CN114438082B - DNA sequence for rapidly identifying related ecology of flowering phase, spring and winter habit of wheat family and application - Google Patents

Abstract

The invention discloses a DNA sequence for rapidly identifying the related ecology of flowering phase and spring and winter habit of wheat family and application thereof, wherein the DNA sequence is a 21bp DNA sequence positioned in a VRN-A1 gene promoter region and has a nucleotide sequence shown as SEQ ID No. 1. The invention is a 21bp DNA sequence which can be used for detecting different ecological types of wheat in wheat crops for the first time. The sequence can be used for molecular marker development. According to existence or nonexistence of the 21bp DNA sequence in the VRN-A1 gene promoter region, most wheat families can be divided into two types of (partial) winter and (partial) spring according to the flowering phase and the spring and winter habit at present, thereby being beneficial to rapid identification of ecological types related to the flowering phase and the spring and winter habit of different wheat materials. The gene editing technology can be used for editing different base sites in the 21bp sequence according to the requirement, so that materials with different growth periods can be created.

Description

DNA sequence for rapidly identifying related ecology of flowering phase, spring and winter habit of wheat family and application

Technical Field

The invention belongs to the field of plant genetic engineering, and discloses a novel DNA sequence and application thereof in rapid identification of ecology related to flowering phase and spring and winter habit of wheat families.

Background

Flowering of plants is a very important biological phenomenon, and has great significance for the reproduction of sexual reproduction species. Meanwhile, the flowering period of crops is comprehensively influenced by multiple factors such as growth temperature, illumination, nutrition, moisture and the like, and has very important influence on the yield of crops. For a long time, this trait has been a breeding goal and has been widely paid attention to crop breeding and geneticists. The current research results show that the flowering of the plant (crop) is simultaneously and comprehensively regulated by genetic factors, epigenetic factors, physiological biochemistry factors and other factors, and the method is a very complex biological process. At present, plant flowering is mainly regulated by classical photoperiod pathway, vernalization pathway, autonomous pathway, gibberellin (GA) pathway, age pathway and other pathways. Wherein, vernalization refers to the phenomenon that plants need to undergo low-temperature exercise for a long time to bloom and fruit. Wheat is one of the main grain crops in the world, and along with the potential threat of population and global climate change to crop yield, wheat yield is one of the main factors affecting the safe production of grains in China and the world, so that the continuous improvement of the unit yield is one of the main targets of modern molecular breeding. Wheat can be classified into spring wheat and winter wheat according to vernalization types. The related research results of the current wheat flowering show that the wheat flowering is mainly regulated by 3 vernalization genes, VRN1, VRN2 and VRN3/FT. Meanwhile, homologous multiple copies of the VRN1 gene in the wheat genome also participate in flowering regulation of wheat. The research results of the regulation network of 3 genes show that the VRN2 gene mainly inhibits the expression of VRN3/FT which promotes flowering, and the VRN1 gene can inhibit the expression of the VRN2 gene, so that the inhibiting effect of the VRN2 gene on the expression of VRN3/FT is relieved, the transcription of VRN3/FT is promoted, and the flowering of wheat is finally promoted. Thus, altering expression of the VRN1 gene in the wheat genome can directly affect wheat flowering time. The VRN1 gene is Transcription Factor (TFs) belonging to MADS-box family, is homologous to AP1 transcription factor family in Arabidopsis genome, mainly controls the transformation of plant from vegetative growth to reproductive growth, and has a certain regulation effect on normal growth and development of plant. Preliminary description, the balance between wheat nutrition and reproductive growth is regulated by regulating and controlling the space-time expression of the VRN1 gene, so that the possibility of improving the wheat yield is improved, and an important theoretical reference is provided for manually editing the gene to improve the wheat yield. In addition, the expression of the VRN1 gene is also regulated and controlled by temperature, so that the wheat can be directly classified into spring wheat and winter wheat according to the low-temperature dependency degree of the VRN1 gene expression in the wheat genome. Thus, the VRN1 gene has the possibility of further developing a molecular marker, thereby realizing the rapid identification of the winter and spring habits of different wheat varieties or strains or germplasm.

