Application of a gene for high thousand-grain weight in common wheat

Abstract

The present invention relates to the technical field of gene synthesis and application, specifically to the application of a high thousand-grain weight gene in common wheat. The open reading frame sequence of this gene is shown in SEQ ID NO.1, and the genomic sequence is shown in SEQ ID NO.2. This gene is named TaCHLI-7A. Complementing the wheat variety Shi 4185 with TaCHLI-7A can significantly increase the leaf width and improve the chlorophyll content; complementing the wheat variety Shi 4185 with TaCHLI-7A can significantly increase the grain size and thousand-grain weight; complementing the wheat variety Shi 4185 with TaCHLI-7A can significantly improve the yield per plant of wheat.

Description

Technical Field

[0001] The present invention relates to the technical field of gene synthesis and application, and specifically to a high thousand-grain weight gene of common wheat, a construction method and an application of this gene. Background Art

[0002] Common wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42) belongs to the genus Triticum in the family Poaceae, and is one of the most important food crops in the world. However, with the rapid growth of the population, the demand for food crops is increasing day by day. Therefore, improving the yield per unit area of wheat has become the key to solving the food problem. So far, a large number of quantitative trait loci (QTLs) related to wheat yield, such as spike length, spikelet number, grains per spike, grain weight per spike, grain length, grain width and thousand-grain weight, etc., have been reported in common wheat.

[0003] The leaves of wheat are not only the main organs for wheat photosynthesis, but also the main factors determining the plant type of wheat. Especially the flag leaf, which is usually considered as the functional leaf, and it contributes 45 - 58% of the photosynthetic products during the grain filling period of wheat. A number of studies have shown that there is a significant positive correlation between the width and area of the flag leaf and the thousand-grain weight of wheat grains. The content of chloroplasts in plant leaves is directly related to photosynthesis. Plants with high chlorophyll content have high photosynthetic efficiency, and at the same time, the photosynthetic products supplied to the grains also increase correspondingly, which helps to increase the size and thousand-grain weight of wheat grains. Therefore, during the improvement process of wheat varieties, for the purpose of increasing the size and thousand-grain weight of wheat grains, breeders usually take two ways, namely, increasing the flag leaf width to increase the flag leaf area and increasing the chlorophyll content in the leaves, to improve the grain traits of wheat. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a high thousand-grain weight gene of common wheat, and through the application of this gene, the leaf width is increased and the chlorophyll content is improved, and finally the purpose of increasing the size and thousand-grain weight of wheat grains is achieved.

[0005] To solve the above technical problem, the open reading frame sequence of the high thousand-grain weight gene of common wheat provided by the present invention is shown in SEQ ID NO.1, and the genomic sequence is shown in SEQ ID NO.2, and this gene is named TaCHLI-7A.

[0006] The construction method of the high thousand-grain weight gene of common wheat of the present invention includes the following steps:

[0007] Step 1) Construct an introgression line population with Xinjiang wheat variety Xinjiang 5, a unique wheat germplasm resource in China, as the donor and the main cultivated wheat variety Shi 4185 as the recipient;

[0008] Step 2) Use the method of reduced-representation genome sequencing to perform genome sequencing on the lines in the introgression line population;

[0009] Step 3) Conduct a genome-wide association study (GWAS) on the introgression line population to determine a QTL locus located at the end of chromosome 7A of common wheat as the dominant genetic locus controlling the grain yield of the main spike of wheat;

[0010] Step 4) After analyzing the grain yield of the main spike of the introgression line population in Step 1), select two introgression lines with significantly increased grain yield of the main spike compared to the parent Shi 4185; further extract the total RNA of the materials at the two-row stage of young spike development of these two introgression lines and the parent Shi 4185, and perform comparative analysis of transcriptome sequencing to find the gene TraesCS7A02G480700, which is not expressed in Shi 4185 but highly expressed in the two introgression lines, and the homologous genes of this gene on the homologous chromosomes 7B and 7D are normally expressed in both Shi 4185 and the two introgression lines;

[0011] Step 5) Design genome-specific primers for the TraesCS7A02G480700 gene, and prove that the TraesCS7A02G480700 gene cannot be amplified in Shi 4185 but can amplify the target band in the two introgression lines, thereby determining TraesCS7A02G480700 as the candidate gene for this QTL locus and naming it TaCHLI-7A.

[0012] One application of the high thousand-grain weight gene of common wheat in the present invention is that complementing the wheat variety Shi 4185 with TaCHLI-7A can significantly increase the leaf width of wheat and improve the chlorophyll content.

[0013] Another application of the high thousand-grain weight gene of common wheat in the present invention is that complementing the wheat variety Shi 4185 with TaCHLI-7A can significantly increase the size and thousand-grain weight of wheat grains.

