A method for cultivating high-yield crops and its dedicated specific genome fragment and specific primer pair

Abstract

The present invention discloses a method for cultivating high-yield crops and its dedicated specific genomic fragment and specific primer pair. The method provided by the present invention for screening rice with different yield traits may include the following steps: (1) detecting the genotype of the rice to be tested based on the specific gene fragment; the specific gene fragment is located in the rice genome and is SUSY2, and there are two allelic forms, SUSY2_a and SUSY2_b, the SUSY2_a is shown as Sequence 1 in the sequence listing, and the SUSY2_b is shown as Sequence 2 in the sequence listing; (2) performing the following determination: under the same conditions, the average yield of the rice population with the genotype homozygous for SUSY2_b is higher than the average yield of the rice population with the genotype homozygous for SUSY2_a. Experiments have shown that the method provided by the present invention can screen rice with different individual yield traits, is simple to operate, has a high accuracy rate, and has important application value in rice breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for cultivating high-yield crops and a dedicated specific genome segment and specific primer pair thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, with more than half of the world's population relying on rice as their staple food. Therefore, breeding high-yield rice varieties is crucial. Identifying superior alleles controlling high-yield traits in rice is crucial for tracking and selecting high-yield rice varieties during the breeding process.

[0003] Rice breeding is a key component of agricultural civilization, having accompanied the development of human society for nearly 10,000 years. Rice is a major source of food in Asia, and its importance is growing in other regions, such as Africa. The rice genome was one of the first to be fully sequenced, and related research has rapidly advanced in recent years.

[0004] In practice, rice breeding encompasses conventional and hybrid breeding. Its fundamental goal is to combine desirable traits within a single variety, enabling the production of high-yield, high-quality rice under a wide range of geographical conditions. Current breeding methods mostly rely on visible traits and / or molecular markers. Due to the numerous and complex factors influencing rice traits, the means to improve them are still very limited. The probability of obtaining a favorable trait combination through various breeding methods remains low.

[0005] Because functional gene research often focuses on mutants, these findings are essential for understanding the biological characteristics of a species, but their guidance for agricultural breeding is often limited. Studies of natural variation within rice populations currently focus on single nucleotide polymorphisms (SNPs). Genome-wide association analysis based on these data can estimate associations between phenotypes and SNPs. The impact of these findings on agricultural breeding remains to be evaluated. The current bottleneck in rice breeding is identifying superior breeding parents and finding a superior genetic base material to ensure that desirable traits are retained from generation to generation under field conditions.

[0006] Based on the basic concepts of quantitative genetics, individuals with higher yields are likely to possess superior genes that influence their phenotype. These superior yield-influencing genes can be obtained from suitable rice materials under field conditions. Traditional rice breeding is essentially based on this principle.

[0007] Advances in molecular biology over the past few decades have led to the rise of molecular breeding, with breeding using specific genes or molecular markers as selection criteria becoming a hallmark of molecular breeding. Antagonistic breeding is the most prominent example. For yield, a limiting factor in molecular breeding is determining which genes or molecular fragments are most suitable for selection. Summary of the Invention

[0008] The invention provides a method for cultivating high-yield crops and a dedicated specific genome segment and specific primer pair.

[0009] The present invention first provides a method for screening rice with different yield traits.

[0010] The method for screening rice with different yield traits provided by the present invention (method A) may comprise the following steps:

[0011] (1) Detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is SUSY2, and has two allelic forms, SUSY2_a and SUSY2_b, wherein the SUSY2_a is shown as Sequence 1 in the sequence listing, and the SUSY2_b is shown as Sequence 2 in the sequence listing;

[0012] (2) The following judgment is made: Under the same conditions, the average yield of the rice population with the SUSY2_b homozygous genotype is higher than the average yield of the rice population with the SUSY2_a homozygous genotype.

