CN114921472A - Gene for controlling shape of sorghum grain and separation and cloning method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of molecular biology, and discloses a gene for controlling sorghum grain shape, a separation method, a cloning method and application thereof, wherein the gene is SbGS-7, and the sorghum grain shape comprises a round grain shape and a long grain shape; wherein, the nucleotide sequence of the gene for controlling the sorghum grain round grain shape is shown as SEQ ID NO: 1, and the nucleotide sequence of the gene for controlling the grain shape of sorghum grains is shown as SEQ ID NO: 2, respectively. The invention separates and clones a gene SbGS-7 capable of controlling grain shape (long/round grain) of sorghum grains, provides a molecular marker M3 associated with the grain shape, and is used for identifying whether the sorghum contains the long/round grain gene SbGS-7. The invention can carry out molecular marker-assisted selection of long/round grain genotypes in the seedling stage by utilizing the marker, thereby ensuring 100 percent of accuracy, greatly reducing the quantity of field planting materials, saving manpower, material resources and financial resources and accelerating the breeding process of the grain shape characters of the sorghum.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a gene for controlling sorghum grain shape, and a separation and cloning method and application thereof.
Background
Sorghum grains are important raw materials for industrial processing in China. The formation process of the grains is a process of mutually regulating and controlling by a plurality of QTL/genes so as to open a physiological and biochemical metabolic network with front and back connection and alternative bypass. The' grain shape (round, elliptical, oval and long) is one of the important traits of the sorghum grain yield, and has become one of the important traits related to sorghum domestication through long-term selection and domestication of breeders, and is also one of the important causes that grain shape traits of different sorghum inbred lines show obvious difference and the grain for cultivating sorghum is far larger than that of wild ancestors of the sorghum. The abundant variation of grain shape characters provides an extremely important resource base for exploring and analyzing the genetic mechanism of the sorghum yield and the constituent factors thereof. Compared with the rapid development of the grain shape characteristic function analysis of gramineous crops such as rice, corn and the like, the successful identification and separation of the grain shape gene in sorghum is reported, and the process of accelerating the genetic improvement of the yield characteristic of sorghum by using the modern molecular biotechnology is seriously hindered.
Through the above analysis, the problems and defects of the prior art are as follows: in the prior art, genes for successfully identifying and separating grain shapes in sorghum are rarely reported, and the process of accelerating genetic improvement of sorghum yield traits by using modern molecular biotechnology is hindered.
The difficulty in solving the above problems and defects is: due to the lack of genetic research on the grain shape of the sorghum, the breeding efficiency of a new sorghum variety suitable for the grain shape of the Maotai-flavor liquor brewing process is influenced by only depending on experience in the breeding of the liquor sorghum.
The significance for solving the problems and the defects is as follows: for the gene clone for controlling the aspect ratio character of sorghum grains, the variation site influencing the character in the gene is determined. By designing the functional markers, round and long sorghum varieties can be effectively distinguished, and theoretical basis is provided for the molecular marker-assisted breeding and the accelerating of the breeding process of new varieties conforming to the shapes of the grains of the sorghum for wine.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a gene for controlling the shape of sorghum grains, and a separation and cloning method and application thereof.
The gene for controlling the shape of the sorghum grains is SbGS-7, and the shape of the sorghum grains comprises a round grain shape and a long grain shape; wherein the nucleotide sequence of the gene for controlling the sorghum grain round grain shape is SEQ ID NO: 1, the nucleotide sequence of the gene for controlling the length and shape of sorghum grains is SEQ ID NO: 2.
the invention also aims to provide protein coded by the gene for controlling the shape of the sorghum grains, and the amino acid sequence of the protein coded by the gene for controlling the shape of the sorghum grains is SEQ ID NO: 3, the protein amino acid sequence coded by the gene for controlling the sorghum grain to grow into granules is SEQ ID NO: 4.
the invention also aims to provide a functional molecular marker of the sorghum long/round grain shape gene SbGS-7 obtained by using the gene for controlling the shape of the sorghum grains, and an acquisition method of the functional molecular marker M3 of the sorghum long/round grain shape gene SbGS-7, which comprises the following steps:
a functional molecular marker M3 for identifying the long/round-grained gene SbGS-7 based on PCR technology was developed based on the insertion/deletion characteristics of 10 bases contained at base 1828 within the 7 th intron, targeting sorghum, and selected from the group consisting of SEQ ID NO: 5, wherein the nucleotide sequence is 5 '→ 3'.
Another object of the present invention is to provide a method for identifying a sorghum long/roundgrain gene SbGS-7 by using a functional molecular marker of the sorghum long/roundgrain gene SbGS-7, wherein the method for identifying the sorghum long/roundgrain gene SbGS-7 comprises:
(1) and amplifying, sequencing and sequence splicing by a conventional PCR method to obtain a sequence of the sorghum long/round granule gene SbGS-7.
(2) Comparing the sequences obtained in the step (1) by using a multi-sequence comparison software tool and NCBI online Blast to obtain 7 specific base differences of the sorghum long/round grain gene SbGS-7, wherein the differences are located in exons 1 and 2 of the gene, and when the sequences are translated, except 3 of the differences are synonymous mutations, 4 of the differences comprise 2 insertions/deletions and 2 non-homologous mutations, so that functional specific amino acid changes are caused, the grain shape of sorghum is finally influenced, and the 7 specific base differences of the intron sequences are located in introns 1, 4 and 7 of the gene and comprise 4 insertions/deletions and 2 base mutations. Based on the 10-base difference between the long-grain and round-grain varieties in the 7 th intron, M3 functional molecular markers were developed.
