein gamma subunit containing 232 amino acids. The complete GS3 protein contains 4 domains, namely an N-terminal plant-specific Organ size regulation domain (OSR), transmembrane domain, TNFR domain (TNFR), and VWFC domain (Von Willebrand factor type C, VWFC). To date, four alleles of GS3 have been found (Mao et al, 2006, Proc Natl Acad Sci USA,107(45): 19579-. Among them, MH63(GS3-3, long grain) has a mutation of the cysteine codon (TGC) at the second exon of GS3 to a stop codon (TGA) resulting in premature translation termination and 178 amino acids deletion at the 3' end of the protein, compared to the GS3 sequences of ZS97(GS3-1, medium grain) and NIP (GS3-2, medium grain), thus the encoded protein loses its function and becomes larger seeds. Whereas the second exon of GS3 in Chuan 7(GS3-4, short grain) is not mutated, but the deletion of one C base at the fifth exon leads to translation early termination, the GS3 protein lacks TNFR and VWFC domains, only one OSR domain is reserved, shows a ultrashort grain phenotype, and is a specific strong functional allelic variation. Further studies showed that the GS3 protein competes for binding to RGB1 through the N-terminal domain, inhibits downstream signals of DEP1/GGC2 to negatively regulate grain length, and stabilizes self-protein through the C-terminal domain. The GS3 protein in ZS97 was more easily degraded because the C-terminal tail was longer, and GS3-4 accumulated more GS3 protein than GS3-1 (Sun et al, 2018, Nat Commun,9: 851).

The function of the cloned granulosa gene is summarized and analyzed, and the function mainly relates to various regulation and control paths such as ubiquitin proteasome pathway, G protein signal pathway, hormone pathway, MAPK signal pathway, polypeptide signal pathway, epigenetic and transcription factor pathway and the like (Fan et al, 2019, Mol Breeding,39: 163-25). Some channels are mutually crossed to form a complex and precise regulation network, and the growth and development of rice seeds are finally influenced. At present, most of cloned rice grain-shaped genes are functional research and application of single genes, polygene genetic interaction research is less, and interaction regulation networks among the grain-shaped genes are further perfected on the cloning and function analysis of the grain-shaped genes.

Disclosure of Invention

The invention aims to identify a new allele GS3-5 of a major gene GS3 for controlling rice grain length by using a map-based cloning method, separate a complete coding segment DNA fragment, improve rice yield and rice quality by using the new allele, and provide a new gene resource for genetic improvement of rice yield and quality.

In order to achieve the purpose, the invention adopts the technical scheme that:

a rice grain length regulation gene is GS3-5, wherein the nucleotide sequence of the GS3-5 coding region is shown in a sequence table SEQ ID NO. 1.

Preferably, the amino acid sequence of GS3-5 is shown in a sequence table SEQ ID NO. 2.

An application of a rice grain length regulation gene GS3-5 in regulating rice grain length.

A functional marker of a rice grain length regulatory gene is GS3-CDS primer, and the sequence of the GS3-CDS primer is shown in a sequence table SEQ ID NO. 3-4.

The application of the functional marker GS3-CDS primer in distinguishing GS3 alleles GS3-1, GS3-2, GS3-4 and GS 3-5.

An InDel molecular marker of a rice grain length regulatory gene is characterized in that the InDel molecular marker is C3L4 and C3L6, wherein a primer sequence of the molecular marker C3L4 is shown as a sequence table SEQ ID NO.5-6, and a primer sequence of the molecular marker C3L6 is shown as a sequence table SEQ ID NO. 7-8.

An InDel molecular marker of a rice grain length regulatory gene is characterized in that the InDel molecular marker is C3S6 and C3S7, wherein a primer sequence of the molecular marker C3S6 is shown in a sequence table SEQ ID NO.9-10, and a primer sequence of the molecular marker C3S7 is shown in a sequence table SEQ ID NO. 11-12.

The application of the InDel molecular marker of the rice grain length regulating gene in the fine positioning GS3-5 and molecular marker assisted breeding.

Compared with the prior art, the invention has the beneficial effects that:

(1) the allele GS3-5 of the major gene GS3 of the rice grain length is identified in ultra-short grain rice, and GS3-5 is the GS3 allele which is discovered at present and has the strongest function and negatively regulates the rice grain length, so that the basis is laid for fully utilizing the GS3 excellent allele in rice yield breeding and quality breeding, and a new direction is provided for exploring a molecular mechanism for forming rice grain shapes and perfecting an interaction regulation network among the grain shape genes.

(2) The invention provides a functional marker GS3-CDS which can distinguish multiple GS3 alleles, can simply, quickly and inexpensively identify different allelic gene types of GS3 to predict rice grain length variation, and also provides a molecular marker for finely positioning GS3-5 to screen ultra-short grain shape gene types.