[0013] The method for screening rice with different yield traits provided by the present invention (method B) may comprise the following steps:

[0014] (1) Using the genomic DNA of the rice to be tested as a template, PCR amplification is performed using a specific primer pair; if there is only one PCR amplification product and it is shown as Sequence 1 in the sequence listing, the genotype of the rice to be tested is SUSY2_a homozygous; if there is only one PCR amplification product and it is shown as Sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_b homozygous;

[0015] (2) The following judgment is made: Under the same conditions, the average yield of the rice population with the SUSY2_b homozygous genotype is higher than the average yield of the rice population with the SUSY2_a homozygous genotype.

[0016] The present invention also provides a rice breeding method (Method C), which may include the following steps:

[0017] (1) Detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is SUSY2, and has two allelic forms, SUSY2_a and SUSY2_b, wherein the SUSY2_a is shown as Sequence 1 in the sequence listing, and the SUSY2_b is shown as Sequence 2 in the sequence listing;

[0018] (2) The rice with the homozygous SUSY2_b genotype was the target rice for breeding.

[0019] To solve the above technical problems, the present invention further provides a rice breeding method (method D), which may include the following steps:

[0020] (1) Using the genomic DNA of the rice to be tested as a template, PCR amplification is performed using a specific primer pair; if there is only one PCR amplification product and it is shown as Sequence 1 in the sequence listing, the genotype of the rice to be tested is SUSY2_a homozygous; if there is only one PCR amplification product and it is shown as Sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_b homozygous;

[0021] (2) The rice with the homozygous SUSY2_b genotype was the target rice for breeding.

[0022] The target rice is a rice with high yield.

[0023] Any of the above-mentioned specific primer pairs may consist of primer 1 and primer 2;

[0024] The primer 1 may be as follows (a1) or (a2):

[0025] (a1) a single-stranded DNA molecule represented by Sequence No. 3 in the Sequence Listing;

[0026] (a2) a single-stranded DNA molecule having the same function as sequence 3 after one or more nucleotides are substituted and / or deleted and / or added;

[0027] The primer 2 may be as follows (b1) or (b2):

[0028] (b1) the single-stranded DNA shown in Sequence No. 4 of the Sequence Listing;

[0029] (b2) A single-stranded DNA molecule having the same function as sequence 4 after one or more nucleotides are substituted and / or deleted and / or added.

[0030] The present invention also protects a specific allele fragment. The specific allele fragment can be SUSY2_a or SUSY2_b;

[0031] The SUSY2_a may be (c1) or (c2) as follows:

[0032] (c1) the DNA molecule shown in Sequence 1 of the Sequence Listing;

[0033] (c2) a DNA molecule in which one or more nucleotides in sequence 1 are substituted and / or deleted and / or added and which has the same function as sequence 1;

[0034] The SUSY2_b may be (d1) or (d2) as follows:

[0035] (d1) DNA molecule shown in Sequence 2 of the Sequence Listing;

[0036] (d2) A DNA molecule having the same function as sequence 2 after one or more nucleotides are substituted and / or deleted and / or added.

[0037] In (c2) or (d2), the position where the "substitution and / or deletion and / or addition" occurs is located in a region outside the three differences between Sequence 1 in the Sequence Listing and Sequence 2 in the Sequence Listing.

[0038] The specific primer pair also falls within the protection scope of the present invention.

[0039] To solve the above technical problems, the present invention further provides a kit comprising the specific primer pair. The kit may further comprise conventional reagents for extracting rice genomic DNA and / or conventional reagents for PCR amplification and / or conventional reagents for sequencing.

[0040] The purpose of the kit is as follows (e1) or (e2) or (e3):

[0041] (e1) screening rice with different yield traits;

[0042] (e2) identifying or assisting in the identification of yield traits of rice;

[0043] (e3) Identifying or assisting in the identification of rice with different yield traits.

[0044] The preparation method of the kit also falls within the scope of protection of the present invention. The preparation method of the kit comprises the steps of individually packaging each primer in the kit.

[0045] The present invention also protects the use of the specific allele fragment or the specific primer pair, which is as follows (e1) or (e2) or (e3):

[0046] (e1) screening rice with different yield traits;

[0047] (e2) identifying or assisting in the identification of rice yield traits;

[0048] (e3) Identifying or assisting in the identification of rice with different yield traits.