(3) Confirming the effectiveness of the functional molecular marker of the SbGS-7 gene in detecting the grain shape of the grain, and verifying the sequence obtained in the step (2) in a natural sorghum group; 24 long-grain strain lines and 25 round-grain strains are selected according to the aspect ratio phenotype, the aspect ratio of specific bands is completely consistent with that of 49 strain grains, and the accuracy is 100%, so that the functional molecular marker of the SbGS-7 gene is determined to have high applicability.
Further, in the step (1), the PCR amplification reaction system includes:
sorghum genome DNA 1ul, forward and reverse primers 1ul, Takara Ex Taq 0.2ul, 10 XEx Buffer2ul, dNTP 2mM 2ul, dd H 2 O12.8 ul, total volume 20 ul;
the reaction was amplified in a BIO-RAD T100TM Thermal cyclePCR instrument, and the reaction flow was as follows: pre-denaturation at 94 ℃ for 5 min; 30 seconds at 94 ℃, 30 seconds at 59 ℃, 30 seconds at 72 ℃ and 35 cycles; 5 min at 72 ℃; storing at 4 ℃.
Further, the forward and reverse primer concentrations were each 10 pmol/L.
Further, in step (2), the multiple sequence alignment software tool comprises MegAlign and DNAMAN software.
Further, the method for genotyping the functional molecular marker of the SbGS-7 gene comprises the following steps:
the amplified PCR products have band difference in polyacrylamide gel to identify the genotype of each sorghum to be detected, i.e. identify which type of the homozygous long-grain and homozygous round-grain genotypes.
The invention also aims to provide a method for separating and cloning a gene for controlling the shape of sorghum grains by applying the gene for controlling the shape of the sorghum grains, which comprises the following steps:
GWAS was analyzed by genome-wide association for scanning the aspect ratio trait of multi-year multipoint sorghum, and the site of significant association within the linkage disequilibrium hotspot segment of chromosome 7, 39.83kb, contained only a single gene, Sobic.007G124200, which encodes a trehalose phosphatase, designated SbGS-7; the full-length genome sequence of the gene is obtained by designing a crossover primer, carrying out PCR amplification, sequencing and sequence splicing.
Comparing with BTx623 reference genome, when the granule shape is round, SbGS-7 gene is normal sequence, and when the granule shape is long, the gene has 5 site mutations and 2 site mutations in 1 st and 2 nd exons; the 1 st mutation site occurring in the 1 st exon is a base group which is 93 th from the translation initiation site, and the G mutation is T, namely the arginine CGG mutation is an arginine CGT mutation, and is a synonymous mutation; the 2 nd mutation site is a 106 th base away from the translation initiation site, and the mutation of A to G, namely the mutation of isoleucine ATC to valine GTC, is a non-synonymous mutation; the 3 rd mutation site is a site which is lack of CCGCCG six bases at the 143 th base away from the translation initiation site, so that 2 alanines are lacked during translation; the 4 th mutation site is formed by inserting CGG three bases at the position 180 th from the translation starting site, so that 1 arginine is added during translation; the 5 th mutation site is 268 th base away from the translation initiation site, and G is mutated into T, namely alanine GCC is mutated into serine TCC which is nonsynonymous mutation; the 327 th base from the 1 st mutation site of the 2 nd exon to the translation initiation site is mutated into T, namely alanine GCG is mutated into alanine GCT, and the mutation is synonymous mutation; the 2 nd mutation site is a synonymous mutation with the 351 nd base from the translation initiation site, wherein G is mutated into C, namely valine GTG is mutated into valine GTC.
The gene has 4 site mutations in the 1 st intron, the 1 st mutation site is 305 th base from the translation initiation site, and C is mutated into T; the 2 nd mutation site is a base 329 th from the translation initiation site, and G is mutated into T; the 3 rd mutation site is a 343 rd base from the translation initiation site, and the T mutation is A; the 4 th mutation site is deletion of ATTC four bases at 358 th base from the translation start site, the 4 th intron has 1 site mutation, the mutation site is deletion of CCAT six bases at 925 th base from the translation start site, the 7 th intron has 1 site mutation, and the mutation site is deletion of CCGCCGCCGT ten bases at 1828 th base from the translation start site.
A functional molecular marker M3 for identifying the long/round granule gene SbGS-7 based on PCR technology was developed based on the insertion/deletion characteristics of 10 bases contained at base 1828 within the 7 th intron; the marker is a nucleotide sequence amplified from sorghum genome DNA by a primer pair M3-F and M3-R, and is used for distinguishing sorghum long grain types and sorghum round grain types after being detected by polyacrylamide gel.
The invention also aims to provide application of the functional molecular marker of the sorghum long/round grain shape gene SbGS-7 in screening and identifying sorghum germplasm resources and serving as a molecular marker for assisting breeding.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a gene for controlling the shape of sorghum grains, in particular to a gene for protecting and controlling the shape of sorghum grains, SbGS-7, development of a functional molecular marker of the gene and a corresponding primer, belonging to the technical field of agricultural biology and being capable of being used for rapid screening of different grain-shaped germplasms of sorghum and molecular marker-assisted breeding containing the SbGS-7 gene.
The invention separates and clones a gene SbGS-7 capable of controlling grain shape (length/round grain) of sorghum grains, and provides a molecular marker associated with the grain shape; the molecular marker M3 obtained by the invention can identify whether the sorghum contains the long/granule gene SbGS-7. The marker can be used for carrying out molecular marker-assisted selection on long/round grain genotypes in the seedling stage, the accuracy rate of 100 percent is ensured, the quantity of field planting materials can be greatly reduced, the manpower, material resources and financial resources are saved, and the breeding process of the grain shape characters of sorghum can be accelerated.
