Rice quality functional gene detection rice core 1 primer set, primer pool, kit and application thereof

Abstract

The application discloses a rice product core No.1 primer group for detecting rice quality functional genes, a primer pool, a kit and application thereof, and belongs to the technical field of biology. The rice product core No.1 primer group comprises a first primer pair to a 40th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 40th primer pair and the reverse primer of the 40th primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO:80 in a sequence table. The rice product core No.1 primer group, the primer pool and the kit provided by the application can be used for detecting rice quality functional genes, and provide a method for rapidly, accurately and low-cost detecting variation information of appearance quality genes and taste quality genes of rice for rice genetic and breeding workers.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a primer set, primer pool, reagent kit, and application of rice quality functional genes (specifically, the Rice Core No. 1 primer set). Background Technology

[0002] China is one of the origins of rice (Oryza sativa) and has a long history of cultivating and consuming it. Half of the world's population consumes rice, mainly in Asia, Southern Europe, and parts of tropical America and Africa. Developing high-yielding, stable-yielding, and high-quality rice varieties is crucial to global food security.

[0003] With economic development and the continuous improvement of people's living standards, the demand for high-quality rice is increasing, making rice quality improvement a key focus of current rice breeding work. The appearance and taste of rice are the most important indicators affecting rice quality grades. Discovering superior rice quality genes and applying them to rice quality genetic improvement is an important research direction now and in the future.

[0004] Several major genes related to the appearance and eating quality of rice have been cloned in rice, but only a small number of these genes possess natural variation sites that can be used for breeding. Currently, various molecular markers, such as SSR markers, have been developed for single-gene variation sites for assisted selection breeding. However, when using SSR markers for PCR amplification, different methods are required to detect multiple gene sites separately, making the detection process cumbersome, time-consuming, costly, and inefficient. Therefore, developing a universal primer set and a precise and efficient multi-gene detection method for rice quality gene detection is crucial for rice breeding and quality improvement. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a primer set (specifically, the rice core 1 primer group), primer pool, reagent kit, and their applications for detecting rice quality functional genes. The technical solution is as follows:

[0006] On one hand, the present invention provides a Rice Core 1 primer set for detecting rice quality functional genes. The Rice Core 1 primer set includes primer pairs 1 to 40, each primer pair including a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 40th primer pair and the reverse primer of the 40th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing.

[0007] On the other hand, embodiments of the present invention provide a primer pool for detecting rice quality functional genes. The primer pool includes primer pool A and primer pool B. Primer pool A includes primer pairs 1 to 26, and primer pool B includes primer pairs 27 to 40. Each primer pair includes a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 40th primer pair, and the reverse primer of the 40th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing.

[0008] Specifically, the primer pool includes primer pool A and primer pool B.

[0009] In another aspect, embodiments of the present invention provide a kit for detecting rice quality functional genes, the kit comprising the above-mentioned Rice Core No. 1 primer set.

[0010] In another aspect, embodiments of the present invention provide an application of the Rice Core 1 primer set for detecting rice quality function genes, the application including: using the above-mentioned Rice Core 1 primer set for detecting rice quality function genes.

[0011] Specifically, the detection of rice functional genes includes: detecting rice appearance quality genes and detecting taste quality genes.

[0012] The beneficial effects of the technical solution provided by the embodiments of the present invention are as follows: The Rice Core No. 1 primer set and primer pool provided by the embodiments of the present invention can perform multiplex PCR amplification in the same reaction system, and the primer pairs will not interfere with each other during amplification. By comparing these primer pairs one by one, primers that can form dimers are screened out, avoiding the formation of primer dimers and reducing non-specific amplification. At the same time, the Rice Core No. 1 primer set and primer pool provided by the embodiments of the present invention can perform multiplex PCR amplification for multiple genes in the same reaction system, which greatly simplifies the operation steps, improves the amplification efficiency, ensures a high success rate, and reduces the false negative rate of quality genes. The Rice Core No. 1 primer set, primer pool, and kit provided by the present invention can be used to detect rice quality functional genes, providing rice geneticists and breeders with a rapid, accurate, and low-cost method for detecting gene variation information of rice appearance quality and eating quality. It can quickly and accurately diagnose the superior alleles carried by existing varieties, backbone parents, and lines, providing guidance for the formulation of breeding strategies and greatly improving the efficiency of breeding. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below.