Through sequence comparison analysis of VRN1 genes in wheat and barley genomes of different ecological types, the first intron of the VRN1 gene is quite conserved in different varieties and contains cis-regulatory elements responding to environmental factors such as temperature, illumination and the like. In addition, the gene has abundant sequence variation, such as point mutation, small fragment insertion or deletion, in addition to introns, on promoter regions of VRN1 genes in different ecological wheat genomes. These results indicate that the coding region of the VRN1 gene is quite conserved. The sequence differences of the first intron and the promoter region have a very important influence on the gene of VRN 1.

In the genome of eukaryote, the gene expression is mainly used for finely regulating and controlling the expression of related genes through the interaction between cis-regulatory elements and reaction factors, so that the normal growth and development of individuals are ensured, and the influence of internal and external environmental stress factors is met. The current research result shows that the mutation of the gene coding region often causes the severe change of the gene expression and most of the gene coding region is harmful change, and compared with the DNA sequence polymorphism of the gene cis-regulatory element, the DNA sequence polymorphism can change the expression quantity and the expression time of related genes in time-space, so that individuals have different phenotypes. Thus, the cis-regulatory element can be used as a target site for gene editing or artificial selection to cultivate or create individuals of a desired trait by altering the polymorphism of the DNA sequence. At present, partial research results show that the DNA sequence of the gene promoter has very important regulation effect on the expression of the VRN1 gene, but the specific mechanism of how the DNA sequence changes to regulate the expression of the VRN1 gene is not very clear, in particular, whether the DNA sequence of the promoter region of the VRN1 gene can be used for developing a high-efficiency DNA molecular marker for large-scale wheat population screening, thereby rapidly identifying the variety or strain or germplasm of wheat with spring and winter habits and guiding agricultural production. In addition, the molecular marker can be used as a target site for gene editing or artificial selection, and can be used for cultivating or creating new varieties or new strains or new germplasm of wheat with different flowering time by changing polymorphism of DNA sequences, so as to be used for large-scale wheat agricultural production or breeding.

Based on this objective, the present invention, by combining the MH-seq technology (Zhao et al 2020; patent No. 201910108157.9) developed autonomously with GWAS, first identified a 21bp DNA sequence located in the promoter region of the VRN-A1 gene in the wheat genome, which has an important regulatory effect on the expression of the VRN-A1 gene, thereby affecting wheat flowering, and being a key functional element for regulating wheat flowering. The regulatory element can be applied to rapid detection and breeding application of the wheat ecological type, greatly shortens the period of cultivation or creation of the agronomic characters related to the flowering phase of the wheat, and has important theoretical and practical application prospects and values.

Disclosure of Invention

The invention aims to provide a small fragment DNA (21 bp) regulatory element and application thereof in rapid identification of ecology related to flowering phase and spring and winter habit of wheat family. The small fragment DNA (21 bp) regulatory element screened by the invention can be used for rapidly detecting the ecology type of wheat, screening or creating wheat varieties with shorter growth cycle or breeding intermediate type materials, and is applied to wheat agricultural production.

The aim of the invention can be achieved by the following technical scheme:

a small fragment DNA regulatory element having a nucleotide sequence as set forth in SEQ ID No. 1. The regulatory element is a 21bp DNA sequence located in the promoter region of the VRN-A1 gene: TGGAAGAGAGGGGAGGAGAGG.

Firstly, specifically amplifying the DNA fragment through PCR, secondly, utilizing the sequence as a mark, and utilizing resequencing data to carry out group scanning on different wheat varieties or germplasm results, thereby screening out wheat materials with the DNA sequence changed in natural materials, and further carrying out phenotype investigation.

Expression cassette, recombinant vector, transgenic cell line or transgenic recombinant bacteria containing the small fragment DNA regulatory element.

And (3) carrying out gene editing on the small fragment DNA regulatory element to obtain a DNA sequence.

The small fragment DNA regulatory element is applied to identifying the flowering phase of wheat crops and the ecological type related to spring and winter habits or molecular breeding of wheat. As a preferable technical scheme, PCR amplification is carried out in different wheat colony materials by utilizing specific primers, the spring and winter habits and flowering periods of detected materials are judged according to the existence of amplified fragments, the wheat variety or strain lacking the fragments is expressed as early flowering spring, and the wheat variety or strain containing the fragments is expressed as late flowering winter or weak spring; the sequence of the specific primer is as follows: f: GTGGTTGGGTGAGGACGT; r: CCTGCCGGAATCCTCGTT.

The expression cassette, the recombinant vector, the transgenic cell line or the transgenic recombinant bacteria containing the small fragment DNA regulatory element are applied to the identification of the ecotype related to the flowering period of wheat crops and the winter and spring habit or the molecular breeding of wheat.