Compared with the traditional map-based cloning technology, the method for controlling the sorghum grain shape gene SbGS-7 is obtained by genome-wide association analysis (GWAS), and has the following advantages: 1) the work of constructing a basic segregation population and a multi-generation segregation large population recombined in a target interval is saved, and the age limit of gene cloning is greatly shortened; 2) the whole genome range can be integrally researched by monitoring thousands of SNPs at one time; 3) the accuracy and precision of the study is greatly improved, for example, the SbGS-7 gene in the present invention is directly defined within the 39.83kb region and comprises only one gene. With the improvement of genome sequencing technology and the reduction of sequencing cost, and the high development of bioinformatics, GWAS becomes one of the most effective methods for digging and analyzing important agronomic trait genes of sorghum and related genetic mechanisms thereof.
According to the invention, a functional molecular marker based on a PCR technology is developed aiming at the insertion/deletion characteristics of 10 bases contained at the 1828 th base of the 7 th intron according to the difference of long/round grain SbGS-7 gene sequences, and polyacrylamide gel is directly used for genotyping, so that the long/round grain shape gene SbGS-7 and alleles thereof of sorghum germplasm resources can be identified simply, quickly and at high flux, and individual plants can be detected from the seedling stage aiming at grain shape properties, non-target individuals are eliminated, thereby realizing molecular marker assisted breeding and remarkably improving the breeding efficiency of sorghum varieties.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a flow chart of a method for separating and cloning a gene for controlling sorghum grain shape according to an embodiment of the present invention.
Fig. 2 is a diagram of long and round grain sorghum kernels provided by the embodiment of the present invention.
FIG. 3 is a Mahattan diagram of sorghum grain shape gene mapping obtained by the MLM method in genome-wide association analysis provided by the embodiment of the present invention.
FIG. 4 is a linkage disequilibrium thermogram of the granule gene SbGS-7 provided in the examples of the present invention.
FIG. 5 is a sequence alignment chart of a sorghum long/round grain gene SbGS-7 with the gene function variation provided by the embodiment of the invention.
FIG. 6 is an amino acid alignment chart of a sorghum long/round grain gene SbGS-7 with the gene function variation provided by the embodiment of the invention.
FIG. 7 is a schematic diagram showing the different haplotypes with the long/circular granule gene SbGS-7 provided by the example of the present invention.
FIG. 8 is a polyacrylamide gel electrophoresis image of functional molecular markers of sorghum grain shape gene SbGS-7 in sorghum variety resources, provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a gene for controlling the shape of sorghum grains, a separation and cloning method and application thereof, and the invention is described in detail below by combining with the attached drawings.
As shown in fig. 1, the method for separating and cloning a gene controlling a sorghum grain shape, provided by the embodiment of the present invention, comprises the following steps:
s101, carrying out whole genome association analysis on the long/round granule genes;
s102, separating an SbGS-7 gene;
s103, sequence comparison;
s104, development of a functional molecular marker of the SbGS-7 gene.
The technical solution of the present invention is further described with reference to the following specific examples.
Example 1
The invention aims to solve the technical problems of separating and cloning a gene SbGS-7 for controlling grain shape (long/round grain) of sorghum grains and providing a molecular marker associated with the grain shape; the molecular marker M3 obtained by the invention can identify whether the sorghum contains the long/circule gene SbGS-7. The marker can be used for carrying out molecular marker-assisted selection of long/round grain genotypes in the seedling stage, the accuracy rate of 100% is ensured, the quantity of field planting materials can be greatly reduced, manpower, material resources and financial resources are saved, and the breeding process of the grain shape characters of sorghum can be accelerated.
In order to solve the technical problem, the invention provides a method for controlling a sorghum grain round grain shape gene SbGS-7: the nucleotide sequence of the gene is shown as SEQ ID NO: 1, the preparation method is as follows.
The invention also provides a protein coded by the granulocyte colony-forming gene SbGS-7, and the amino acid sequence of the protein is shown as SEQ ID NO: 3, the preparation method is as follows.
Correspondingly, the nucleotide sequence of the SbGS-7 gene of the sorghum vulgare is shown as SEQ ID NO: 2, and the protein coded by the protein has the sequence shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.
The invention also provides a functional molecular marker M3 for identifying the long/round granule gene SbGS-7 based on PCR technology, which is developed according to the insertion/deletion characteristic of 10 bases contained at the 1828 th base in the 7 th intron, and takes sorghum as a research object, wherein the molecular marker is selected from the following primer pairs, the nucleotide sequence of which is 5 '→ 3',
the forward primer is (M3-F): GTGCCGTCTCTTTCGTTC
The reverse primer is (M3-R): ACGTCACCAAGGAGAGAC
Note: underlined are primer linkers.
The invention also provides a technical scheme for realizing the purpose:
the invention discloses a separation and cloning control sorghum grain shape gene SbGS-7. Scanning for the aspect ratio trait of multi-year multipoint sorghum by Genome-wide Association Study (GWAS) contained only a single gene, sobic.007g124200, encoding trehalose phosphatase, designated SbGS-7, at a site significantly associated within the 39.83kb linkage disequilibrium hotspot segment of chromosome 7. The full-length genome sequence of the gene is obtained by designing a crossover primer, carrying out PCR amplification, sequencing and sequence splicing. In contrast to the BTx623 reference genome, the SbGS-7 gene is a normal sequence when the granule shape is a circular granule, and 5 site mutations and 2 site mutations in exons 1 and 2, respectively, when the granule shape is a long granule. The 1 st mutation site occurring in the 1 st exon is a 93 th base away from the translation initiation site, and the G mutation is T, namely the CGG (arginine) mutation is CGT (arginine) mutation is a synonymous mutation; the 2 nd mutation site is 106 th base away from the translation initiation site, and the mutation of A to G, namely the mutation of ATC (isoleucine) to GTC (valine), is nonsynonymous mutation; the 3 rd mutation site is a site which is lack of six bases of CCGCCG at the 143 th base away from the translation initiation site, so that 2 alanines are lacked during translation; the 4 th mutation site is formed by inserting CGG three bases at the position 180 th base away from the translation starting site, so that 1 arginine is added during translation; the 5 th mutation site is 268 th base away from the translation initiation site, and G is mutated into T, namely GCC (alanine) is mutated into TCC (serine), and the mutation is nonsynonymous; the 327 th base from the 1 st mutation site of the 2 nd exon to the translation initiation site is mutated into T, namely GCG (alanine) is mutated into GCT (alanine), and the mutations are synonymous mutations; the 2 nd mutation site is a synonymous mutation with the 351 nd base from the translation initiation site, wherein G is mutated into C, namely GTG (valine) is mutated into GTC (valine).