[0014] Example 1

[0015] This invention provides a Rice Core 1 primer set for detecting rice quality function genes. The Rice Core 1 primer set includes primer pairs 1 to 40, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 40, and the reverse primer of primer pair 40 are shown in SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing, as detailed in Table 1.

[0016] Table 1 shows the sequences of 80 primer pairs and their corresponding primer pools.

[0017]

[0018]

[0019] Example 2

[0020] This invention provides a primer pool for detecting rice quality functional genes. The primer pool includes primer pool A, primer pool B, and primer pool C. Each primer pair includes a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 40th primer pair, and the reverse primer of the 40th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing.

[0021] Specifically, the Daopin 1 primer set includes primer pool A and primer pool B, as shown in Table 1.

[0022] Example 3

[0023] This invention provides a kit for detecting functional genes in rice quality, the kit including the rice core 1 primer set provided in Example 1.

[0024] Example 4

[0025] This invention provides an application of the Rice Core 1 primer set for detecting rice quality function genes. The application includes using the Rice Core 1 primer set provided in Example 1 to detect rice quality function genes.

[0026] Furthermore, the detection of rice functional genes includes: Primer pool A is used to detect rice appearance quality genes, which include: the Chalk5 gene to reduce chalkiness; the WCR1 gene to reduce heart whitening; the WBR7 gene to reduce belly whitening; the GFR1 gene to control low grain filling rate; the OsSPL13 / GLW7 and GE / CYP78A13 / BG2 genes to increase grain size; the qNGR2 / GRF4 gene to increase grain size and nitrogen fertilizer use efficiency; the GS6 / DLT / OsGRAS-32 / SMOS2 / D62 gene to decrease grain size; the TGW6 gene to increase grain weight; and the gene to increase... Genes that increase grain length include GS2, GS3, GL3.1 / qGL3, GL3.3 / qTGW3, OsSNB, OsLG3, and qLGY3 / OsMADS1 / GW3p6 / OsLG3b; gene that decreases grain length includes GL3.2 / CYP78A5; gene that increases grain thickness, length, and yield includes qLGY3 / OsMADS1 / GW3p6 / OsLG3b; gene that increases grain width and weight includes GW2 and GW8 / OsSPL16; and gene that decreases grain width includes GS5, GW5 / GSE5 / qSW5, and GWY10.

[0027] Primer pool B was used to detect taste quality genes, which included: the Waxy / GBSSI gene affecting amylose content; the ALK / SSSII gene increasing gelatinization temperature; the OsAAP6 gene increasing protein and amino acid content; the OsGluA2 gene increasing protein content; the OsACS6 / SSG6 gene increasing L-asparagine; the qPAL6 gene increasing fatty acid C16:0 content; the qLIN6 gene decreasing fatty acid C18:0 content; and the fgr / Badh2 and OsODC1 genes increasing aroma.

[0028] In this embodiment, the rice sample to be tested can be a rice seed, seedling, leaf or panicle, etc., preferably a rice seed or rice leaf.

[0029] In this embodiment, 10 rice varieties were selected as the rice varieties to be tested. The specific varieties included: Efeng Simiao, Meixiang Xinzhan, Huaqianghui No. 1, R8612, E Nongxiang Liangyou No. 1, E Nongxiang Liangyou Chuxiang 89, E Nongxiang Liangyou 1300, Towada, Lemont, and ZH11. The above rice varieties were provided by the Institute of Food Crops, Hubei Academy of Agricultural Sciences.

[0030] Whole genome DNA was extracted from the rice samples to be tested.

