CN114717349A

CN114717349A - Molecular marker for rice plant type and genotype and application thereof

Info

Publication number: CN114717349A
Application number: CN202110009947.9A
Authority: CN
Inventors: 韩斌; 司丽珍; 吕威; 赵强; 朱静洁; 上官颖颖
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2021-01-05
Filing date: 2021-01-05
Publication date: 2022-07-08

Abstract

The invention discloses a molecular marker of rice plant type and application thereof. The SNP marker of the invention is closely related to the rice plant type, and can be effectively used for breeding rice, determining the rice genotype and breeding rapidly.

Description

Molecular marker for rice plant type and genotype and application thereof

Technical Field

The invention relates to a molecular marker of rice plant type and application thereof. In particular to a SNP molecular marker of rice plant type and application thereof.

Background

The rice plant type is one of the important factors determining the rice yield, the plant type is determined by the height of the plant, the width of leaves, the shape of ears, the tillering angle and the like, and the rice plant type is also one of the targets of rice breeding.

The use of the semi-dwarf gene sd1 was responsible for the "green revolution" in crop breeding (Sasaki et al, 2002). This gene consists of three exons and two introns and is involved in gibberellin synthesis. The gene SD1 has strong expression in leaves, stems and unopened flowers, and the plant height is reduced after mutation, but the influence on the yield is small; meanwhile, the strain is high and short, and is not easy to fall down, which is the reason for increasing the yield (Spielmeyer et al, 2002).

The identification and cloning of the ideal plant type IPA1 provide important gene information reserve for breeding rice. IPA1 belongs to SPL family gene, has highly conserved DNA binding domain, and is regulated by OsmiR 156. In the vegetative growth period of rice, IPA1 reduces tillering of rice and simultaneously makes rice stalks become thick and strong; during the development of the ear, IPA1 promotes the development of the branches and stalks of the ear, increasing the number of grains per ear (Jiao et al 2010). Besides promoting the growth and development of plants under normal conditions, the immune response can be improved when the rice blast is infected, which can increase the yield and improve the resistance of the rice blast, and breaks through the view that a single gene cannot simultaneously consider the yield and the disease resistance (Wang et al 2018).

No molecular marker for the gene is reported, so that the field still needs to find the molecular marker for identifying the genotype of the gene, thereby quickly locking the genotype of rice at the early stage and accelerating the breeding speed of breeding excellent rice varieties.

Disclosure of Invention

The invention provides an SNP marker which is related to the rice plant type and can be effectively used for detecting the rice plant type, and a high-efficiency and sensitive rice plant type specific primer and a probe are designed by utilizing the SNP marker to detect the DNA of a sample, thereby realizing the judgment of the rice plant type and effectively solving the problem of quickly breeding the progeny rice.

In a first aspect of the present invention, there is provided an isolated nucleic acid molecule from the rice SD1 gene, comprising a first SNP marker, wherein the first SNP marker is: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 1 and 2 as primers is A or G.

In one or more embodiments, the nucleic acid molecule is a fragment of at least 5bp of the rice SD1 gene in length. In one or more embodiments, the nucleic acid molecule is at least 10bp, 15bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb in length. In one or more embodiments, the nucleic acid molecule is 10bp-600bp, 50-500bp, 100-400bp, 150-300bp, or 200-250bp in length.

In one or more embodiments, the nucleotide sequence of the nucleic acid molecule includes the sequence set forth in SEQ ID NO 3.

The first aspect of the present invention also provides an isolated nucleic acid molecule from the rice SD1 gene, which comprises a deletion marker: the nucleotide sequence at position 194 and 195 of SEQ ID NO. 6 or the nucleotide sequence at position 194 and 577 of SEQ ID NO. 7.

In one or more embodiments, the nucleic acid molecule is a fragment of SEQ ID NO 6 comprising its nucleotide sequence at position 194 and 195 and having a length of at least 5 bp. In one or more embodiments, the nucleic acid molecule is at least 10bp, 15bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb in length. In one or more embodiments, the nucleic acid molecule is 10bp-600bp, 50-500bp, 100-400bp, 150-300bp, or 200-250bp in length.