The gene has 4 site mutations in the 1 st intron, the 1 st mutation site is 305 th base from the translation initiation site, and C is mutated into T; the 2 nd mutation site is a base 329 th from the translation initiation site, and G is mutated into T; the 3 rd mutation site is a 343 rd base from the translation initiation site, and the T mutation is A; the 4 th mutation site is deletion of ATTC four bases at 358 th base from the translation initiation site, the 4 th intron has 1 site mutation, the mutation site is deletion of CCAT six bases at 925 th base from the translation initiation site, the 7 th intron has 1 site mutation, and the mutation site is deletion of CCGCCGCCGT ten bases at 1828 th base from the translation initiation site.
A functional molecular marker M3 for the identification of the long/circular granule gene SbGS-7 was developed based on the insertion/deletion characteristics of the 10 bases contained at base 1828 within the 7 th intron using PCR technology. The marker can distinguish long grain and round grain types of sorghum after a nucleotide sequence amplified from sorghum genome DNA by a primer pair M3-F and M3-R is detected by polyacrylamide gel electrophoresis.
The method for identifying the sorghum long/round granule gene SbGS-7 by using the functional molecular marker provided by the embodiment of the invention comprises the following steps:
(1) amplifying, sequencing and splicing the sequence by a conventional PCR method to obtain a sequence of a sorghum long/round grain gene SbGS-7;
(2) comparing the sequences obtained in the step (1) by using a multi-sequence comparison software tool (such as Meg Align or DNAMAN software) and NCBI online Blast to obtain specific 7 base differences of the sorghum long/round grain gene SbGS-7, wherein the differences are positioned in exons 1 and 2 of the gene, and when translation is carried out, except 3 of the differences are synonymous mutations, 4 of the differences comprise 2 insertions/deletions and 2 non-homologous mutations, so that functional specific amino acid changes are caused, and finally grain shape of sorghum is influenced, thereby developing the M3 functional molecular marker.
(3) Confirming the effectiveness of the functional molecular marker of the SbGS-7 gene in detecting the grain shape of the grain, and verifying the sequence obtained in the step (2) in a natural sorghum group; 24 long-grain strain lines and 25 round-grain strains are selected according to the aspect ratio phenotype, the aspect ratio of specific bands is completely consistent with that of 49 strain grains, and the accuracy is 100%, so that the functional molecular marker of the SbGS-7 gene is determined to have high applicability.
The PCR amplification reaction system of the SbGS-7 gene functional molecular marker provided by the embodiment of the invention is as follows:
1ul of sorghum genome DNA, 1ul of forward and reverse primers, 0.2ul of Takara Ex Taq, 2ul of 10 XEx Buffer, 2ul of dNTP (2mM), 12.8ul of dd H2O 12.8 and 20ul of total volume; in the 1ul primer, the forward and reverse primer concentrations were each 10 pmol/L.
The reaction was amplified in a BIO-RAD T100TM Thermal cyclePCR instrument, and the reaction flow was as follows: pre-denaturation at 94 ℃ for 5 min; 30 seconds at 94 ℃, 30 seconds at 59 ℃, 30 seconds at 72 ℃ and 35 cycles; 5 min at 72 ℃; storing at 4 deg.C.
The genotype typing method of the SbGS-7 gene functional molecular marker provided by the embodiment of the invention comprises the following steps:
the amplified PCR products have different bands in polyacrylamide gel electrophoresis, and the genotype of each sorghum to be detected is identified (namely, the genotype belonging to the homozygous long-grain shape and the homozygous round-grain shape, which type in the 2 types of genotypes is identified).
The application of the functional molecular marker of the sorghum long/round grain shape gene SbGS-7 comprises screening and identifying sorghum germplasm resources and serving as a molecular marker for assisted breeding.
Compared with the traditional map-based cloning technology, the method has the beneficial effects that: the control of the sorghum grain shape gene SbGS-7 is obtained by genome-wide association analysis (GWAS), and has the following advantages: 1) the work of constructing a basic segregation population and a multi-generation segregation large population recombined in a target interval is saved, and the age limit of gene cloning is greatly shortened; 2) the whole genome range can be integrally researched by monitoring thousands of SNPs at one time; 3) the accuracy and precision of the study is greatly improved, for example, the SbGS-7 gene in the present invention is defined directly within the 39.83kb region and contains only one gene. With the improvement of genome sequencing technology and the reduction of sequencing cost and the high development of bioinformatics, GWAS becomes one of the most effective methods for mining and analyzing important agronomic trait genes of sorghum and related genetic mechanisms thereof.
According to the difference of long/round grain SbGS-7 gene sequences, the invention develops a functional molecular marker based on a PCR technology aiming at the insertion/deletion characteristics of 10 bases contained at the 1828 th base of the 7 th intron, directly utilizes polyacrylamide gel electrophoresis to carry out genotyping, can simply, rapidly and high-flux identify the long/round grain shape gene SbGS-7 and allele thereof of sorghum germplasm resources, and can also aim at grain shape characters, detect single plants from the seedling stage and eliminate non-target individuals, thereby realizing molecular marker assisted breeding and obviously improving the breeding efficiency of sorghum varieties.