Drawings

FIG. 1 is a technical flowchart of embodiment 1 of the present invention;

FIG. 2 is a graph showing the result of allele identification of GS3-5 in example 1 of the present invention, wherein 2-a is a graph showing the grain length phenotype of ZML and Chuan 7; FIGS. 2-b and 2-c are ZML/Chuan 7F₂A grain length phenotype map of the parents of the genetic population; FIG. 2-d is a map-based clone of the GS3-5 gene, and FIG. 2-e is a statistical graph of grain length phenotypes for two homozygous genotypes;

FIG. 3 is a graph showing the results of allele identification of GS3-5 in example 1 of the present invention, wherein 3-a is a graph showing the phenotype of the particle length of NIP and core 478; FIGS. 3-b and 3-c show NIP/core 478F₂A grain length phenotype map of the parents of the genetic population; FIG. 3-d is a map-based clone of the GS3-5 gene, and FIG. 3-e is a statistical plot of grain length phenotypes for two homozygous genotypes;

FIG. 4 is a graph of the results of a PAGE test to distinguish the various GS3 alleles using the functional marker GS 3-CDS;

FIG. 5 shows a comparison of the gene and protein sequences of the five allelic forms of GS 3;

FIG. 6 is a seed length phenotype plot of five rice varieties corresponding to five alleles of GS 3.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 map-based cloning and validation of GS3-5

The technical flow chart of map-based cloning and verification of GS3-5 gene is shown in the attached figure 1, and the specific method is as follows:

construction of GS3-5 location genetic population and determination of rice grain length and grain width

Selecting an ultra-short grain parent ZML (53.2mm, derived from farmer varieties) and a short grain variety Sichuan 7(58.69mm) for hybridization, wherein the grain length phenotype graphs of the ZML and the Sichuan 7(Chuan7 or C7) are shown in the attached figure 2-a; simultaneously selecting ultra-short parental core 478(55.42mm, derived from farmer variety) and conventional medium variety NIP (78.67mm) for hybridization, wherein the grain length phenotype graphs of core 478(HX478) and NIP are shown in FIG. 3-a. The resulting hybrids were planted in 2 rows (12 plants per row). According to the SSR standard for identifying the authenticity of rice varieties, true and false hybrid identification is carried out on each single plant by means of polymorphic molecular markers, and 1F is selected₁Planting the progeny of the single plant into a large group of 16 rows (12 plants in each row) to obtain F₂A genetic population.

F₂Selecting representative 10 plump seeds from each individual plant in the random population, sequentially arranging the seeds end to end in the same direction length or in a shoulder-shoulder manner in the same width without overlapping and leaving gaps, reading ten-grain length or ten-grain width data by using a vernier caliper, repeating for 3 times, and taking the average value as phenotype data. When examining the grain shape phenotype, individual plants with abnormal individual grain shape phenotype should be removed, and uniform and representative seeds are selected, so as to avoid influence on result accuracy caused by experimental error. Wherein ZML/Chuan 7F₂The grain growth phenotype of the genetic population is shown in FIGS. 2-b and 2-c, NIP/core 478F₂The grain length phenotype of the genetic population is shown in FIGS. 3-b and 3-c

2, combining BSA method and Rice6k gene chip technology to carry out preliminary location analysis on QTL GS3-5

Investigation of ZML/Chuan 7F₂After the grain length phenotype of 130 individuals in the genetic population is reached, selectingTaking 30 single plants with the grain length of less than 55.50mm and 30 single plants with the grain length of more than 60.38mm, and respectively establishing two extreme grain length phenotype DNA mixing pools. Simultaneous survey of NIP/core 478F₂After the individual plants of 110 individual plants in the genetic population have the grain length phenotype, 26 individual plants with the grain length smaller than 61.51mm and 21 individual plants with the grain length larger than 72.01mm are selected, and two extreme grain length phenotype DNA mixing pools are respectively established. Selecting 5 seeds for mixed germination of each plant, mixing and sampling after germination for 7-10 days, and ensuring that all germinated samples are taken, wherein the length of the leaf is 2 cm. And grinding a blade sample of each pool by using liquid nitrogen to form a DNA mixing pool, recording corresponding numbers, sending the DNA mixing pools to the Wuhan life science technology center of China seed group for Rice6k gene chip detection and drawing an SNP chip detection diagram, and finding that signals are dense in the middle of No.3 chromosome of Rice according to the SNP detection result of the gene chip, so that the target section is preferentially verified.