In one or more embodiments, the nucleic acid molecule is a fragment of SEQ ID NO 7 comprising the nucleotide sequence at position 194 and 577 thereof. In one or more embodiments, the nucleic acid molecule is at least 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb in length.

Also provided in the first aspect of the invention are primers comprising

(1) A primer for detecting a first SNP marker in a rice genome, wherein the first SNP marker is: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 1 and 2 as primers is A or G, and/or

(2) A primer for detecting a deletion marker in a rice genome, wherein the deletion marker is: the nucleotide sequence at position 194 and 195 of SEQ ID NO. 6 or the nucleotide sequence at position 194 and 577 of SEQ ID NO. 7.

In one or more embodiments, (1) the primer can amplify a sequence amplified by SEQ ID NOS 1 and 2 as primers or a fragment thereof including nucleotide 186 from the 5' end of SEQ ID NO 3.

In one or more embodiments, (1) the amplification product of the primer comprises a sequence amplified by using SEQ ID NOS 1 and 2 as primers or a fragment thereof comprising the 186 th nucleotide from the 5' end of SEQ ID NO. 3.

In one or more embodiments, the amplification product of the primer of (1) comprises SEQ ID NO. 3 or a fragment thereof comprising nucleotide 186 from the 5' end of SEQ ID NO. 3.

In one or more embodiments, the primer of (1) hybridizes to SEQ ID NO. 3 under stringent conditions.

In one or more embodiments, (1) the primer is selected from the group consisting of: (1) 1 and 2 or a sequence that hybridizes under stringent conditions to SEQ ID No. 3, (2) a sequence that is at least 90% identical to (1); and (3) mixtures of the sequences described in (1) and (2).

In one or more embodiments, (2) the primers are capable of amplifying the sequences amplified by using SEQ ID NOS.4 and 5 as primers or fragments thereof comprising the nucleotide sequence at position 194 and 195 of SEQ ID NO. 6 or the nucleotide sequence at position 194 and 577 of SEQ ID NO. 7.

In one or more embodiments, (2) the amplification product of the primer comprises SEQ ID NO 6 or a fragment thereof comprising the nucleotide sequence at position 194 and 195 of SEQ ID NO 6; or (2) the amplification product of the primer comprises SEQ ID NO. 7 or a fragment thereof comprising the nucleotide sequence at position 194-577 of SEQ ID NO. 7.

In one or more embodiments, (2) the amplification products of the primers span the nucleotide sequences 194-195 of SEQ ID NO. 6 or the nucleotide sequences 194-577 of SEQ ID NO. 7.

In one or more embodiments, the primer of (2) hybridizes to SEQ ID NO 6 or 7 under stringent conditions.

In one or more embodiments, (2) the primer is selected from the group consisting of: (1) sequences shown in SEQ ID NO. 4 and 5 or sequences that hybridize under stringent conditions to SEQ ID NO. 6 or 7, (2) sequences that are at least 90% identical to (1); and (3) mixtures of the sequences described in (1) and (2).

The first aspect of the invention also provides a probe for detecting the first SNP marker of the rice genome, wherein the first SNP marker is as follows: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 1 and 2 as primers is A or G.

In one or more embodiments, the probe comprises:

(1) a probe recognizing SEQ ID NO 3 or a fragment thereof comprising the 186 th base from the 5' end of SEQ ID NO 3, and/or

(2) The complementary sequence of (1).

In one or more embodiments, the probe comprises:

(a1) a probe recognizing SEQ ID NO 3 or a fragment thereof comprising the 186 th base from the 5' end of SEQ ID NO 3, the 186 th base being A, and/or

(a2) The complement of (a 1).

In one or more embodiments, the probe further comprises:

(b1) a probe recognizing SEQ ID NO 3 or a fragment thereof comprising the 186 th base from the 5' end of SEQ ID NO 3, the 186 th base being G, and/or

(b2) (b 1).