Example 2: isolation and cloning of the granulosa Gene SbGS-7
(1) Whole genome association analysis of long/round granule genes
An aspect ratio character survey is carried out in Guizhou, Hangzhou, Ling water and Ledong by using 248 shares of variety resources of grain sorghum from different provinces in China, and correlation analysis is carried out on the character, and a site which is obviously correlated in a linkage disequilibrium hotspot section of 39.83kb of a 7 th chromosome only comprises a single gene Sobic.007G124200 which codes trehalose phosphatase and is named as SbGS-7.
(2) Isolation of the SbGS-7 Gene
Through designing a crossover primer, carrying out PCR amplification, sequencing and sequence splicing in 10 extreme long-grain varieties and 9 short-grain varieties to obtain the full-length genome sequence of the gene. In order to verify the accuracy of the sequence, young ears of round grain variety BTx623 and long grain variety white glutinous sorghum in the reproductive growth development period are simultaneously selected, RNA is extracted and reversely transcribed into cDNA, and then PCR amplification and sequencing are carried out to obtain the full-length CDS of the gene.
(3) Sequence alignment
The results of comparing the sequences of 10 extremely long grain varieties and 9 round grain varieties with the reference genome of the sorghum sequencing variety BTx623 and the results of comparing the full-length CDS sequence of the round grain variety BTx623 and the long grain variety white glutinous sorghum show that when the grain shape is round grain, the sequence of the SbGS-7 gene is completely identical to that of the reference genome and is a normal sequence, and when the grain shape is long grain, the gene has 5-site mutation and 2-site mutation in the 1 st exon and the 2 nd exon respectively. The 1 st mutation site occurring in the 1 st exon is a base 93 th from the translation initiation site, and the G mutation is T, namely the CGG (arginine) mutation is a synonymous mutation; the 2 nd mutation site is 106 th base away from the translation initiation site, and the mutation of A to G, namely the mutation of ATC (isoleucine) to GTC (valine), is nonsynonymous mutation; the 3 rd mutation site is a site which is lack of CCGCCG six bases at the 143 th base away from the translation initiation site, so that 2 alanines are lacked during translation; the 4 th mutation site is formed by inserting CGG three bases at the position 180 th base away from the translation starting site, so that 1 arginine is added during translation; the 5 th mutation site is 268 th base away from the translation initiation site, and G is mutated into T, namely GCC (alanine) is mutated into TCC (serine), and the mutation is nonsynonymous; the 1 st mutation site of the 2 nd exon is a 327 th base away from the translation initiation site, and the G mutation is T, namely the GCG (alanine) mutation is GCT (alanine), and is a synonymous mutation; the 2 nd mutation site is a synonymous mutation with the 351 nd base from the translation initiation site, wherein G is mutated to C, namely GTG (valine) is mutated to gtc (valine). 2 insertions/deletions and 2 non-synonymous mutations result in a change in the functionally specific amino acids, which ultimately affects the grain shape of sorghum.
The gene has 4 site mutations in the 1 st intron, the 1 st mutation site is 305 th base from the translation initiation site, and C is mutated into T; the 2 nd mutation site is a 329 th base away from the translation initiation site, and G is mutated into T; the 3 rd mutation site is a 343 rd base from the translation initiation site, and the T mutation is A; the 4 th mutation site is deletion of ATTC four bases at 358 th base from the translation start site, the 4 th intron has 1 site mutation, the mutation site is deletion of CCAT six bases at 925 th base from the translation start site, the 7 th intron has 1 site mutation, and the mutation site is deletion of CCGCCGCCGT ten bases at 1828 th base from the translation start site.
(4) Development of functional molecular marker of SbGS-7 gene
A functional molecular marker M3 for identifying the long/round granule gene SbGS-7 based on PCR technology is developed according to the insertion/deletion characteristic of 10 bases contained at the 1828 th base in the 7 th intron, and the nucleotide sequence amplified from sorghum genomic DNA by a primer pair M3-F and M3-R can distinguish sorghum long granules from round granules after being detected by polyacrylamide gel electrophoresis.
The nucleotide sequence of the primer pair is shown as SEQ ID NO: and 5, as follows:
the forward primer is (M3-F): GTGCCGTCTCTTTCGTTC
The reverse primer is (M3-R): ACGTCACCAAGGAGAGAC
Example 3: detection of SbGS-7 gene functional molecular marker on sorghum varieties
The present invention utilizes 49 sorghum variety sources of different origin with known aspect ratio phenotype (see table 1) to verify the applicability of molecular marker M3.
TABLE 1
The identification method comprises the following steps:
(1) PCR amplification was carried out using SbGS-7 gene functional molecular marker M3 on the test materials shown in Table 1, and the PCR amplification products were obtained using genomic DNA as a template.
The PCR amplification reaction system is as follows:
sorghum genome DNA 1ul, forward and reverse primers 1ul each, Takara Ex Taq 0.2ul, 10 XEx Buffer2ul, dNTP (2mM)2ul, dd H 2 O12.8 ul, the total volume is 20 ul;
the reaction is amplified in a BIO-RAD T100TM Thermal cycler PCR instrument, and the reaction flow is as follows: pre-denaturation at 94 ℃ for 5 min; 30 seconds at 94 ℃, 30 seconds at 59 ℃, 30 seconds at 72 ℃ and 35 cycles; 5 min at 72 ℃; storing at 4 ℃.
(2) Polyacrylamide gel electrophoresis detection of PCR products
Performing polyacrylamide gel detection on the PCR product directly, wherein if the product only contains 83bp fragments, the insertion deletion site corresponding to the M3 marker contains 10-base deletion; the grain shape of the sorghum to be detected is a round grain shape;
if the product only contains a 93bp fragment, the insertion deletion site corresponding to the M3 marker contains 10-base deletion; the grain shape of the sorghum to be detected is a long grain shape;
the result is shown in fig. 8, in the identification of the grain shape of 49 sorghum varieties, the statistical result of the specific bands of the functional molecular marker M3 is completely consistent with the length-width ratio of grains, and the accuracy is 100%, which indicates that the molecular marker has strong applicability, and the grain shape can be quickly identified and screened by using the marker in the practical breeding application.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.