[0014] The third application of the high thousand-grain weight gene of common wheat in the present invention is that complementing the wheat variety 4185 with TaCHLI-7A can significantly increase the yield per plant of wheat. Description of the Drawings

[0015] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments:

[0016] Figure 1 Schematic diagram of a new dominant genetic locus for controlling the grain yield of the main spike;

[0017] Figure 2 Schematic diagram for the cloning of TaCHLI-7A based on transcriptome sequencing analysis;

[0018] Figure 3 Schematic diagram for the comparative analysis of leaf width and chlorophyll content of the TaCHLI-7A complemented wheat variety Shijia 4185 lines;

[0019] Figure 4 Schematic diagram for the comparative analysis of grain traits of the TaCHLI-7A complemented wheat variety Shijia 4185 lines;

[0020] Figure 5 Schematic diagram for the analysis of other agronomic traits of the TaCHLI-7A complemented wheat variety Shijia 4185 lines. Detailed implementation manners

[0021] The present invention provides a high thousand-grain weight gene of common wheat, which is named TaCHLI-7A, and its open reading frame sequence is shown as SEQ ID NO.1, specifically as follows:

[0022]

[0023] The genomic sequence of TaCHLI-7A is shown in SEQ ID NO.2 as follows:

[0024]

[0025] The construction method of the high thousand-grain weight gene TaCHLI-7A of common wheat in the present invention comprises the following steps:

[0026] Step 1) Construct an introgression line population with Xinjiang wheat No. 5, a unique wheat germplasm resource in China, as the donor and the main cultivated wheat variety Shimai 4185 as the recipient.

[0027] Step 2) Adopt the method of reduced-representation genome sequencing to perform genome sequencing on the lines in the introgression line population.

[0028] Step 3) Perform a genome-wide association study (GWAS) on the introgression line population to determine that a QTL locus located at the end of chromosome 7A of common wheat is a dominant genetic locus controlling the grain yield of the main spike of wheat.

[0029] Step 4) After analyzing the grain yield of the main spike of the introgression line population in Step 1), select two introgression lines with significantly increased grain yield of the main spike compared to the parent Shimai 4185; further extract the total RNA of the materials at the two-ridge stage of young spike development of these two introgression lines and the parent Shimai 4185, and perform transcriptome sequencing and comparative analysis to find the gene TraesCS7A02G480700, which is not expressed in Shimai 4185 but highly expressed in the two introgression lines, and the homologous genes of this gene located on the homologous chromosomes of 7B and 7D are normally expressed in both Shimai 4185 and the two introgression lines.

[0030] Among them, in the process of RNA transcriptome sequencing analysis, three biological replicates are set for each of the two introgression lines IL194734 and IL194915 and Shimai 4185, a total of 9 sequencing samples. Subsequently, after comparing each introgression line with Shimai 4185 respectively, genes with a difference in expression level greater than twofold are obtained. Then, the overlapping genes are selected from the two parts of differentially expressed genes as candidate differentially expressed genes.

[0031] Step 5) Design genome-specific primers for the TraesCS7A02G480700 gene, prove that the TraesCS7A02G480700 gene cannot be amplified in Shimai 4185 but target bands can be amplified in the two introgression lines, thereby determining TraesCS7A02G480700 as a candidate gene for this QTL locus and naming it TaCHLI-7A.

[0032] For primer detection, two pairs of specific molecular markers for the TaCHLI-7A gene were designed, namely 480700RTF3 / 480700RTR3 and 480700RTF3 / 480700RTR4. Using these two pairs of primers, PCR amplification detection was performed on the introgression lines and Shi 4185, followed by agarose gel electrophoresis detection. It was found that there was no target band in Shi 4185, while there was a target band in the introgression lines with large main spike grain yields.

[0033] Example 1: A new dominant genetic locus controlling the main spike grain yield

[0034] An introgression line population was constructed with Xinjiang wheat variety Xinjiang 5, a unique wheat germplasm resource in China, as the donor and wheat variety Shi 4185 as the recipient (as shown in Figure 1 A). In 2019 and 2020, the agronomic trait phenotypes of each line in the introgression line population were investigated and analyzed at the Beijing farm for two consecutive years. Using the method of reduced-representation genome sequencing, the genomes of 166 lines in the population were further sequenced. Based on the genome sequencing data, GWAS analysis was performed, and the results showed that a QTL locus at the end of chromosome 7A was significantly associated with the main spike grain yield trait (as shown in Figure 1 B). Further analysis of the agronomic traits of each introgression line showed that all the lines containing heterozygous target introgressed fragments had main spike grain yields comparable to those of the lines with homozygous introgressed fragments (as shown in Figure 1 C). Therefore, this locus was determined to be a dominant genetic locus controlling the main spike grain yield of wheat.