In one or more embodiments, the probe further comprises:

(1) a probe recognizing SEQ ID NO 6 or a fragment thereof comprising bases 194 and 195 of SEQ ID NO 6 and/or a probe recognizing SEQ ID NO 7 or a fragment thereof comprising bases 194 and 577 of SEQ ID NO 7 and/or

(2) The complementary sequence of (1).

In the first aspect of the present invention, there is also provided a kit comprising reagents for detecting a first SNP marker and optionally a deletion marker in the rice genome, wherein the first SNP marker is: taking rice genome DNA as a template, and taking SEQ ID NO 1 and 2 as primers to carry out PCR amplification to obtain 186 th nucleotide from the 5' end of an amplification product, wherein the 186 th nucleotide is A or G; wherein the deletion marker is: the nucleotide sequence at position 194 and 195 of SEQ ID NO. 6 or the nucleotide sequence at position 194 and 577 of SEQ ID NO. 7.

In one or more embodiments, the kit comprises:

(1) a primer for detecting the first SNP marker and optionally the deletion marker, and

optionally (2) a probe for detecting the first SNP marker and the optional deletion marker, and

optionally (3) a nucleic acid molecule having a first SNP marker and optionally a deletion marker.

In one or more embodiments, the kit comprises: a primer as described in any embodiment of the first aspect herein, optionally a probe as described in any embodiment of the first aspect herein and optionally a nucleic acid molecule as described in any embodiment of the first aspect herein.

The first aspect of the present invention also provides a method for identifying the plant type of rice, which is the height of a plant, or the SD1 genotype, comprising,

(1) detecting a first SNP marker in a rice genome, wherein the first SNP marker is: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 1 and 2 as primers,

(2) detecting a deletion marker in the rice genome, the deletion marker being: the 194 nd and 195 th nucleotide sequences of SEQ ID NO. 6 or the 194 nd and 577 th nucleotide sequences of SEQ ID NO. 7,

(3) and identifying the plant type of the rice according to the first SNP marker and the deletion marker, wherein the first SNP marker is GG, the deletion marker is the nucleotide sequence at the 194 th and 577 th positions of the SEQ ID NO. 7 and is identified as a highstalk, the first SNP marker is AA, the deletion marker is the nucleotide sequence at the 194 th and 577 th positions of the SEQ ID NO. 7 and is identified as a semi-dwarfstalk, the first SNP marker is GG, and the deletion marker is the nucleotide sequence at the 194 th and 195 th positions of the SEQ ID NO. 6 and is identified as a dwarfstalk.

In one or more embodiments, the detecting comprises PCR, more preferably, the detecting is fluorescent quantitative PCR, HRM detection, gel electrophoresis, or sequencing.

In one or more embodiments, the detection of step (1) comprises PCR amplification of the rice genome using primers as described in any of the embodiments of the first aspect herein or SEQ ID NOs 1 and 2 as primers.

In one or more embodiments, the detection of step (2) comprises PCR amplification of the rice genome using primers as described in any of the embodiments of the first aspect herein or SEQ ID NOS: 4 and 5 as primers.

The first aspect of the present invention also provides a use of a reagent for detecting a first SNP marker and optionally a deletion marker in a rice genome in identifying a rice plant type or identifying a SD1 genotype, or in preparing a kit for identifying a rice plant type or identifying a SD1 genotype, wherein the first SNP marker is: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by taking the rice genome DNA as a template and adopting SEQ ID NO 1 and SEQ ID NO 2 as primers is marked as a deletion marker: the nucleotide sequence at position 194 and 195 of SEQ ID NO. 6 or the nucleotide sequence at position 194 and 577 of SEQ ID NO. 7.

In one or more embodiments, the nucleotide 186 is a or G.

In one or more embodiments, the reagent comprises a primer as described in any embodiment of the first aspect herein and optionally a probe as described in any embodiment of the first aspect herein and optionally a nucleic acid molecule as described in any embodiment of the first aspect herein.