The application of the DNA sequence obtained by editing the small fragment DNA regulatory element in the cultivation (or creation) of new varieties (or new germplasm) of wheat in the flowering phase and the spring and winter habit change of the small fragment DNA regulatory element or the research of molecular regulatory mechanism. The 21bp DNA sequence can be subjected to gene editing through CRISP and other technologies, so that new wheat materials related to the flowering period can be created or bred, and the novel wheat materials can be used for agricultural production or breeding of wheat and research on molecular regulation and control mechanisms.

A method for identifying the related ecology of flowering period and spring and winter habit of wheat crops or molecular breeding of wheat, which is any one of (1) to (4):

(1) The small fragment DNA regulatory element is applied to identification or screening of ecology related to important agronomic characters such as flowering phase, spring and winter habit and the like of wheat crops through developing molecular markers or probes, so that the breeding process of wheat molecules is accelerated;

(2) The small fragment DNA regulatory element is used as a target site for gene editing, and the new germplasm or variety of wheat with changed flowering period and spring and winter habit thereof is changed or created through editing (insertion, replacement, deletion and the like of bases);

(3) The small fragment DNA regulatory element or the edited DNA sequence is used for culturing new germplasm or variety of wheat with changed flowering period and spring and winter habit through genetic transformation means (transgene or transient expression, etc.);

(4) The natural wheat materials with the small fragment DNA regulatory elements changed are hybridized and bred with other wheat varieties with different ecological types, so that new wheat materials relevant to flowering time are created or cultivated in a short time, and the novel wheat materials are used for wheat agricultural production or breeding.

The beneficial effects of the invention are that

The invention is a 21bp DNA sequence which can be used for detecting different ecological types of wheat in wheat crops for the first time. Meanwhile, the sequence can be used for molecular marker development. The VRN-A1 constructed transgenic vector without the 21bp DNA fragment is transferred into common wheat, so that the flowering gene is expressed in advance, and the transgenic material is flowering early, thereby creating wheat varieties or strains with shortened growth period. The gene editing technology can be also utilized to edit different base sites in the 21bp sequence according to the requirement to create materials with different growth periods, so that the method has very important production value and greatly promotes agricultural production.

Drawings

FIG. 1 shows agarose gel electrophoresis results of PCR amplification products of the VRN-A1 specific primer pair C22 and the Chinese spring VRN-A1 promoter region.

FIG. 2 shows the results of flowering phase comparison among wheat varieties C22 containing a natural 21bp sequence deletion and common China Spring (CS).

FIG. 3 shows the results of partial resequencing of other wheat varieties or lines.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

EXAMPLE 1 design of specific primers amplification of the DNA sequence of the VRN-A1 promoter region in the wheat genome

Wheat is a monocot whose genomic sequences have been assembled. The 21bp DNA sequence of the VRN-A1 gene promoter region was obtained by the inventors according to the MH-seq (Zhao et al 2020) technique in combination with GWAS. The sequence is as follows: TGGAAGAGAGGGGAGGAGAGG.

The DNA fragment was cloned into pMD19-T from the wheat genome, sequenced and the sequences aligned. Then, PCR amplification was performed in different wheat group materials using primers (GTGGTTGGGTGAGGACGT) and R (CCTGCCGGAATCCTCGTT) specific to the DNA fragment, and the flowering habit and the approximate flowering time of the detected material were determined based on the presence or absence of the amplified fragment.

The polymorphism of the DNA sequences of the C22 wheat variety and the Chinese Spring (CS) wheat variety in the promoter region of the VRN-A1 gene is detected by amplifying the 21bp sequence through a VRN-A1 specific PCR primer, as shown in figure 1.