[0031] The extraction was performed using the CTAB (Cetyltrimethylammonium Bromide) method, the SDS (Sodium Dodecyl Sulfate) method, or existing commercially available whole genome DNA extraction kits and their extraction methods.

[0032] In this embodiment, the CTAB method was used to extract whole-genome DNA from the sample to be tested. Specifically, about 200 mg to 300 mg of rice leaves to be tested were placed in a 2.0 mL centrifuge tube and thoroughly ground. 500 μL of CTAB extraction solution preheated to 65 °C was added to each centrifuge tube and thoroughly mixed. The tubes were then incubated in a 65 °C water bath for 30 min, during which time each centrifuge tube was gently inverted to mix. Then, an equal volume of chloroform / isoamyl alcohol (chloroform to isoamyl alcohol volume ratio of 24:1) was added to each centrifuge tube. After thorough mixing, the tubes were allowed to stand for 10 min and centrifuged at 12000 rpm for 10 min. The supernatant was collected. The supernatant was transferred to a new centrifuge tube and an equal volume of pre-cooled isopropanol was added. The tubes were gently inverted to mix and incubated at -20 °C for 30 min, followed by centrifugation at 4 °C and 12000 rpm for 10 min. Discard the supernatant, retain the precipitate, and wash the precipitate with 70% ethanol. In this example, wash twice to obtain the whole genome DNA of the rice to be tested. Dry the whole genome DNA of the rice to be tested at room temperature, add 100 μL of water, and dissolve it completely before use.

[0033] The obtained whole genome DNA of the rice to be tested was amplified by multiplex PCR (Polymerase Chain Reaction) using the Rice Core No. 1 primer set provided in the embodiments of the present invention.

[0034] Specifically, the rice core 1 primer set for the multiplex PCR amplification reaction consisted of a mixture of all 40 primer pairs. The multiplex PCR amplification reaction was performed using KAPA2G FAST Multiplex PCR Kits (purchased from Kapa Biosystems). In this example, the multiplex PCR amplification reaction system was 20 μL. Each reaction tube contained 50 ng of the whole genomic DNA of the rice sample, 10 μL of 2multiplex PCR mix, and 0.5 μL of each primer pool (primer pools A-I). ddH2O was added to bring the total volume to 20 μL.

[0035] The conditions for the first round of multiplex PCR amplification were as follows: preheating at 95℃ for 3 min; (annealing at 95℃ for 20 s, annealing at 60℃ for 30 s, extension at 72℃ for 2 min) × 25 cycles; holding at 72℃ for 5 min.

[0036] After the multiplex PCR amplification reaction is completed, the multiplex PCR amplification product is obtained and then purified. Specifically, the multiplex PCR amplification product is purified using the AgencourtAMPure XP Kit purchased from Beckman Coulter to remove excess primers and obtain the purified multiplex PCR amplification product. Refer to the kit's instruction manual for detailed procedures.

[0037] Library construction and sequencing of multiplex PCR amplification products (detection of important rice trait gene loci).

[0038] The purified multiplex PCR amplification products were used to construct sequencing libraries using Illumina, MGI, or other commercially available library construction kits, and then sequenced for analysis.

[0039] In this embodiment, NEBNext is used. ® Ultra™ II DNA Library Prep Kit for Illumina ® (Purchased from New England BioLabs) Next-generation sequencing libraries were constructed. Specifically, the purified multiplex PCR amplification products were first quantified. 30 ng of the purified multiplex PCR amplification products were used for DNA fragment end repair and A addition, followed by adapter ligation to obtain ligation products. These ligation products were then purified using magnetic beads to obtain the next-generation sequencing library. The constructed next-generation sequencing library was quantified using qPCR, and after quality control, it was sequenced using a next-generation sequencing platform. The sequencing results are shown in Table 2.

[0040] Table 2 shows the sequencing results.

[0041]

[0042] In Table 2, A represents allele, R represents reference genotype, H represents heterozygous genotype, and "-" indicates not detected.

[0043] Bioinformatics analysis of sequencing data.