In one or more embodiments, the rice plant type is identified according to the detected first SNP marker, wherein the first SNP marker is GG, the deletion marker is nucleotide sequence at position 194 and 577 of SEQ ID NO:7, the first SNP marker is AA, the deletion marker is nucleotide sequence at position 194 and 577 of SEQ ID NO:7, the first SNP marker is semi-dwarf, the first SNP marker is GG, and the deletion marker is nucleotide sequence 195 at position 194 and 195 of SEQ ID NO:6, the first SNP marker is dwarf.

In a second aspect of the present invention, there is provided an isolated nucleic acid molecule from rice IPA1 gene comprising a second SNP marker, wherein the second SNP marker is: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C.

In one or more embodiments, the nucleic acid molecule is a fragment of rice IPA1 gene at least 5bp in length. In one or more embodiments, the nucleic acid molecule is at least 10bp, 15bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb in length. In one or more embodiments, the nucleic acid molecule is 10bp-600bp, 50-500bp, 100-400bp, 150-300bp, or 200-250bp in length.

In one or more embodiments, the nucleotide sequence of the nucleic acid molecule includes the sequence set forth in SEQ ID NO. 10.

In the second aspect of the present invention, there is also provided a primer for detecting a second SNP marker in the rice genome, wherein the second SNP marker is: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C.

In one or more embodiments, the primers are capable of amplifying the sequences amplified by SEQ ID NO 8 and 9 as primers or a fragment thereof comprising nucleotide 125 from the 5' end of SEQ ID NO 10.

In one or more embodiments, the amplification product of the primer comprises a sequence amplified from SEQ ID NO 8 and 9 as a primer or a fragment thereof comprising nucleotide 125 from the 5' end of SEQ ID NO 10.

In one or more embodiments, the amplification product of the primer comprises SEQ ID NO. 10 or a fragment thereof comprising nucleotide number 125 from the 5' terminus of SEQ ID NO. 10.

In one or more embodiments, the primer hybridizes to SEQ ID NO. 10 under stringent conditions.

In one or more embodiments, the primer is selected from the group consisting of: (1) the sequences shown in SEQ ID NO 8 and 9 or the sequences hybridizing with SEQ ID NO 10 under stringent conditions, (2) the sequences having at least 90% identity to (1); and (3) mixtures of the sequences described in (1) and (2).

In the second aspect of the invention, the invention also provides a probe for detecting the second SNP marker in the rice genome, wherein the second SNP marker is as follows: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C.

In one or more embodiments, the probe comprises

(1) A probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10, and/or

(2) The complementary sequence of (1).

In one or more embodiments, the probe comprises:

(a1) a probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10, said 125 th base being T, and/or

(a2) The complement of (a 1).

In one or more embodiments, the probe further comprises:

(b1) a probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10, said 125 th base being C, and/or

(b2) (b 1).

In the second aspect of the present invention, there is also provided a kit for identifying IPA1 genotype, which comprises reagents for detecting a second SNP marker in the rice genome, the second SNP marker being: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C.

In one or more embodiments, the kit comprises a test

(1) A primer for the second SNP marker, and

optionally (2) a probe for detecting a second SNP marker, and

optionally (3) a nucleic acid molecule having a second SNP marker.

In one or more embodiments, the kit comprises: a primer as described in any embodiment of the second aspect herein, optionally a probe as described in any embodiment of the second aspect herein and optionally a nucleic acid molecule as described in any embodiment of the second aspect herein.

In one or more embodiments, the kit comprises:

(i) (1) sequences shown as SEQ ID NO 8 and 9 or sequences that hybridize to SEQ ID NO 10 under stringent conditions, (2) sequences that are at least 90% identical to (1); and (3) mixtures of the sequences described in (1) and (2), and

optionally (ii) (a1) a probe recognizing SEQ ID NO 10 or a fragment thereof comprising base 125 from the 5' end of SEQ ID NO 10, said base 125 being a T, and/or the complement of (a2) (a 1); and optionally (b1) a probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10, said 125 th base being C, and/or the complementary sequence of (b2) (b1), and

(iii) optionally (iii) a nucleic acid molecule comprising the sequence shown in SEQ ID NO 10.