[0035] Example 2: Cloning of the TaCHLI-7A gene

[0036] In the introgression line population, two introgression lines with high main spike grain yields (IL194734 and IL194915, which were obtained by crossing Xinjiang 5 as the donor and Shi 4185 as the recipient, and then backcrossing 5 generations with Shi 4185 as the recurrent parent and self-crossing 4 generations to obtain two stable and homozygous introgression lines (BC 5 F 4 )) were selected. Samples were taken from their young ear development two-ridge stage materials and those of Shi 4185, and total RNA extraction and transcriptome comparative analysis were performed (as shown in Figure 2 A). Through analysis, it was found that there were 116 common differentially expressed genes (gene expression differences exceeding 2-fold) in the two comparison combinations of IL194734 vs. Shi 4185 and IL194915 vs. Shi 4185, including 54 genes with consistent up-regulation and 60 genes with consistent down-regulation (as shown in Figure 2as shown in Figure B). Further analysis showed that 26 of these differentially expressed genes were located in the chromosomal regions mapped by QTL, and one of the genes, TraesCS7A02G480700, was not expressed in Shi 4185 but highly expressed in the two introgression lines (as shown in Figure 2 Figure C). At the same time, the homologous genes of TraesCS7A02G480700 located on the homologous chromosomes of 7B and 7D were normally expressed in Shi 4185 and the two introgression lines (as shown in Figure 2 Figure D).

[0037] Further design of genome-specific primers for the TraesCS7A02G480700 gene for detection showed that TraesCS7A02G480700 was not amplified in Shi 4185, but the target bands could be amplified in the introgression lines IL194734 and IL194915 (as shown in Figure 2 Figure E). Therefore, the TraesCS7A02G480700 gene was identified as the candidate gene for this QTL locus. Since it encodes the magnesium chelatase subunit I, it was named TaCHLI-7A.

[0038] Example 3: Complementation of TaCHLI-7A in wheat variety Shi 4185 significantly increases leaf width and chlorophyll content

[0039] To further verify the function of TaCHLI-7A, a binary vector expressing the TaCHLI-7A gene using its own promoter was constructed P TaCHLI-7A ::TaCHLI-7A . Through genetic transformation, the TaCHLI-7A gene was complemented in Shi 4185 to construct transgenic lines (as shown in Figure 3 Figure A). The analysis results showed that the leaf widths of the transgenic plants were significantly increased (as shown in Figure 3 Figure B and Figure 3 Figure C), the leaf lengths did not change significantly (as shown in Figure 3 Figure D), and the leaf surface areas were also significantly increased (as shown in Figure 3 Figure E). Further detection of the chlorophyll content in the leaves showed that the chlorophyll content in the leaves of the transgenic plants was significantly increased compared with that in the wild-type Shi 4185 (as shown in Figure 3 Figure F).

[0040] Example 4: Complementation of TaCHLI-7A in wheat variety Shi 4185 significantly increases grain size and thousand-grain weight

[0041] Analysis of grain traits showed that for the two complementation lines ( P TaCHLI-7A ::TaCHLI-7A -4 and PTaCHLI-7A ::TaCHLI- 7A -5), the seed length, width, and seed size were significantly increased compared to those of the wild-type Shi 4185 (as shown in Figure 4 A to Figure 4 D). Further comparing the accumulation changes of fresh grain weight during seed filling from the 2nd day to the 32nd day after flowering, the results showed that the seed weight of the transgenic plants significantly exceeded that of the wild-type from 5 to 8 days after seed flowering, and was significantly increased compared to wild-type Shi 4185 from 11 to 20 days. This result indicates that during the process of seed filling, the filling rate of the transgenic lines was significantly higher than that of wild-type Shi 4185 (as shown in Figure 4 E). Eventually, the 1000-grain weight of the transgenic lines was significantly increased compared to that of the wild-type, with the final increase reaching 33.37% (#4) and 24.62% (#5) respectively (as shown in Figure 4 F).

[0042] Example 5: Complementation of TaCHLI-7A in wheat variety Shi 4185 significantly increases the yield per plant

[0043] Further comparative analysis of other agronomic traits of the TaCHLI-7A complementation lines and wild-type Shi 4185 (as shown in Figure 5 ). The results showed that there were no significant changes in plant height, number of spikes per plant, spike length, number of spikelets, and number of grains per spike (as shown in Figure 5 A to Figure 5 F). However, the grain yield per plant of the two transgenic lines was significantly increased compared to that of wild-type Shi 4185 (as shown in Figure 5 G).

[0044] In summary, the present invention is not limited to the above specific embodiments. Those skilled in the art can make several changes or modifications without departing from the technical solution of the present invention, and the above changes or modifications all fall within the protection scope of the present invention.

Claims

1. Application of a gene for high thousand-grain weight in common wheat, characterized in that the open reading frame sequence of this gene is as shown in SEQ ID NO.1, and this gene is named TaCHLI-7A; complementing the wheat variety Shi 4185 with TaCHLI-7A significantly increases the leaf width and improves the chlorophyll content.

2. Application of the gene for high thousand-grain weight in common wheat according to claim 1, characterized in that complementing the wheat variety Shi 4185 with TaCHLI-7A further significantly increases the grain size and thousand-grain weight.

3. Application of the gene for high thousand-grain weight in common wheat according to claim 1, characterized in that complementing the wheat variety Shi 4185 with TaCHLI-7A further significantly improves the yield per plant.

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