CN106957920B - SNP (single nucleotide polymorphism) locus related to length of unhiazhou rice grains and application thereof - Google Patents

Abstract

The invention discloses an SNP locus related to the length of rice grains and application thereof. The application provided by the invention is specifically the application of the single nucleotide polymorphism of the following SNP sites in the African rice genome or the substance for detecting the single nucleotide polymorphism of the following SNP sites in the African rice genome in identification or auxiliary identification of the seed length of the African rice to be detected; the SNP site corresponds to the 64 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in an African rice genome; the nucleotide at the SNP site is C or T. The grain length of the African rice with the SNP site being C homozygote is obviously longer than that of the African rice with the SNP site being T homozygote. The method has important significance for screening and cultivating new varieties of African rice with high harvest yield.

Description

SNP (single nucleotide polymorphism) locus related to length of unhiazhou rice grains and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to an SNP (single nucleotide polymorphism) locus related to the length of rice grains and application thereof.

Background

Rice (Oryza L.) plants contain two extremely important cultivars, Oryza sativa (o.satival.) and Oryza glaberrima Steud, which originate independently from asia and africa, respectively.

African cultivated rice was cultivated rice that had independently originated in africa about 3000 years ago, and oryza sativa was domesticated from a different ancestral species. In the middle of the 16 th century, Portuguese introduced Asian rice into West Africa, causing the species of African rice to decrease rapidly, so the current planting area and area is much smaller than that of Asian rice. Presently, cultivated rice in asia has been widely grown worldwide, providing a food source for nearly half of the world's population. The African cultivated rice is distributed in the west of Africa, is one of the main local food crops, and plays a positive role in the civilization of Africa and the local economy. However, research on African rice is very poor compared to Asian rice, which has been extensively studied, and the population genetic structure and evolutionary history of African rice are so far poorly understood.

However, the African cultivated rice has the characteristics of early maturity, disease resistance, drought resistance and the like, and has certain production advantages in special ecological environment. Therefore, the screening of the high-yield African cultivated rice has certain application value.

Disclosure of Invention

The invention discloses an SNP locus related to the length of rice grains and application thereof.

The application provided by the invention is specifically the application of the single nucleotide polymorphism of the following SNP sites in the African rice genome or the substance for detecting the single nucleotide polymorphism of the following SNP sites in the African rice genome in identification or auxiliary identification of the seed length of the African rice to be detected;

the SNP site corresponds to the 64 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in an African rice genome; the nucleotide at the SNP site is C or T.

The "substance for detecting a single nucleotide polymorphism at a SNP site in the African rice genome" may be a primer set or a kit as described below.

The invention protects a primer pair and a kit for identifying or assisting in identifying the seed length of African rice to be detected.

The primer pair for identifying or assisting in identifying the seed length of the African rice to be detected is a primer pair capable of obtaining a DNA fragment with a nucleotide sequence shown as a sequence 1 in a sequence table by using African rice genome DNA as a template through amplification.

Further, the DNA fragment is obtained by performing PCR amplification by using African rice genome DNA as a template and adopting a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.

More specifically, the primer pair is a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.

The kit for identifying or assisting in identifying the length of the seeds of the African rice to be detected, provided by the invention, contains the primer pair, dNTP and DNA polymerase.

The application of the primer pair or the kit in identification or auxiliary identification of the length of the seeds of the African rice to be detected also belongs to the protection scope of the invention.

The invention also provides a method for identifying or assisting in identifying the length of the seeds of the African rice to be detected.

The method for identifying or assisting in identifying the length of the seeds of the African rice to be detected provided by the invention specifically comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the African rice to be detected, determining the genotype of the African rice to be detected, and determining the seed length of the African rice to be detected according to the genotype of the African rice to be detected as follows: and C, the kernel length of the African rice to be detected of the genotype C is larger than or is candidate to be larger than that of the African rice to be detected of the genotype T.

The SNP site corresponds to the 64 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in an African rice genome; the nucleotide at the SNP site is C or T;

the C genotype is homozygous at the 64 th site which is C and corresponds to the nucleotide sequence shown as the sequence 1 in the sequence table in the African rice genome;

the T genotype is homozygous for T at the 64 th site corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table in the African rice genome.