The second aspect of the present invention also provides a method of identifying IPA1 genotype, the method comprising,

(1) detecting a second SNP marker in the rice genome, wherein the second SNP marker is as follows: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by taking the rice genome DNA as a template and SEQ ID NO 8 and 9 as primers,

(2) and identifying the rice IPA1 genotype according to the second SNP marker, wherein the second SNP marker is CC, TT or CT.

In one or more embodiments, the detecting comprises PCR, more preferably, the detecting is fluorescent quantitative PCR, HRM detection, or sequencing.

In one or more embodiments, the detection of step (1) comprises PCR amplification of the rice genome using SEQ ID NOS: 8 and 9 as primers.

In a second aspect, the present invention also provides a method for breeding rice, the method comprising,

(2) identifying IPA1 genotype according to the second SNP marker, wherein,

if the second SNP marker is CC then the IPA1 genotype is homozygous for C,

if the second SNP marker is TT then the IPA1 genotype is homozygous for T,

obtaining the hybrid type if the second SNP marker is CT and performing the step (3),

optionally, (3) selfing the hybrid plant type and subjecting the progeny rice to steps (1) and (2) to obtain rice with the second SNP marker of CC or TT.

In one or more embodiments, the amplification is fluorescent quantitative PCR, HRM detection, or sequencing.

The second aspect of the invention also provides the use of a reagent for detecting a second SNP marker in a rice genome for identifying the IPA1 genotype or breeding rice or for preparing a kit for identifying the IPA1 genotype or breeding rice, wherein the second SNP marker is: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers.

In one or more embodiments, the nucleotide at position 125 is T or C.

In one or more embodiments, the reagent comprises a primer as described in any embodiment of the second aspect herein and optionally a probe as described in any embodiment of the second aspect herein and optionally a nucleic acid molecule as described in any embodiment of the second aspect herein.

In one or more embodiments, IPA1 genotype is identified or rice is bred based on the detected second SNP marker, wherein the second SNP marker for C-type homozygous IPA1 genotype is CC and the second SNP marker for T-type homozygous IPA1 genotype is TT.

In a fourth aspect of the present invention, there is also provided a kit for detecting the plant type and/or genotype of rice, which comprises reagents for detecting (1) a first SNP marker and optionally a deletion marker, and/or (2) a second SNP marker in the genome of rice,

the first SNP marker was: the 186 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 1 and 2 as primers is A or G,

the deletion markers are: the 194 nd and 195 th nucleotide sequences of SEQ ID NO. 6 or the 194 nd and 577 th nucleotide sequences of SEQ ID NO. 7,

the second SNP marker was: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C.

In one or more embodiments, the kit comprises

(1) A primer for detecting the first SNP marker and optionally the deletion marker, and/or the second SNP marker, and

optionally (2) a probe for detecting the first SNP marker and optionally the deletion marker, and/or the second SNP marker, and

optionally (3) a nucleic acid molecule having a marker selected from the group consisting of a first SNP marker and optionally a deletion marker, and/or a second SNP marker.

In one or more embodiments, the kit comprises: a primer as described in any embodiment of the first and/or second aspect herein, optionally a probe as described in any embodiment of the first and/or second aspect herein and optionally a nucleic acid molecule as described in any embodiment of the first and/or second aspect herein.

Detailed Description

The inventor compares specific sequences of rice plant types through research on multiple varieties of rice, and identifies the rice plant types and rapidly breeds rice with required plant types through detection of nucleic acid sequences of specific sites of rice with different plant types.

Specifically, the invention relates to an SNP marker related to a rice plant type, a primer and a kit for detecting the SNP marker, application of the SNP marker, the primer and the kit in rice plant type detection, and a method for detecting the rice plant type.

Herein, SNP (single nucleotide polymorphism) is a type of molecular genetic marker, mainly referring to DNA sequence polymorphism caused by variation of single nucleotide on genome level. SNPs typically exhibit polymorphisms that involve only single base variations, such as transitions, transversions, insertions and deletions.