3. Molecular marker detection analysis of genotype

Designing a corresponding molecular marker according to a local rice database RiceVarMap website (http:// RiceVarMap. ncpgr. cn/v1/), and specifically comprising the following steps: inputting the chromosome of the target section and the corresponding physical position, obtaining all InDel marks in the section, screening the InDel marks with 3-6bp difference in the parental sequence, and designing the primers with difference by using the website primer design function. Wherein the final PCR amplified fragment is preferably controlled to be about 80-150bp, and the specificity is detected by Blast. The synthesized InDel primer needs to use two random mixing pools of parents and populations for polymorphism screening, and a polymorphism marker with good amplification is selected to carry out polyacrylamide gel electrophoresis (PAGE) detection on a positioning population for genotype analysis.

Wherein the reaction system of the PCR comprises: 4 μ l of the small sample DNA +8 μ l of ddH₂O + 20. mu.l mineral oil + 2. mu.l 10 XPCR buffer + 0.35. mu.l dNTPs + 0.2. mu.l Primer F + 0.2. mu.l Primer R + 0.1. mu. l R-Taq enzyme + 5.15. mu.l ddH₂And O. PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 30 cycles (the specific annealing temperature and cycle number are adjusted according to the corresponding primer); extending for 7min at 72 ℃; keeping the temperature at 25 ℃ for 1 min. PCR product after amplification is inSilver staining was performed after separation on 4% polyacrylamide gel. Primers showing band differences in parents can be used to further detect the molecular marker genotype of individual strains of the population. This example is used for primary and further fine localization of molecular markers of GS3-5, and the corresponding primer sequences and functions are shown in Table 1.

TABLE 1 primer sequences and functions used in example 1

4. Co-segregation verification GS3-5 QTL effect

For ZML/Chuan 7F₂The genetic population is analyzed by the primary location markers C3L1 and C3L2 (see Table 1 for primer sequences), and the two homozygous genotypes of each marker in the target segment of chromosome 3 can obviously distinguish the grain length phenotype, i.e., the phenotype is co-segregated with the genotype. Of these, 5 recombinant individuals supported the left border C3L1 marker and 10 recombinant individuals supported the right border C3L2 marker, thus initially locating the associated grain length QTL within the physical interval of about 1Mb of the two molecular markers C3L1 and C3L2 (as shown in FIG. 2-d). And t-test analysis of grain length phenotype of the two homozygous genotypes found P<0.001, indicating that the grain length phenotype of the two homozygous genotypes was very significantly different (as shown in figure 2-e).

For NIP/core 478F at the same time₂The genetic population primary localization markers C3S1 and C3S2 (primer sequences are shown in Table 1) were analyzed, and the phenotype and genotype of each marker in the target segment were co-segregated. Among them, 3 recombinant individuals supported the left border C3S1 marker and 7 recombinant individuals supported the right border C3S2 marker, thus preliminarily locating the associated grain length QTL within the physical interval of about 7Mb of the two molecular markers C3S1 and C3S2 (as shown in FIG. 3-d). And t-test analysis of grain length phenotype of the two homozygous genotypes found P<0.001, indicating that the grain length phenotype difference between the two homozygous genotypes was extremely significant (as shown in FIG. 3-e))。

Fine localization of GS3-5 allelic type

To further locate the major QTL of grain length, molecular markers are encrypted in the initially located target sections C3L1 and C3L2 (the primer sequences corresponding to the molecular markers are shown in Table 1), and finally ZML/Chuan 7F₂The genetic population finely locates the grain length QTL in a physical interval (shown in figure 2-d) of about 20kb of two molecular markers of C3L4 and C3L6 (the primer sequences are shown in table 1 and sequence table SEQ ID NO. 5-8); NIP/core 478F₂The genetic population finely locates the grain length QTL in a physical interval of about 18kb (shown in figure 3-d) of two molecular markers of C3S6 and C3S7 (the primer sequences are shown in table 1 and sequence table SEQ ID NO. 9-12). By querying Rice Genome annotation plan data website Rice Genome annotation Project (http:// Rice plant biology. msu. edu /), it was found that only 1 Open Reading Frame (ORF), namely the cloned GS3 gene, is contained in each of the above two intervals.

Comparative sequencing of the GS3-5 allelic type

In order to exclude the interference of other alleles of GS3, a functional marker GS3-CDS is designed at the position of one base deletion mutation of GS3-4, and the sequence of a GS3-CDS primer is shown in a sequence table SEQ ID NO. 3-4. The result of genotyping the ultrashort-grained parent material by PAGE technique is shown in fig. 4. The results show that the genotype of ZML and core 478 is different from that of the existing GS3-1, GS3-2 and GS3-4, and the ZML and the core 478 are a new GS3 allelic gene type and are named as GS 3-5. And the functional marker GS3-CDS can distinguish GS3-1(88bp, corresponding to ZS97 variety), GS3-2(91bp, corresponding to NIP variety), GS3-4(87bp, corresponding to Sichuan 7 variety) and GS3-5(78bp, corresponding to core 478 and ZML variety).