The method for detecting the nucleotides at the following SNP sites in the African rice genome to be detected specifically comprises the following two steps:

(1) taking the genomic DNA of the African rice to be detected as a template, and carrying out PCR amplification by adopting the primer pair to obtain a PCR product;

(2) sequencing the PCR product to determine the nucleotide at the SNP site.

The application of the primer pair or the kit or the method in African rice breeding also belongs to the protection scope of the invention.

The African rice breeding is particularly breeding for improving harvest yield of African rice. In the application, the African rice with the C: C genotype can be selected as a parent to carry out breeding, or a bred variety with the C: C genotype is selected.

In one embodiment of the invention, the african rice to be tested is specifically the african rice variety shown in table 1. In another embodiment of the invention, the african rice to be tested is specifically filial generation (such as generation F2) of african rice varieties PI450352 and PI 450489.

Experiments prove that the SNP site of C766T located in GL4 gene is closely related to the length of African rice grains. The grain length of the African rice with the C homozygote site is obviously longer than that of the African rice with the T homozygote site. The method has important significance for screening and cultivating new varieties of African rice with high harvest yield.

Drawings

FIG. 1 shows a comparison of kernel length between line GIL25 and African rice variety IRGC 102305.

FIG. 2 is a schematic diagram of the gene mapping of GL 4.

FIG. 3 shows the results of PCR identification of transgenic lines transformed with pGL4-2 vector.

FIG. 4 shows the grain length comparison of transgenic lines transformed with pGL4-2 vector and African rice variety IRGC 102305. In the figure, pGL4-2 represents a transgenic line transformed with the pGL4-2 vector.

FIG. 5 is the comparison of the grain size of the transgenic plant with that of the unloaded plant and that of the receptor material.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

pCAMBIA1300 vector: described in "Zhu Zuofeng, Tan Lubin, Fu Yongcai, Liu Fengxia, Cai Hongwei, Xie Daoxin, Wu Feng, Wu Jianzhong, Matsumoto Takashi, SunChuanqing. genetic control of flowable architecture failure. Nature Commun,2013,4:2200 doi: 10.1038/ncommmsm.A., publicly available from the applicant and only available for use in experiments with duplicate inventions.

African rice variety IRGC102305, african wild rice W1411 and african rice varieties in table 1: wherein the African oryza sativa beginning with the "PI" number is available from the United states department of agriculture under the website: http:// www.ars-grin. gov/npgs/pi _ books/scans/pi188pt2. pdf; or https:// npgscheb.ars-grid.gov/gringlobal/search.aspx? (ii) a Other numbers are available from the international rice research institute under the following websites: comprises the following steps: http:// www.irgcis.irri.org:81/grc/irgcishome. html; or https:// shigen. nig. ac. jp/rice/oryzae base/strain/wildCore/list.

Example 1 location of African rice grain size regulating gene and determination of SNP site

Positioning of gene for regulating and controlling size of African rice grains

A African rice variety IRGC102305 is hybridized with African wild rice W1411, then IRGC102305 is used as a recurrent parent, a strain GIL25 (figure 1) with obvious grain size difference with IRGC102305 is obtained by multiple backcrosses, and the strain is hybridized with IRGC102305 to construct an F2 segregation population containing 186 individuals. In this F2 population, the kernel length was tightly linked to markers RM3335 and RM5608, and the GL4 gene was located between these two genes. The segregating population was then expanded to 6500 individuals and primers were designed based on the sequence between markers RM3335 and RM5608, finally mapping the GL4 gene to 5.9kb between markers M3 and M4 (FIG. 2). Predicted according to the sequence of African rice IRGC96717(CG14) on website http:// ensembl. Therefore, the gene is the GL4 gene and controls the grain length of the African rice. The strain GIL25 has the same sequence as the GL4 gene of African wild rice W1411.