The inventors found that the first SNP marker and the deletion marker were associated with the height of rice plants. Specifically, the first SNP marker is 186 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID NO. 3, and the deletion marker is 194 th and 195 th nucleotide sequences of SEQ ID NO. 6 or 194 th and 577 th nucleotide sequences of SEQ ID NO. 7. The first SNP marker is GG, the deletion marker is the nucleotide sequence of No. 194 and 577 of SEQ ID NO. 7 and is identified as a high stalk, the first SNP marker is AA, the deletion marker is the nucleotide sequence of No. 194 and 577 of SEQ ID NO. 7 and is identified as a semi-dwarf, the first SNP marker is GG, and the deletion marker is the nucleotide sequence of No. 194 and 195 of SEQ ID NO. 6 and is identified as a dwarf.

The inventors also found that the second SNP marker is related to rice IPA1 gene. Specifically, the second SNP marker is the 125 th base T or C from the 5' end of the nucleotide sequence shown in SEQ ID NO. 10. The second SNP marker of rice with C-type homozygous IPA1 genotype is CC, and the second SNP marker of rice with T-type homozygous IPA1 genotype is TT.

A "sample" as described herein is any type of polynucleotide-containing sample from a subject. Preferably, the sample described herein is derived from or comprises rice plant organs, tissues, cells, nucleic acids or products comprising rice plant organs, tissues, cells, nucleic acids including, but not limited to, rice leaves, roots, stems, flowers, fruits, seeds, cells, DNA, RNA, rice, broken rice, rice bran, rice hulls, processed or unprocessed rice food such as rice flour, rice noodles. The DNA may be genomic DNA.

The term "nucleic acid" or "polynucleotide" refers to a Deoxyribonucleotide (DNA) or ribonucleotide polymer (RNA) in either single-or double-stranded form, and the complement thereof. Nucleic acids contain synthetic, non-natural or altered nucleotide bases. The nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single-and double-stranded DNA, single-and double-stranded RNA, and hybrid molecules having a mixture of single-and double-stranded DNA and RNA. The DNA may be the coding strand or the non-coding strand. In one or more embodiments, the sample comprises fragmented genomic DNA. Methods for obtaining genomic DNA and fragmenting are well known in the art.

The basic unit of DNA is deoxyribonucleotide, which is condensed by phosphodiester bond to form a long chain molecule. Each deoxyribonucleotide consists of a phosphate, a deoxyribose, and a base. Bases (bp) of DNA are mainly adenine (A), guanine (G), cytosine (C) and thymine (T). In the double-helix structure of double-stranded DNA, A is hydrogen-bonded to T, and G is hydrogen-bonded to C. The form of DNA includes cDNA, genomic DNA, fragmented DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be of any length, such as 50-500bp, 100-400bp, 150-300bp or 200-250 bp.

As used herein, a "primer" refers to a nucleic acid molecule having a specific nucleotide sequence that directs the synthesis at the initiation of nucleotide polymerization. The primer composition comprises one or more primers. Primers are typically two oligonucleotide sequences synthesized artificially, one complementary to one DNA template strand at one end of the target region and the other complementary to the other DNA template strand at the other end of the target region, and function as an initiation point for nucleotide polymerization. Primers designed artificially in vitro are widely used in Polymerase Chain Reaction (PCR), qPCR, sequencing, probe synthesis, and the like. The primer may be of any length, for example 5-200bp, 10-100bp, 20-800bp or 25-50 bp.

The primer of the invention is used for detecting SNP. The primer may be a nucleic acid molecule recognizing any one of SEQ ID NOs 3, 6, 7, 10. Illustratively, the primer includes one or more selected from the group consisting of: (1) primers capable of amplifying fragments amplified from SEQ ID NOS: 1 and 2 as primers, (2) primers capable of amplifying fragments amplified from SEQ ID NOS: 4 and 5 as primers, and (3) primers capable of amplifying fragments amplified from SEQ ID NOS: 8 and 9 as primers. In one or more embodiments, the amplification product of the primer comprises one or more selected from the group consisting of: (1) fragments amplified from SEQ ID NOS: 1 and 2 as primers, (2) fragments amplified from SEQ ID NOS: 4 and 5 as primers, and (3) fragments amplified from SEQ ID NOS: 8 and 9 as primers. In one or more embodiments, the amplification product of the primer comprises one or more selected from the group consisting of: (1) 3 or a fragment thereof comprising the 186 th nucleotide from the 5 'terminus of SEQ ID NO 3, (2) the fragment of SEQ ID NO 6 spanning the 194 th and 195 th nucleotide sequences of SEQ ID NO 6 or the fragment of SEQ ID NO 7 spanning the 194 th and 577 th nucleotide sequences of SEQ ID NO 7, and (3) the fragment of SEQ ID NO 10 or a fragment thereof comprising the 125 th nucleotide from the 5' terminus of SEQ ID NO 10.