[0049] The application of the specific allele fragment, the specific primer pair, the kit or any of the above methods in rice breeding also falls within the scope of protection of the present invention. The breeding goal of the rice breeding is to obtain high-yield rice.

[0050] Any of the above-mentioned yields may be per plant yield. Any of the above-mentioned yields may be grain yield.

[0051] The present invention also provides a method for identifying dominant alleles, comprising the following steps: determining alleles with dominant traits by comparing differences in biological traits among different groups; wherein each of the different groups is composed of individuals homozygous for the alleles.

[0052] The organism may be a sexually reproducing organism, specifically a plant, and more specifically rice.

[0053] The trait may be a measurable trait, specifically a yield trait, more specifically a grain yield trait.

[0054] Experiments have shown that the method provided by the present invention can be used to screen rice with different individual yield traits. The operation is simple and the accuracy is high, and the method has important application value in rice breeding. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0056] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0057] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0058] Example 1. Design and synthesis of primers

[0059] Sucrose is an important transport substance in rice. In the rice genome, multiple genes have been annotated as encoding sucrose synthase (EC 2.4.1.13), such as the gene encoding sucrose synthase 2 (JN944363.1; SUSY2) (hereinafter referred to as the SUSY2 gene). The SUSY2 gene is located on rice chromosome 3. Through a large number of preliminary experiments and sequence alignments, the inventors of the present invention discovered that there are two allele fragments in the SUSY2 gene (one allele fragment is shown in sequence 1 of the sequence table, named allele fragment SUSY2_a; the other allele fragment is shown in sequence 2 of the sequence table, named allele fragment SUSY2_b), which are correlated with rice yield per plant.

[0060] A specific primer pair was designed based on the two allele fragments mentioned above, consisting of primer 1 and primer 2.

[0061] Primer 1 (sequence 3 in the sequence listing): 5'-CCAGACTCAGACATATACTGGACGA-3';

[0062] Primer 2 (sequence 4 in the sequence listing): 5'-CTGCACGAAGGCACCATGAGT-3'.

[0063] Example 2: Establishment of a typing method based on the allele fragments in rice

[0064] The establishment method is as follows:

[0065] 1. Using the genomic DNA of the rice to be tested (about 10-100 ng) as a template, PCR amplification was performed using a specific primer pair consisting of primer 1 and primer 2 to obtain a PCR amplification product.

[0066] The reaction program of PCR amplification was as follows: 95°C for 5 minutes; 35 cycles of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute; and 72°C for 8 minutes.

[0067] 2. After completing step 1, the PCR amplification products are sequenced, and the following judgment is made based on the sequencing results: if there is only one PCR amplification product, and it is shown as sequence 1 in the sequence listing, the genotype of the rice to be tested is SUSY2_a homozygous; if there is only one PCR amplification product, and it is shown as sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_b homozygous; if there are two PCR amplification products, one as shown in sequence 1 in the sequence listing and the other as shown in sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_a / SUSY2_b heterozygous.

[0068] Since all rice varieties are cultivated varieties or farmer varieties, the proportion of individuals with homozygous genotypes is high.

[0069] Example 3: Association analysis between genotypes based on the allele fragments and rice yield per plant in rice

[0070] 1. Statistical analysis of per-plant yield of different rice varieties

[0071] In 2014, several rice varieties were planted in Sanya, Hainan Province (see Tables 1, 2, and 3; see column 2 for the rice variety name, column 3 for the rice variety origin, and column 6 for the 2009 serial numbers of some rice varieties). A completely randomized block experiment design was used in the field, with three replications. After the rice matured, the plants were harvested, weighed, and the average yield per plant was calculated (see columns 5, 5, and 6, for the results in Tables 1, 2, and 3).

[0072] 2. According to the typing method established in Example 2, the genotypes of various rice varieties were detected (results are shown in Tables 1, 2 and 3, column 4).