To determine the gene structure of GS3-5, primers (see GS3-cDNA in table 1 for details of primer sequences) were designed based on the known full-length cDNA sequence of GS3 from NIP and the sequences of core 478 and ZML were aligned to the sequences of the three known GS3 alleles. A comparison of the gene and protein sequences for each allele is shown in FIG. 5, in which the black boxes represent exons, the shaded boxes represent UTRs, and the thin lines between the black boxes represent introns. The black triangles represent 3bp insertion (NIP), 1bp deletion (Chuan 7) and 10bp deletion (ZML), respectively, with a four-pointed asterisk at the stop codon. Below each genomic DNA fragment is its corresponding protein structure, where the NIP and ZS97 allele-encoding domains are shown in the figure, the numbers following the domains indicate the number of amino acids encoding the protein, and the five-pointed star indicates the protein sequence position corresponding to the mutation of the different allele type.

Wherein core 478 is identical to the allelic sequence of ZML, the allele being GS 3-5. As can be seen from FIG. 6, the GS3-5 variation position occurs on the fifth exon of GS3, the CDS total length is 686bp (see the sequence table SEQ ID NO.1 in detail), and 146 amino acids are encoded (see the sequence table SEQ ID NO.2 in detail). Compared with the ZS97(GS3-1) sequence, core 478 and ZML have a 10 base deletion upstream of the GS3-4 mutation site, resulting in a frame shift mutation; compared with the sequence of Sichuan 7(GS3-4), the core 478 and ZML GS3 fifth exon sequence has a 9bp deletion, the stop codon position is the same after frame shift mutation, the protein sequence lacks 3 amino acids, the core 478 and the GS3 protein of ZML lack all TNFR and VWFC structural domains at the C end, a shorter protein C end is generated compared with GS3-4, and GS3-5 has stronger function than GS3-4 and generates shorter seeds (as shown in FIG. 6). Based on the fact that GS3-4 is the GS3 allele with the strongest function reported previously, GS3-5 is the GS3 allele which is found to have the strongest function and negatively controls the grain length of rice at present.

In addition, the functional marker GS3-CDS is used for detection, and only the core 478 of the 533 core germplasm resources is of the GS3-5 genotype, shows the rarity of the GS3-5 genotype and is not fully utilized by a breeder, so the allele has great potential for rice grain shape genetic improvement.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

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Claims

1. A molecular marker for finely positioning a rice grain length regulation gene GS3-5 is characterized in that the nucleotide sequence of a GS3-5 coding region is shown as a sequence table SEQ ID NO. 1; the molecular markers are C3L4 and C3L6 or C3S6 and C3S7, wherein the primer sequence of the molecular marker C3L4 is shown in sequence tables SEQ ID NO.5-6, the primer sequence of the molecular marker C3L6 is shown in sequence tables SEQ ID NO.7-8, the primer sequence of the molecular marker C3S6 is shown in sequence tables SEQ ID NO.9-10, and the primer sequence of the molecular marker C3S7 is shown in sequence tables SEQ ID NO. 11-12.

2. The use of the molecular marker of claim 1 for fine localization of rice grain length regulatory gene GS 3-5.

3. The method for locating the rice grain length regulatory gene GS3-5 according to claim 1, wherein the method comprises:

the ZML variety of rice is hybridized with the Chuan7 variety to obtain the ZML/Chuan 7F₂A genetic group, a QTL fine positioning method is adopted to position a regulatory gene GS3-5 in two molecular markers of C3L4 and C3L6, then an open reading frame in the molecular markers is searched by a rice genome annotation plan data website to obtain a rice grain length regulatory gene GS3-5 shown as SEQ ID NO.1,

wherein the primer sequence of the molecular marker C3L4 is shown in sequence tables SEQ ID NO.5-6, and the primer sequence of the molecular marker C3L6 is shown in sequence tables SEQ ID NO. 7-8;

or crossing the rice NIP variety and the core 478 variety to obtain NIP/core 478F₂A genetic group, a QTL fine positioning method is adopted to position a regulatory gene GS3-5 in two molecular markers of C3S6 and C3S7, then an open reading frame in the molecular markers is searched by a rice genome annotation plan data website to obtain a rice grain length regulatory gene GS3-5 shown as SEQ ID NO.1,

wherein the primer sequence of the molecular marker C3S6 is shown in sequence tables SEQ ID NO.9-10, and the primer sequence of the molecular marker C3S7 is shown in sequence tables SEQ ID NO. 11-12.