Example 2

By using the 21bp DNA sequence and combining a resequencing result, scanning different wheat groups, screening out wheat materials with changed sequences in a natural wheat group, and using the wheat materials for flowering phenotype investigation, wherein the main steps are as follows:

1) Obtaining different types of wheat resequencing data from NCBI/EBI/GSA and other databases;

2) After quality control treatment such as removing joints is carried out on the obtained retested data, the retested data is compared with the China spring reference genome V1.0 by BWA comparison software;

3) Filtering out the resequencing results of the alignment quality MAPQ < 20;

4) Performing indel calculation on the high-quality comparison file by using GATK, and filtering out low-confidence indel sites;

5) And (3) selecting an indel locus and 20bp upstream and downstream of a deletion point coordinate (chr 5A: 587423376) for carrying out association analysis, and simultaneously combining flowering phenotype observation according to the existence of the 21bp DNA sequence, so that the flowering habit and the flowering time of the detected wheat material are accurately judged. By facilitating the re-sequencing, the wheat variety C22 is screened, the 21bp sequence is deleted in the VRN-A1 gene promoter region, and by comparing C22 with the flowering period of Chinese Spring (CS) wheat, C22 is found to be about 40 days earlier than Chinese spring, as shown in figure 2. Meanwhile, we also compare the sequencing results of other wheat varieties or lines, and find that the fragment deletion and the premature flower have stronger correlation indeed, as shown in figure 3.

There are a number of wheat genomics databases such as: the SNP hub portal of the wheat multi-group center and Chinese agricultural university already contains the prepared DNA sequence polymorphism information file, so that the DNA sequence polymorphism information of the VRN-A1 gene promoter in different wheat sequencing materials can be rapidly obtained through comparative analysis. By correlating with the flowering period of the corresponding material, effective target sites are provided for creating or cultivating new wheat varieties in different breeding periods by manually editing (inserting, replacing, deleting and the like of bases) the 21bp DNA sequence.

The invention screens a brand new DNA sequence and uses the DNA sequence in the rapid identification of important agronomic character related ecology such as the flowering phase of wheat and the spring and winter habits thereof, and provides proper new varieties and new germplasm materials for different wheat ecological planting areas. This sequence is found in hexaploid wheat, but is also present in a large amount in tetraploid wheat, and is common. Therefore, the DNA sequence can be used for developing a stable molecular marker, and has the following application prospects: (1) According to existence or nonexistence of the 21bp DNA sequence in the VRN-A1 gene promoter region, most wheat families can be divided into two types of (partial) winter and (partial) spring according to the flowering phase and the spring and winter habit at present, thereby being beneficial to rapid identification of ecological types related to the flowering phase and the spring and winter habit of different wheat materials. (2) The 21bp DNA sequence of the VRN-A1 gene promoter region influences flowering mainly by inhibiting the expression of the gene, so that the sequence is modified by manual editing (base insertion, replacement, deletion and the like) through a gene editing means, and the research on new wheat varieties or new intermediate wheat breeding materials in different breeding periods and molecular regulation mechanisms is hopeful to be created or cultivated.

Reference is made to:

Zhao HN,Zhang WL,Zhang T,Lin Y,Hu YD,Fang C,Jiang JM.2020.Genome-wide MNase hypersensitivity assay unveils distinct classes of open chromatin associated with

H3K27me3 and DNA methylation in Arabidopsis thaliana.Genome Biology 21:24。

sequence listing

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Claims (3)

1. The application of the specific primer for detecting the small fragment DNA regulatory element in identifying the flowering phase and the spring and winter habit related ecology of wheat crops is characterized in that the nucleotide sequence of the small fragment DNA regulatory element is shown as SEQ ID No. 1.

2. The use according to claim 1, characterized in that: carrying out PCR amplification in different wheat colony materials by utilizing specific primers, judging the spring and winter habits and flowering periods of the detected materials according to the existence of SEQ ID No.1 in the amplified fragment, wherein the lack of the fragment shows early flowering spring, and the presence of the fragment shows late flowering winter or weak spring; the sequence of the specific primer is as follows: f: GTGGTTGGGTGAGGACGT; r: CCTGCCGGAATCCTCGTT.

3. A method for identifying ecology related to flowering phase and spring and winter habit of wheat crops, which is characterized by comprising the following steps: the method comprises the following steps:

the small fragment DNA regulatory element with the nucleotide sequence shown as SEQ ID No.1 is applied to identification or screening of the flowering period and the spring and winter habit related ecology of wheat crops by developing molecular markers or probes, so that the breeding process of wheat molecules is accelerated;

carrying out PCR amplification in different wheat colony materials by utilizing specific primers, judging the spring and winter habits and flowering periods of the detected materials according to the existence of SEQ ID No.1 in the amplified fragment, wherein the lack of the fragment shows early flowering spring, and the presence of the fragment shows late flowering winter or weak spring; the sequence of the specific primer is as follows: f: GTGGTTGGGTGAGGACGT; r: CCTGCCGGAATCCTCGTT.