[0073] Table 1

[0074]

[0075]

[0076]

[0077] The results in Table 1 show that 50 of the 84 rice varieties were homozygous for SUSY2_a, with an average yield per plant of 31.27±1.51 g; 34 of the 84 rice varieties were homozygous for SUSY2_b, with an average yield per plant of 41.54±2.35 g; the significance test was P<0.001.

[0078] Table 2

[0079]

[0080]

[0081] The results in Table 2 show that 42 of the 53 rice varieties were homozygous for SUSY2_a, with an average yield per plant of 20.41±1.33 g; 11 of the 53 rice varieties were homozygous for SUSY2_b, with an average yield per plant of 31.16±4.54 g; the significance test was P<0.01.

[0082] Table 3

[0083]

[0084]

[0085] The results in Table 3 show that the genotype of 28 of the 36 rice varieties is homozygous for SUSY2_a, and the average yield per plant of these 28 varieties is 20.37±1.54 grams; the genotype of 8 of the 36 rice varieties is homozygous for SUSY2_b, and the average yield per plant of these 8 varieties is 32.24±3.72 grams; the significance test P<0.01.

[0086] 3. Establishing a method for screening rice with different individual yield traits

[0087] The method for screening rice with different single-plant yield traits is:

[0088] (1) Detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is SUSY2, and has two allelic forms, SUSY2_a and SUSY2_b, wherein the SUSY2_a is shown as Sequence 1 in the sequence listing, and the SUSY2_b is shown as Sequence 2 in the sequence listing;

[0089] (2) The following judgment is made: Under the same conditions, the average yield per plant of the rice population with the SUSY2_b homozygous genotype is higher than the average yield per plant of the rice population with the SUSY2_a homozygous genotype. <110> Institute of Botany, Chinese Academy of Sciences <120> A method for cultivating high-yield crops and its dedicated specific genome fragment and specific primer pair <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1132 <212> DNA <213> Artificial sequence <220> <223> <400> 1 ctgcacgaag gcaccatgag tatcagctat gtagcgatag agctcaccat tgcgagccct 60 gttcgtctga gccgagatcc aacggaactg cccaaacaag ttataggtct taataagttc 120 atgcatcttc tcaatctctg cgatttctc tctgtccttg gatttcttga catcgttata 180 gccagcaacc acgacaaggt ttaccagctc cctcagccta gcgttttttag cataagcttc 240 aaccaaccct gttatgttct taactcggtc aagtcttgcc atggaaaga gatgggttt 300 tgatctgtca tccaggtgtc cactacaaaa agtagttac atcattagaa acacgagag 360 tcacatgtta catgccacaa ctatctactt ttccacat cctgttagta tctctaaaat 420 atgtgtctaa ttgtgtacat tctagtatca cccattcatg ataacctggt aaatatataa 480 aatctagaaa aagacacagc aagtaaccct gtcatcaagc aatgagaga gaccaagga 540 cataaaactc aaccttaaag tgccgcaaaa agaagtaaga taacagaat tactcacata 600 tgttcgtcat ttgctctgg gtcagaaatc aagttttcaa gtgaaccatg aagcgaagtg 660 agccgcttgg cctttcagt gtacggaag tatatagaca tgtctgctcc tggagact 720 atgttgaact ttgggtcaa aacatcaatc ccatggacaa tacgatacag accaggaga 780 gtaaatgcag tatggctc atactgtcca actgtgtttt tgctgaagaa aaggacaaaa 840 100. 100. gcatgtgcgc 900. 100. gcatgtgcgc cagatttcac atgctactat gcgtgacaa atcaatatc gaatatga acgtgatga aaaaaaactt taacagccaa caggcaga gagaataaa cctgccagca atttcctggt atgtgctggt gattaaaa tcagcattgt tcatggcgat fathercagct gtgaactgac aggagaaatg gtacttctca tcgtacttcg tccagtatat gtctgagtct gg <210> 2 <211> 1133 <212> DNA <213> The snowstorm <220> <223> <400> 2 ctgcacgag gcaccatgag tatcagctat gtagcgatag agctcaccat tgcgagccct gttcgtctga gccgagatcc aacggaactg cccaaacaag ttataggtct taataagttc atgcatcttc tcaatctctg cgatttcttc tctgtccttg gattcttga catcgtata gccagcaacc acgacaaggt ttaccagctc cctcagccta gcgtttttag cataagcttc aaccaaccct gttatgttct taactcggtc aagtcttgcc atggaaaaga gaatgggttt tgatctgtca tccaggtgtc cactacaaaa agtagttac atcattagaa acacgagag 360 tcacatgtta catgccacaa ctatctactt ttccacat cctgttagta tctctaaaat 420 atgtgtctaa ttgtgtacat tctagtatca cccattcatg ataacctggt aaatatataa 480 aatctagaaa aagagacagc aagtaaccct gtcatcaagc aatgagaga gaccaagga 540 cataaaactc aaccttaaag tgccgcaaaa agaagtaaga taacagaat tactcacata 600 tgttcgtcat ttgctctgg gtcagaaatc aagttttcaa gtgaaccatg aagcgaagtg 660 agccgcttgg cctttcagt gtacggaag tatatagaca tgtctgctcc tggagact 720 atgttgaact ttgggtcaa aacatcaatc ccatggacaa tacgatacag accaggaga 780 gtaaatgcag tatggctc atactgtcca actgtgtttt tgctgaagaa aaggacaaaa 840 ttaagaatca gatcaggat accctaagaa tagccatgaa taactagtag gcatgtgcgc 900 cagatttcac atgctactat gcgtgaacaa atcaataatc gatataatgaa acgtgatgaa 960 aaaaaaaact ttaacagcca aaaggcaga agagaataa acctgccagc aatttcctgg 1020 tatgtgctgg tgattataaa atcagcattg ttcatggcga ttatatcagc tgtgaactga 1080 caggagaaat ggtacttctc atcgtacttc gtccagtata tgtctgagtc tgg 1133 <210> 3 <211> 25 <212> DNA <213> Artificial sequence <220> <223> <400> 3 ccagactcag acatatactg gacga 25 <210> 4 <211> 21 <212> DNA <213> Artificial sequence <220> <223> <400> 4 ctgcacgaag gcaccatgag t 21