Sequence listing
<110> Guizhou province drought grain research institute (Guizhou province sorghum research institute) (Guizhou province corn engineering technology research center) Guizhou university
<120> gene for controlling sorghum grain shape and separation and cloning method and application thereof
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atgacgaagc agggcgtggt ggtgccggtg ccggaggcgg ccgtggcggt gccgcccaac 60
acggcgccgc tgttccagta cccgccgccg cgggccgcgc cgggcatcgc cgtgcgcaag 120
aagtacctgc agatgggcgc cgccgccggt gccggcgccg gggcgcgcat cggcggcggc 180
ggctgggtgg agtccatgcg cgcgtcctcg cccacgcacg ccagggcggc cgccgcgctc 240
gccgcgggcg tcgacgagga gcgctacgcc gagtggatgg tgaagcaccc gtcggcgctg 300
ggcatgttcg accaggtggt ggcggcgtcc aaggggaagc agatcgtcgt gttcctcgac 360
tacgacggca cgctgtcccc catcgtcgac gaccccgacg ccgcctacat gtcggacacg 420
atgcggcggg cggtgcggag cgtcgccaag cacttcccga cggcgatcgt gagcgggcgg 480
tgccgcgaca aggtgttcga gttcgtgaag ctggctgagc tgtactacgc cggcagccac 540
ggcatggaca tcaagggccc cgcgaaaggg tcccggcaca ccaaggccgc caaggccaaa 600
ggcgttctct ttcagccggc cagccagttc ctgcccatga tagagcaggt gcacgattct 660
ctggtggaaa agaccaagtg catacctgga gccaaggtgg agaacaacaa gttttgtgtg 720
tctgtccact tcagatgcgt cgatgagaag agctggagca cattggctga cctggtgaag 780
tcggtgctga cggactaccc gaagctgaag ctgacgcagg ggcggatggt gttcgaggtc 840
cgccccacca tcaagtggga caagggcaag gccctggagt tcctcctcga gtccctgggc 900
ttcgccgact gcaccgacgt gctccccgtc tacatcggcg acgaccgcac cgacgaggac 960
gccttcaagg tgctccgcaa gcgcggccag ggcgtgggca tcctcgtctc caagcacccc 1020
aaggacacct ccgcctccta ctcgctgcag gagcccgccg aggtgatgga gttcctgctg 1080
cggcttgtcg agtgggagcg cctctccaag gcccgccccg ccaagtggtg a 1131
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aagtacctgc agatgggcgc cggtgccggc gccggggcgc gcatcggcgg cggcggcggc 180
tgggtggagt ccatgcgcgc gtcctcgccc acgcacgcca gggcggccgc cgcgctcgcc 240
gcgggcgtcg acgaggagcg ctactccgag tggatggtga agcacccgtc ggcgctgggc 300
atgttcgacc aggtggtggc ggcttccaag gggaagcaga tcgtcgtctt cctcgactac 360
gacggcacgc tgtcccccat cgtcgacgac cccgacgccg cctacatgtc ggacacgatg 420
cggcgggcgg tgcggagcgt cgccaagcac ttcccgacgg cgatcgtgag cgggcggtgc 480
cgcgacaagg tgttcgagtt cgtgaagctg gctgagctgt actacgccgg cagccacggc 540
atggacatca agggccccgc gaaagggtcc cggcacacca aggccgccaa ggccaaaggc 600
gttctctttc agccggccag ccagttcctg cccatgatag agcaggtgca cgattctctg 660
gtggaaaaga ccaagtgcat acctggagcc aaggtggaga acaacaagtt ttgtgtgtct 720
gtccacttca gatgcgtcga tgagaagagc tggagcacat tggctgacct ggtgaagtcg 780
gtgctgacgg actacccgaa gctgaagctg acgcaggggc ggatggtgtt cgaggtccgc 840
cccaccatca agtgggacaa gggcaaggcc ctggagttcc tcctcgagtc cctgggcttc 900
gccgactgca ccgacgtgct ccccgtctac atcggcgacg accgcaccga cgaggacgcc 960
ttcaaggggc tccgcaagcg cggccagggc gtgggcatcc tcgtctccaa gcaccccaag 1020
gacacctccg cctcctactc gctgcaggag cccgccgagg tgatggagtt cctgctgcgg 1080
cttgtcgagt gggagcgcct ctccaaggcc cgccccgcca agtggtga 1128
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Val Pro Pro Asn Thr Ala Pro Leu Phe Gln Tyr Pro Pro Pro Arg Ala
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Ala Gly Ala Gly Ala Gly Ala Arg Ile Gly Gly Gly Gly Trp Val Glu
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Ser Met Arg Ala Ser Ser Pro Thr His Ala Arg Ala Ala Ala Ala Leu
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Ala Ala Gly Val Asp Glu Glu Arg Tyr Ala Glu Trp Met Val Lys His
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Pro Ser Ala Leu Gly Met Phe Asp Gln Val Val Ala Ala Ser Lys Gly
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130 135 140
Val Arg Ser Val Ala Lys His Phe Pro Thr Ala Ile Val Ser Gly Arg
145 150 155 160
Cys Arg Asp Lys Val Phe Glu Phe Val Lys Leu Ala Glu Leu Tyr Tyr
165 170 175
Ala Gly Ser His Gly Met Asp Ile Lys Gly Pro Ala Lys Gly Ser Arg
180 185 190
His Thr Lys Ala Ala Lys Ala Lys Gly Val Leu Phe Gln Pro Ala Ser
195 200 205
Gln Phe Leu Pro Met Ile Glu Gln Val His Asp Ser Leu Val Glu Lys
210 215 220
Thr Lys Cys Ile Pro Gly Ala Lys Val Glu Asn Asn Lys Phe Cys Val