The GL4 gene sequence of African wild rice W1411 is shown as sequence 4 in the sequence table. The GL4 gene sequence of African rice variety IRGC102305 is shown as sequence 5 in the sequence table. The sequence alignment of the coding regions of the GL4 genes of African wild rice W1411 and African oryza sativa variety IRGC102305 revealed a total of five variations: G294A, C766T, C1466T, A1515G and GCCGCC681-686 deletion (variation is for African cultivar IRGC102305 relative to African wild rice W1411). The sequence alignment of GL4 genes of 16 parts of wild rice and 67 parts of African rice shows that the C766T has the highest correlation degree with kernel length. The specific test scheme is as follows:

first, construction of expression vector

Two primer pairs pGL4-2Af/pGL4-2aR and pGL4-2bF/pGL4-2bR, in which the underlined and bold bases were artificially introduced in order to recover the C766T mutation, and the underlined and italicized portions were the pCAMBIA1300 vector sequence, were designed using the genome of the African cultivar IRGC102305 as a template.

pGL4-2aF：5’-

GCTGGCCGTAGAAGTCAAG-3’；

pGL4-2aR：5’-CGACGACGGCGGCGGCT

AGGAGGAGGTGGGTGGTG-3’。

pGL4-2bF：5’-AGCCGCCGCCGTCGTCGCTGCA-3’；

pGL4-2bR：5’-

GTATAGCTCAGCACGAGC-3’。

The genome of African cultivated rice IRGC102305 is used as a template, pGL4-2Af/pGL4-2aR and pGL4-2bF/pGL4-2bR are amplified by primer pairs respectively to obtain fragments with the lengths of about 3.1kb and 1.8kb respectively, after gel cutting recovery, the two fragments are connected with a pCAMBIA1300 vector by homologous recombinase to obtain a recombinant vector containing GL4 upstream 2kb and downstream 560bp, and the recombinant vector is named as pGL4-2 after correct sequencing. The inserted fragment of pGL4-2 vector has the same sequence as IRGC102305 except that the position of C766T is different from that of the African rice IRGC 102305. At position C766T, vector pGL4-2 is base C and Nokia rice cultivar IRGC102305 is base T.

The structure of the pGL4-2 vector is described: the DNA fragment shown in the sequence 6 in the sequence table is subjected to homologous recombination with a pCAMBIA1300 vector (the upstream and downstream homologous arms are respectively the 1-18 th site and the 4906-4931 th site of the sequence 6).

Wherein, the 1 st-18 th site and the 4906-4001 th site of the sequence 6 are sequences on a pCAMBIA1300 vector, the 19 th-2340 th site is a promoter sequence of GL4 gene endogenous to the IRGC102305 of the African cultivated rice variety, and the 2341-40046 th site is GL4 gene (the 3169-4007 th site is an intron sequence).

Second, transforming African cultivated rice and PCR identification

Transforming the vector pGL4-2 constructed in the step one into an African rice cultivar IRGC102305 with short and non-shattered grains by a gene gun method, carrying out 2 rounds of screening by using an NB culture medium containing hygromycin, and then carrying out differentiation to obtain a transgenic plant. The experiment was also set up with a control for the transfer of pCAMBIA1300 empty vector into African cultivar IRGC 102305.

Transgenic plants were identified by PCR. The specific operation is as follows: taking the transformed plants in the growth period, the empty vector control plants and the leaves of the African cultivated rice variety IRGC102305, extracting total DNA by using a CTAB method, and carrying out PCR identification on the hygromycin resistance gene carried by the pCAMBIA1300 vector by using the following primers:

HptF：5’-gtctccgacctgatgcagctctcgg-3’；

HptR：5’-gtccgtcaggacattgttggag-3’。

the identification result is shown in FIG. 3, lane 1 is DNA Marker III (product of Beijing Tuolingfang science and technology Co., Ltd.), and the size of the target band is 520 bp. Lanes 2-15 are transgenic positive plants, lane 16 is double distilled water, lane 17 is non-transgenic African oryza sativa variety IRGC102305, and lane 18 is the positive control pCAMBIA1300 vector.

Functional identification of transgenic rice

And obtaining 15 transgenic plants through hygromycin resistance screening and PCR identification in the second step. The 15 transgenic plants, a control transferred into pCAMBIA1300 empty vector and a non-transgenic African cultivated rice variety IRGC102305 are taken as experimental materials. The grain phenotype of each experimental material was observed and the grain length was counted for 3 transgenic lines, 3 empty-load control lines and african cultivar IRGC102305, 10 lines per line.