In some embodiments, the primer has (1) a nucleotide sequence set forth in any one of SEQ ID NOs 1, 2, 4, 5, 8, 9 or a mutant having at least 70% sequence identity thereto, or (2) the complement of (1). In one or more embodiments, the primers are primer pairs having the sequences shown in SEQ ID NO 1 and 2 or SEQ ID NO 4 and 5 or SEQ ID NO 8 and 9, respectively. In discussing primers, the term "recognition" as used herein means that the primers hybridize to the template sequence under stringent or highly stringent conditions and that the fragment amplified by the pair of primers (1) encompasses the 186 th base from the 5 'end of SEQ ID NO:3, (2) spans the 194 th and 195 th nucleotide sequences of SEQ ID NO:6 or the fragment of SEQ ID NO:7 and spans the 194 th and 577 th nucleotide sequences of SEQ ID NO:7, and/or (3) encompasses the 125 th base from the 5' end of SEQ ID NO: 10.

Stringent conditions for hybridization of the nucleic acids described herein are known to those skilled in the art. Preferably, the conditions are such that the sequences are at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other, typically remaining hybridized to each other. Non-limiting examples of stringent hybridization conditions are hybridization in a high salt buffer containing 6XSSC, 50mM Tris-HCl (pH7.5), 1m MEDTA, 0.02% PVP, 0.02% Ficolll, 0.02% BSA and 500mg/ml denatured salmon sperm DNA at 65 ℃ and optionally washed once or twice in 0.2XSSC, 0.01% BSA at 50 ℃.

The present invention may also employ probes to detect SNPs described herein. A "probe" as used herein is a nucleic acid sequence (DNA or RNA) that recognizes a sequence of interest (complementary to the sequence of interest). The probe is combined with the target gene through molecular hybridization to generate a hybridization signal, thereby displaying the target gene. The probe may include the entire sequence of interest or may be a fragment of the sequence of interest. The probe may be DNA or RNA transcribed therefrom. Typically, the probe carries a detectable label, such as a fluorescent label. Such fluorescent labels include, but are not limited to FAM, CY5, and VIC. Fluorescent labels suitable for use with the probes herein and methods of attaching them to the probes are known in the art.

Herein, the probe includes a probe recognizing any one selected from SEQ ID NOS 3, 6 and 9 or a fragment thereof including the 186 th base from the 5 'end of SEQ ID NO 3 or the 125 th base from the 5' end of SEQ ID NO 10.

Illustratively, the probe comprises one or more selected from the group consisting of: (1) 3 or a fragment thereof comprising base 186 from the 5' end of SEQ ID No. 3, said base being a; and/or a probe recognizing SEQ ID NO 3 or a fragment thereof comprising the 186 th base from the 5 'end of SEQ ID NO 3 which is C, (2) a probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10 which is A; and/or a probe recognizing SEQ ID NO 10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO 10 which is G, (3) a probe recognizing SEQ ID NO 6 or a fragment thereof comprising the 194 nd 195 th base of SEQ ID NO 6; and/or a probe recognizing SEQ ID NO 7 or a fragment thereof comprising bases 194-577 of SEQ ID NO 7.