Claims

1. Application of specific allele sequence in rice breeding, characterized by: The breeding is to screen rice with high yield; The specific allele sequence is SUSY2_b; The specific primer pair is used to amplify the specific allele sequence; The specific primer pair is composed of DNA shown in sequence 3 and sequence 4 in the sequence listing; the PCR amplification product of the specific primer pair in the rice genome is SUSY2_a or SUSY2_b; The SUSY2_a is a DNA molecule shown in Sequence 1 in the sequence table; The SUSY2_b is the DNA molecule shown in Sequence 2 in the sequence table; Under the same conditions, the average yield of the rice population with the homozygous SUSY2_b genotype was higher than that of the rice population with the homozygous SUSY2_a genotype.

2. A method for screening rice with different yield traits, comprising the following steps: (1) Using the genomic DNA of the rice to be tested as a template, PCR amplification is performed using a primer pair; if there is only one PCR amplification product and it is shown as sequence 1 in the sequence listing, the genotype of the rice to be tested is SUSY2_a homozygous; if there is only one PCR amplification product and it is shown as sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_b homozygous; the primer pair is composed of the DNA shown as sequence 3 and sequence 4 in the sequence listing; (2) Make the following judgment: Under the same conditions, the average yield of the rice population with the homozygous genotype of SUSY2_b is higher than the average yield of the rice population with the homozygous genotype of SUSY2_a.

3. A rice breeding method comprising the following steps: (1) Using the genomic DNA of the rice to be tested as a template, PCR amplification is performed using a primer pair; if there is only one PCR amplification product and it is shown as sequence 1 in the sequence listing, the genotype of the rice to be tested is SUSY2_a homozygous; if there is only one PCR amplification product and it is shown as sequence 2 in the sequence listing, the genotype of the rice to be tested is SUSY2_b homozygous; the primer pair is composed of the DNA shown as sequence 3 and sequence 4 in the sequence listing; (2) The rice with the homozygous SUSY2_b genotype was the target rice for breeding.

Images