The results show that:

compared with the African rice cultivar IRGC102305 which is subjected to no-load control and no transgenic treatment, 15 transgenic plants obtained through the secondary hygromycin resistance screening and PCR identification are all longer seeds (figure 4).

The grain length of 3 transgenic lines, 3 no-load control lines and African rice cultivar IRGC102305 was counted, and the results are shown in FIG. 5. Wherein pGL4-2-1, pGL4-2-2 and pGL4-2-3 represent 3 randomly selected transgenic lines, vector-1, vector-2 and vector-3 represent 3 randomly selected empty control lines, and IRGC102305-1 represents a non-transgenic treated African oryza sativa variety IRGC 102305. As can be seen, the grain length of the African cultivated rice IRGC102305 is 8.25 cm, the grain length of the positive single plant transferred into the empty vector is 8.21-8.27 cm, and the grain length of the positive single plant transferred into the pGL4-2 vector is 9.24-9.28 cm. The grain length of the positive single plant transferred into pGL4-2 is obviously improved compared with that of the unloaded positive single plant and the receptor material IRGC102305 (t test, P is less than 0.05). Therefore, C766T is an SNP capable of increasing the length of African cultivated rice seeds. The SNP site can be described as the 64 th site in the African rice genome corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table; the nucleotide at the SNP site is C or T.

Example 2 application of SNP site C766T in identification of seed length of existing African rice variety

First, experimental material

67 African rice varieties with sources distributed in multiple countries in the west of Africa are used as experimental materials (see Table 1 specifically), and the application of the SNP site C766T in the selection of the length of the African rice seeds is identified.

Second, Experimental methods

After each test plant is subjected to plate-hanging sampling at the seedling stage, extracting genome DNA of each test plant, taking the genome DNA as a template, performing PCR amplification by using a primer F and a primer R to obtain a PCR product, then performing sequencing, and determining the genotype of the single plant to be tested according to the sequencing result by the following method: if the obtained PCR product contains a nucleotide sequence shown in the sequence 1 and the 64 th position of the sequence 1 is C homozygote, the single plant to be detected is C: C genotype; if the obtained PCR product contains the nucleotide sequence shown in the sequence 1 and the 64 th position of the sequence 1 is T homozygote, the single plant to be detected is T: T genotype.

F: 5'-AAGCCTCTCGTCGCGCAGCA-3' (SEQ ID NO: 2);

r: 5'-GAGCTCAGCTCGCTGCGAGGT-3' (SEQ ID NO: 3).

Meanwhile, after the African rice is mature, the seed length of each individual plant is examined and counted.

Third, experimental results

The results are shown in table 1, where it can be seen that: in total, 4 seeds in 5 African cultivated rice with C: C genotype are longer than 8.5 cm, and 47 seeds in 62 African cultivated rice with T: T genotype are shorter than 8.5 cm. 80% of the African cultivation rice seeds with the C: C genotype are longer than 8.5 cm, and 75.8% of the African cultivation rice seeds with the T: T genotype are shorter than 8.5 cm. The result shows that the grain length of the African rice with the genotype C: C is obviously longer than that of the African rice with the genotype T: T.

Table 167 African cultivars of Oryza sativa with grain length and genotype of C766T

Example 3 application of SNP site C766T in identification of grain length of filial generation of African rice parent

First, experimental material

F450352 (male, C: C genotype) and PI450489 (female, T: T genotype) hybridized with PI450352 (male, C: C genotype) in Table 1₂The SNP site C766T is identified to be applied to the selection of the length of the African rice seeds instead of experimental materials. Two parental controls were set simultaneously.