[0044] The sequences of gene loci related to rice yield were obtained and sequenced, and the obtained bioinformatics were analyzed. Specifically, low-quality reads were removed from the raw sequencing data, and the remaining reads were aligned to the whole genome reference sequence of the rice variety Nipponbare using BWA software to identify variations between different rice varieties and the whole genome reference sequence. The results were then analyzed using Samstools software, and the analysis results are shown in Table 3.

[0045] Table 3 shows the gene locus detection results for 10 rice varieties tested.

[0046]

[0047] Table 3 shows that the sequencing results of the 10 rice varieties tested in this embodiment showed an average detection efficiency of 91.67% and an average detection depth of 3278.49 for the target gene loci. This indicates that using the Rice Core No. 1 primer set provided in this embodiment can effectively avoid mutual interference between primer pairs. Simultaneously, the Rice Core No. 1 primer set can efficiently detect rice quality function genes and the gene composition of backbone parents and hybrid progeny lines, greatly improving the detection efficiency of rice hybrid breeding. The superior alleles of the 10 rice varieties tested were determined using multiplex PCR amplification and bioinformatics analysis, providing guidance for formulating breeding strategies.

[0048] The rice variety tested in this embodiment of the invention has the same detection results as those provided in Table 2, indicating that the rice variety core 1 primer set, primer pool, and reagent kit provided in this embodiment of the invention are accurate. Furthermore, during breeding, users can select superior genes corresponding to the primer pairs according to their breeding objectives and screen them using the primer pairs to obtain high-quality rice varieties. Taking primers PA063 and PA064 as examples, they represent relative phenotypes of increasing and decreasing grain length, respectively. Users can select varieties that increase or decrease grain length according to their breeding objectives.

[0049] The Daopinxin No. 1 primer set and primer pool provided in this embodiment of the invention can perform multiplex PCR amplification in the same reaction system without primer interference. By comparing these primers pairwise, primers that can form dimers are eliminated, thus avoiding primer dimer formation and reducing non-specific amplification. Furthermore, using the Daopinxin No. 1 primer set and primer pool provided in this embodiment of the invention allows for multiplex PCR amplification of multiple genes in the same reaction system, greatly simplifying the operation steps, improving amplification efficiency, ensuring a high success rate, and reducing the false negative rate of genes. More importantly, the Daopinxin No. 1 primer set and primer pool can be used to directly construct sequencing libraries and are compatible with existing mainstream next-generation sequencing platforms, such as various sequencing platforms of Illumina or MGI. The rice core 1 primer set and primer pool provided by this invention can be used to detect functional genes in rice. It provides rice geneticists and breeders with a rapid, accurate, and low-cost method to detect gene variations in important plant architecture, yield, flowering time, resistance to biotic and abiotic stresses, fertility, nutritional efficiency, and rice quality. It can quickly and accurately diagnose the superior alleles carried by existing varieties, core parents, and lines, providing guidance for the formulation of breeding strategies and greatly improving breeding efficiency.

[0050] The above description is merely an optional embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rice core primer set for detecting rice quality function genes, characterized in that, The rice core 1 primer set includes primer pairs 1 to 40, each primer pair including a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 40th primer pair, and the reverse primer of the 40th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing.

2. A primer pool for detecting functional genes for rice quality, characterized in that, The primer pool includes at least one of primer pool A and primer pool B, primer pool A includes primer pairs 1 to 26, primer pool B includes primer pairs 27 to 40, each primer pair includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 40th primer pair, and the reverse primer of the 40th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 80 in the sequence listing.

3. The primer pool according to claim 2, characterized in that, The primer pools include primer pool A and primer pool B.

4. A kit for detecting functional genes for rice quality, characterized in that, The kit includes the rice core primer set 1 as described in claim 1.

5. The application of a rice variety core 1 primer set for detecting rice quality function genes, characterized in that, The application includes using the rice core primer set as described in claim 1 to detect rice quality function genes.

6. The application according to claim 5, characterized in that, The detection of rice quality function genes includes: detection of rice appearance quality genes and taste quality genes.

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