225 230 235 240
Ser Val His Phe Arg Cys Val Asp Glu Lys Ser Trp Ser Thr Leu Ala
245 250 255
Asp Leu Val Lys Ser Val Leu Thr Asp Tyr Pro Lys Leu Lys Leu Thr
260 265 270
Gln Gly Arg Met Val Phe Glu Val Arg Pro Thr Ile Lys Trp Asp Lys
275 280 285
Gly Lys Ala Leu Glu Phe Leu Leu Glu Ser Leu Gly Phe Ala Asp Cys
290 295 300
Thr Asp Val Leu Pro Val Tyr Ile Gly Asp Asp Arg Thr Asp Glu Asp
305 310 315 320
Ala Phe Lys Val Leu Arg Lys Arg Gly Gln Gly Val Gly Ile Leu Val
325 330 335
Ser Lys His Pro Lys Asp Thr Ser Ala Ser Tyr Ser Leu Gln Glu Pro
340 345 350
Ala Glu Val Met Glu Phe Leu Leu Arg Leu Val Glu Trp Glu Arg Leu
355 360 365
Ser Lys Ala Arg Pro Ala Lys Trp
370 375
<210> 4
<211> 374
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Met Thr Lys Gln Gly Val Val Val Pro Val Pro Glu Ala Ala Val Ala
1 5 10 15
Val Pro Pro Asn Thr Ala Pro Leu Phe Gln Tyr Pro Pro Pro Arg Ala
20 25 30
Ala Pro Gly Val Ala Val Arg Lys Lys Tyr Leu Gln Met Gly Ala Gly
35 40 45
Ala Gly Ala Gly Ala Arg Ile Gly Gly Gly Gly Gly Trp Val Glu Ser
50 55 60
Met Arg Ala Ser Ser Pro Thr His Ala Arg Ala Ala Ala Ala Leu Ala
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Ala Gly Val Asp Glu Glu Arg Tyr Ser Glu Trp Met Val Lys His Pro
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Ser Ala Leu Gly Met Phe Asp Gln Val Val Ala Ala Ser Lys Gly Lys
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Gln Ile Val Val Phe Leu Asp Tyr Asp Gly Thr Leu Ser Pro Ile Val
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Arg Ser Val Ala Lys His Phe Pro Thr Ala Ile Val Ser Gly Arg Cys
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Arg Asp Lys Val Phe Glu Phe Val Lys Leu Ala Glu Leu Tyr Tyr Ala
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Val His Phe Arg Cys Val Asp Glu Lys Ser Trp Ser Thr Leu Ala Asp
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Leu Val Lys Ser Val Leu Thr Asp Tyr Pro Lys Leu Lys Leu Thr Gln
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Lys Ala Leu Glu Phe Leu Leu Glu Ser Leu Gly Phe Ala Asp Cys Thr
290 295 300
Asp Val Leu Pro Val Tyr Ile Gly Asp Asp Arg Thr Asp Glu Asp Ala
305 310 315 320
Phe Lys Gly Leu Arg Lys Arg Gly Gln Gly Val Gly Ile Leu Val Ser
325 330 335
Lys His Pro Lys Asp Thr Ser Ala Ser Tyr Ser Leu Gln Glu Pro Ala
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Glu Val Met Glu Phe Leu Leu Arg Leu Val Glu Trp Glu Arg Leu Ser
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<212> DNA
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Claims (10)
1. The gene for controlling the shape of the sorghum grains is characterized in that the gene for controlling the shape of the sorghum grains is SbGS-7, and the shape of the sorghum grains comprises a round grain shape and a long grain shape; the nucleotide sequence of the gene for controlling the sorghum grain round grain shape is SEQ ID NO: 1, the nucleotide sequence of the gene for controlling the sorghum grain growth is SEQ ID NO: 2.
2. the protein encoded by the sorghum grain shape controlling gene according to claim 1, wherein the amino acid sequence of the protein encoded by the sorghum grain round grain shape controlling gene is SEQ ID NO: 3, the protein amino acid sequence coded by the gene for controlling the sorghum grain to grow into granules is SEQ ID NO: 4.
3. a functional molecular marker M3 of a sorghum long/round grain shape gene SbGS-7 obtained by using the gene for controlling the shape of a sorghum grain according to claim 1, wherein the method for obtaining the functional molecular marker M3 of the sorghum long/round grain shape gene SbGS-7 comprises the following steps:
a functional molecular marker M3 for identifying the long/round-grained gene SbGS-7 based on PCR technology was developed based on the insertion/deletion characteristics of 10 bases contained at base 1828 within the 7 th intron, targeting sorghum, and selected from the group consisting of SEQ ID NO: 5, wherein the nucleotide sequence is 5 '→ 3'.