Second, Experimental methods

After each test plant is subjected to plate-hanging sampling at the seedling stage, genome DNA of each test plant is extracted and used as a template, a primer F and a primer R (a sequence 2 and a sequence 3) are used for PCR amplification to obtain a PCR product, then sequencing is carried out, and the genotype of the single plant to be tested is determined according to the sequencing result by the following method: if the obtained PCR product contains a nucleotide sequence shown in the sequence 1 and the 64 th position of the sequence 1 is C homozygote, the single plant to be detected is C: C genotype; if the obtained PCR product contains a nucleotide sequence shown in the sequence 1 and the 64 th position of the sequence 1 is T homozygote, the single plant to be detected is T, namely T genotype; if the obtained PCR product contains the nucleotide sequence shown in the sequence 1 and the 64 th site of the sequence 1 is a C-T hybrid type, the to-be-detected single plant is a C: T genotype.

Meanwhile, after the African rice is mature, the seed length of each individual plant is examined and counted.

Third, experimental results

The result shows that the average length of the kernel of the male parent PI450352 of the C: C genotype is 8.88 cm, and the average length of the kernel of the female parent PI450489 of the T: T genotype is 7.78 cm. F₂Of the 47 plants, the average length of the seeds is 8.87 cm, the average length of the seeds is 8.41 cm, the average length of the seeds is 52 plants, the average length of the seeds is 7.82 cm. The result shows that the grain length of the African rice hybrid progeny of the C: C genotype is obviously longer than that of the African rice hybrid progeny of the T: T genotype, and the C760T locus can be used as a marker for molecular assisted selection when the variety is bred.

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tcttggattt gattagcagc ggtacggtaa cacctctagc ttgcatttgc atggtagggt 600

gatttggcta ggagagcagg atcatccacg cattcaattt tcctcatttc tttttgcagg 660

ctcaatagag gagaggaggg gcctagctag ccccaagata atgcagcaaa aaggagagaa 720

gagaacatgt catccagtcc aaagtgcaaa cgtctctata caaatggaac atatggagtg 780

gacctaagcc tagttgcttc tgtgtcccaa gctcccaagt gtcaaagcct taatgtgcaa 840

aaccacatct gtggggactc ctgaaagcaa actaaatgat tgcaaaagag ttcgtttatt 900

cacctattta tttatttatt ttattttctt tccatgtgtc tgttgtttga tactttgatt 960

gatcatgtgc taaactgcag aaaaaaccaa caggcccaga gtaccggcga aaaggctcct 1020

cgatgaaaag tgcagggatt ctgggggagt tgccagtgcg ccaccgttgg atttttgcac 1080

acatataaac agggctctgc tgcacctctc acatagtaac atatataggg acggttccac 1140

caagcacgtt tgggaaatgg ttttttgagc aacatctcat gcgaacaatt taggcactat 1200

ttacttgtac cttcaccttg tacgtgtgct ccatcccata gctgcattat ttgcataaat 1260

ccttttaatt gcatatatac atactctctc catatttttt ttaataaatg gcgtatttca 1320

ctttttatca caccatgtct tttaataaga cgaaatatca aatgtgtgac aaaaaaatca 1380

ataatgtcat ctattaaaag acagattaag tatatgagag gcatttaatt atttgccacc 1440

tttgatgttt tacaatttat cactcgtgtg acatagacgc atagggtaac aaattgcgaa 1500

acatcaagta atctgtgagt agcaaatact taaatatctc ctaatatgtt agcattgcac 1560

ccaactttat atcaaaactc tataactata ttgagctttg tcctgcccat ggtaaagtgt 1620

ggggttaaag tgttaaaaag ctattggtag cagtatctag tatggcgtgg caggccggtg 1680

cagataaaag gggccccggg ttttgcccgg aaatgaggag aggacgcccc ggcagctagc 1740

ggacgctttg catttgcatg catggtgctc tctacttctc ccctccgttc ccacggcaaa 1800

tgcaaaacgc atgctagtat ttggcaccat tagcctaaga tagtagtatc agagtgttac 1860

agaaagaata taattatgta gtagtatagt aattaattaa agcctgcgat taaggtaaag 1920

cagcagtgca gttacaaacc taaacaaaca agagtcagcg aactgaaaag aagaccggga 1980

agaagaaggg aaaaaaaaaa gacccgaaca gagttttgat gagcagtgtc aggtctgtca 2040

catgagatga gagcgaagta gctcagctca gctcagcccc cactcgctca catggaacgc 2100

ctctgctccc cgcgactact taaccagcta aacgctcggt tgattaggag agaagaaaaa 2160

aacgagggaa aaaacggtgg aaacacacgc aaaacacaca acgccgtgag gccttgtaaa 2220

tacgggcgac gccgacgcgc atcgctaccc gaacaccaaa cgcctcagct tgccttggct 2280

ctcgcgagtc gctacgcccg cgacgacacg gccgcgccgc gcgcgcgcga gcgagcgagc 2340

atgtcgggct cctctgctgc cgacccctcg ccatccgcgt cgaccgcggg ggcggcggcc 2400

tcgccgctcg cgctgctccg cgcgcacggg cacgggcacc tcacgccgcc gtcggcggcg 2460

acggggccgg cgccgccgcc gccgtcgccg gcgtcggggt cggcgccgcg ggactaccgc 2520

aaggggaact ggacgctcca cgagacgctc atcctcatca ccgccaagcg tctcgacgac 2580

gaccgccgcg ccggcgttgg gggtggcgcg gcggctggtg gcggtggcgc cggatcgccg 2640

ccgacgccga ggtcggcgga gcagcggtgg aagtgggtgg agaactactg ctggaagaac 2700

ggctgcctcc gcagccagaa ccagtgcaat gacaagtggg acaacctcct ccgcgactac 2760

aagaaggtcc gcgactacga gtcccgcgtc gccgccgccg ccgtcaccgg cggcgcggcc 2820

gccgccaact ccgcccccct cccgtcgtac tggacgatgg agcggcacga gcgcaaggac 2880

tgcaacctcc ccaccaacct ggcgccggag gtctacgacg cgctctccga ggtgctctcc 2940

cgccgcgcgg cgcgacgcgg cggcgccacg atcgcgccca ccccgccgcc accaccgctc 3000

gcgctgccgc cgccgccgcc gccgccctcg ccgccgaagc ctctcgtcgc gcagcagcac 3060

caccaccatc acggccatca ccaccaccca cctcctcctc agccgccgcc gtcgtcgctg 3120

cagctccctc cggcggtcgt ggctccgccg ccggcgtccg tttccggtaa tggtcggtgc 3180

gcgcaccgta cacttaatac tcgtagtagc tgttacatcc cctcccctcc aaaccattta 3240

ctactgttct ctcacactga catgtggggc ccacctcgca gcgagctgag ctccgccact 3300

atacgttatt aaaagcccgc gttatgattg ggctagttac gttgttgagt tgagctggtc 3360

gtaattattt actaccgcta cttttttttt acctttttac cgtggggttc gggagagggt 3420

ggtcgcggta ataataatgt cctcaactca ggggttggga gaataaagct gcgtgcagtg 3480

tggtgcagtt catgcatggg aaaggtgatg cgaatccgga tattttatgg gggtttaatt 3540

gaaagattta ctccacgacg atactaccct gtactcctgc catgctgcaa gcatgcgtaa 3600

tgcgttacat tgcgaaatca ctcgctttga aagaaaaaaa agcctaaaac tttggagcaa 3660

aaaaaaagca ccttttgttt tctcctcgtg catgcatgcc gcgctgccta tcttgaacta 3720

ctttggactt ttgtatcgat cagcaaaact atacctatat tagcagtaat taatactaca 3780

tttgtagata tcctttgact gttctatctt atttttgata attaaaaaaa ttagttacat 3840

ttaaaaatgc tatttatatt ttatcatcta ataacaataa aagtattagt taaatattaa 3900

acgttggata tgaatagttt aaaactgtat tgttttgggg cggagggagt aattgattga 3960

tggattaatt aagtggttga ctaatgtgtg tgtgatttat ttgtgtagcg gaggaggaga 4020

tgtcggggtc gtcggagtcg ggggaggagg aggaggggtc gggcggggag ccggaggcga 4080

agcggcggcg gctgagccgg ctggggtcga gcgtggtgag gagcgcgacg gtggtggcga 4140

ggacgctggt ggcgtgcgag gagaagcggg agcgccggca ccgggagctg ctgcagctgg 4200