4. A method for identifying a sorghum growth/telomere gene, SbGS-7, by using a functional molecular marker of the sorghum growth/telomere gene, SbGS-7, as defined in claim 3, wherein the method for identifying the sorghum growth/telomere gene, SbGS-7, comprises:
(1) amplifying, sequencing and sequence splicing by a conventional PCR method to obtain a sequence of a sorghum long/round granule gene SbGS-7;
(2) and (2) comparing the sequences obtained in the step (1) by using a multi-sequence comparison software tool and NCBI online Blast to obtain specific 7 base differences of exon sequences of the sorghum long/round grain gene SbGS-7, wherein the differences are located in exons 1 and 2 of the gene, and when translation is carried out, except 3 of the differences are synonymous mutations, the rest 4 of the differences comprise 2 insertions/deletions and 2 non-homologous mutations, so that functional specific amino acid changes are caused, the grain shape of the sorghum is finally influenced, and the specific 7 base differences of intron sequences are located in introns 1, 4 and 7 of the gene, and comprise 4 insertions/deletions and 2 base mutations. Developing an M3 functional molecular marker according to the difference of 10 bases of long-particle-shaped and round-particle-shaped varieties in the 7 th intron;
(3) confirming the effectiveness of the functional molecular marker of the SbGS-7 gene in detecting the grain shape of grains, and verifying the sequence obtained in the step (2) in a natural sorghum group; 24 long-grain strains and 25 round-grain strains are selected according to the aspect ratio phenotype, the statistical result of the specific bands is completely consistent with the aspect ratio characters of 49 strains, and the accuracy is 100%, so that the functional molecular marker of the SbGS-7 gene is determined to have strong applicability.
5. The method for identifying the sorghum long/round grain shape gene SbGS-7 according to claim 4, wherein in the step (1), the PCR amplification reaction system comprises:
sorghum genome DNA 1ul, forward and reverse primers 1ul each, Takara Ex Taq 0.2ul, 10 XEx Buffer2ul, dNTP 2mM 2ul, dd H 2 O12.8 ul, total volume 20 ul;
the reaction is amplified in a BIO-RAD T100TM Thermal cycler PCR instrument, and the reaction flow is as follows: pre-denaturation at 94 ℃ for 5 min; 30 seconds at 94 ℃, 30 seconds at 59 ℃, 30 seconds at 72 ℃ and 35 cycles; 5 min at 72 ℃; storing at 4 deg.C.
6. The method for identifying the sorghum long/round grain granule gene SbGS-7 according to claim 5, wherein the forward and reverse primer concentrations are each 10 pmol/L.
7. The method for identifying the sorghum long/round grain gene SbGS-7 as claimed in claim 4, wherein in the step (2), the multiple sequence alignment software tools comprise MegAlign and DNAMAN software.
8. The method for identifying the sorghum long/round grain shape gene SbGS-7 as claimed in claim 4, wherein the SbGS-7 gene functional molecule marker genotyping method comprises the following steps:
the amplified PCR products have band difference in polyacrylamide gel electrophoresis to identify the genotype of each sorghum to be detected, namely identifying which type of the homozygous long-grain and homozygous round-grain genotypes.
9. The method for separating and cloning genes for controlling the shape of sorghum grains according to claim 1, wherein the method for separating and cloning genes for controlling the shape of sorghum grains comprises the following steps:
GWAS scanning the aspect ratio trait of multi-year multipoint sorghum by genome-wide association analysis, a site significantly associated within the linkage disequilibrium hot spot segment of 39.83kb on chromosome 7 contained only a single gene sobic.007gx124200, which encodes a trehalose phosphatase designated SbGS-7; through designing a crossover primer, carrying out PCR amplification, sequencing and sequence splicing to obtain a full-length genome sequence of the gene;
comparing with BTx623 reference genome, when the granule shape is round, SbGS-7 gene is normal sequence, and when the granule shape is long, the gene has 5 site mutations and 2 site mutations in 1 st and 2 nd exons; the 1 st mutation site occurring in the 1 st exon is a base 93 th from the translation initiation site, and G mutation is T, namely the arginine CGG mutation is an arginine CGT mutation which is a synonymous mutation; the 2 nd mutation site is 106 th base away from the translation initiation site, and the mutation of A to G, namely the mutation of isoleucine ATC to valine GTC, is nonsynonymous mutation; the 3 rd mutation site is formed by deleting six bases of CCGCCG at the 143 th base away from the translation initiation site, so that 2 alanines are deleted during translation; the 4 th mutation site is formed by inserting three bases of CGG at the position 180 th base away from the translation starting site, so that 1 arginine is added during translation; the 5 th mutation site is 268 th base from the translation initiation site, and G is mutated into T, namely alanine GCC is mutated into serine TCC, and the mutation is nonsynonymous mutation; the 327 th base from the 1 st mutation site of the 2 nd exon to the translation initiation site is mutated into T, namely alanine GCG is mutated into alanine GCT, and the mutation is synonymous mutation; the 2 nd mutation site is a synonymous mutation with the 351 nd base from the translation initiation site, wherein G is mutated into C, namely valine GTG is mutated into valine GTC.
The gene has 4 site mutations in the 1 st intron, the 1 st mutation site is 305 th base from the translation initiation site, and C is mutated into T; the 2 nd mutation site is a base 329 th from the translation initiation site, and G is mutated into T; the 3 rd mutation site is a base 343 th from the translation initiation site, and T is mutated into A; the 4 th mutation site is deletion of ATTC four bases at 358 th base from the translation initiation site, the 4 th intron has 1 site mutation, the mutation site is deletion of CCAT six bases at 925 th base from the translation initiation site, the 7 th intron has 1 site mutation, and the mutation site is deletion of CCGCCGCCGT ten bases at 1828 th base from the translation initiation site.
A functional molecular marker M3 for identifying the long/circular granule gene SbGS-7 based on PCR technology was developed based on the insertion/deletion characteristics of 10 bases contained at base 1828 in intron 7; the marker is a nucleotide sequence amplified from sorghum genome DNA by a primer pair M3-F and M3-R, and is used for distinguishing sorghum long grain types and sorghum round grain types after being detected by polyacrylamide gel.
10. The use of the functional molecular marker of the sorghum long/round grain shape gene SbGS-7 as claimed in claim 3 in screening and identifying sorghum germplasm resources and as a molecular marker for assisted breeding.
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| CN105603072A (en) * | 2016-01-22 | 2016-05-25 | 辽宁省农业科学院 | Gene SbAn-1 capable of controlling existence of sorghum awn and SNP mutation site and application |
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