aggagcggcg gctgcgcctc gaggaggagc gcaccgaggt ccgccgccag ggcttcgccg 4260

gcctcatcgc cgccgtcaac agcctctcct ccgccatcca cgccctcgtc tccgaccacc 4320

gcagcggcga ctcctccggc cgatgatcgc cattgcaatc atatgcaatg cagcaaggac 4380

gatcgatgta aataacccat ggagatgcat ggatcgaggc atcggattat tatgtttgga 4440

atggctgcaa gaagaggagt agctaggcta ggggattaat tttttttttt ttgagtgtgc 4500

atcgccatcg cgtcgtctgc gagtattggg agtacggtgc attgcatgca caacgcctcc 4560

gtttcttgat ttctttcttt ctctcctgtg tcctgtgatt tttttgttgt ttattctttt4620

cgtgcaatta gtggagagac tggcaggtgt gtggtgtgaa tgatcgaaat ggttagtgtg 4680

gctgctgctg gtggtgctgt tgctgttgct gcgttttttc ttctcgggtg ttgggtttgt 4740

cgtggacggc gatcattagc ggcacagtgg atgagagagc tgagctctag ctgcaggtcg 4800

cttggtctcg ctgcgctgtc atgtgatcac tactgcgcat gggggccacg gttaccgcta 4860

tagctgctgc aactgcgtgc gagaacgagc tcgtgctgag ctatacaagt ggcactggcc 4920

gtcgttttac a 4931

Claims (9)

1. The application of the substance for detecting the single nucleotide polymorphism of the following SNP sites in the African rice genome in identifying or assisting in identifying the seed length of the African rice to be detected;

the SNP site corresponds to the 64 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in an African rice genome; the nucleotide at the SNP site is C or T.

2. Use according to claim 1, characterized in that: the substance for detecting the single nucleotide polymorphism of the following SNP sites in the African rice genome is a primer pair or a kit for identifying or assisting in identifying the grain length of the African rice to be detected;

the primer pair is a primer pair which can amplify a DNA fragment with a nucleotide sequence shown in a sequence 1 in a sequence table by taking African rice genome DNA as a template;

the kit contains the primer pair, dNTP and DNA polymerase.

3. Use according to claim 2, characterized in that: the DNA fragment is obtained by performing PCR amplification by using African rice genome DNA as a template and adopting a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.

4. Use according to claim 2 or 3, characterized in that: the primer pair is composed of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.

5. The primer pair for identifying or assisting in identifying the seed length of the African rice to be detected is a primer pair consisting of two single-stranded DNAs shown as a sequence 2 and a sequence 3 in a sequence table.

6. A kit for identifying or assisting in identifying the kernel length of African rice to be detected, which comprises the primer pair of claim 5, dNTP and DNA polymerase.

7. A method for identifying or assisting in identifying the grain length of African rice to be detected comprises the following steps: detecting nucleotides at the following SNP sites in the genome of the African rice to be detected, determining the genotype of the African rice to be detected, and determining the seed length of the African rice to be detected according to the genotype of the African rice to be detected as follows: c, the kernel length of the African rice to be detected of the genotype C is larger than or is candidate to be larger than that of the African rice to be detected of the genotype T;

the SNP site corresponds to the 64 th site of a nucleotide sequence shown in a sequence 1 in a sequence table in an African rice genome; the nucleotide at the SNP site is C or T;

the C genotype is homozygous at the 64 th site which is C and corresponds to the nucleotide sequence shown as the sequence 1 in the sequence table in the African rice genome;

the T genotype is homozygous for T at the 64 th site corresponding to the nucleotide sequence shown in the sequence 1 in the sequence table in the African rice genome.

8. The method of claim 7, wherein: the method for detecting the nucleotides at the following SNP sites in the genome of African rice to be detected comprises the following two steps:

(1) carrying out PCR amplification by using the genomic DNA of the African rice to be detected as a template and the primer pair of claim 5 to obtain a PCR product;

(2) sequencing the PCR product to determine the nucleotide at the SNP site.

9. Use of the primer pair according to claim 5 or the kit according to claim 6 or the method according to claim 7 or 8 for breeding oryza africana.

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