The term "variant" or "mutant" as used herein refers to a polynucleotide that has a nucleic acid sequence altered by insertion, deletion or substitution of one or more nucleotides compared to a reference sequence, while retaining its ability to hybridize to other nucleic acids. A mutant according to any of the embodiments herein comprises a nucleotide sequence having at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence (SEQ ID NOS: 1-12 as described herein) and retaining the biological activity of the reference sequence. Sequence identity between two aligned sequences can be calculated using, for example, BLASTn from NCBI. Mutants also include nucleotide sequences that have one or more mutations (insertions, deletions, or substitutions) in the reference sequence and in the nucleotide sequence, while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to within 1-10, such as 1-8, 1-5, or 1-3. The substitution may be a substitution between purine nucleotides and pyrimidine nucleotides, or a substitution between purine nucleotides or between pyrimidine nucleotides. The substitution is preferably a conservative substitution. For example, conservative substitutions with nucleotides of similar or analogous properties are not typically made in the art to alter the stability and function of the polynucleotide. Conservative substitutions are, for example, exchanges between purine nucleotides (A and G), exchanges between pyrimidine nucleotides (T or U and C). Thus, substitution of one or more sites with residues from the same in the polynucleotides of the invention will not substantially affect their activity. When referring to mutants having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% sequence identity to a primer (e.g.SEQ ID NO:1-2, 4-5, 7-8) or probe according to the invention, preferably such mutants will hybridize under high stringency conditions to the corresponding DNA sequences comprising SEQ ID NO:3, 6, 9. The high stringency conditions can be hybridization and membrane washing at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS.

In another aspect, the present invention provides a method for detecting the plant type of rice in a sample, comprising determining or quantifying the plant type of rice by performing the SNP marker detection described herein on a sample to be detected. The method further comprises: (1) extracting DNA of a sample to be detected; (2) determining or quantifying the genotype of the SNP markers described herein in the DNA using the primers and/or probes described herein; and (3) determining or quantifying the rice plant type based on the results of (2). Wherein the first SNP mark is GG, the deletion mark is the nucleotide sequence of position 194 and 577 of SEQ ID NO. 7, the first SNP mark is AA, the deletion mark is the nucleotide sequence of position 194 and 577 of SEQ ID NO. 7, the first SNP mark is GG, the deletion mark is the nucleotide sequence of position 194 and 195 of SEQ ID NO. 6, the first SNP mark is dwarf; the second SNP marker of (1) is CC, and is identified as rice with C-type homozygous IPA1 genotype, and the second SNP marker of (TT) is identified as rice with T-type homozygous IPA1 genotype.

The rice plant type can be detected and identified by conventional methods for SNP detection in the art, such as fluorescent quantitative probe method or HRM high resolution melting curve method or DNA sequencing, the procedures of these methods and the reagents used are well known in the art. DNA sequencing includes first, second and third generation sequencing.

Herein, the method for extracting DNA in a sample is not particularly limited, and DNA extraction methods suitable for use herein are well known in the art.

SNP marker detection methods suitable for use herein are well known in the art and include, but are not limited to: sequencing, single strand conformation polymorphism polymerase chain reaction (PCR-SSCP), real-time fluorescence quantitative PCR and high resolution melting curve analysis (HRM), fluorescent probe quantitative PCR, restriction fragment length polymorphism polymerase chain reaction (PCR-restriction fragment length polymorphism, PCR-RFLP), flight time mass spectrum and the like. Other reagents than primers and/or probes are known in the art as may be required in SNP marker detection methods.

According to some embodiments of the present invention, the method for determining or quantifying the plant type of rice by performing the detection of the SNP marker described herein on a sample to be tested further comprises: extracting DNA in a sample; carrying out PCR of DNA by using primers SEQ ID NO. 1-2, 4-5 and 7-8 to obtain an amplification product; subjecting the amplification product to HRM analysis or DNA sequencing analysis (e.g., 3730 sequencer sequencing) to obtain the genotype of the SNP markers described herein in the DNA; and determining or quantifying the plant type of the rice based on the genotype of the SNP marker.

The invention also provides a kit which contains a reagent for detecting one or more SNP markers in the rice genome. The reagent may be a primer and/or a probe as described in any embodiment herein. Optionally, the kit further comprises a nucleic acid molecule (i.e., amplification product) of the invention, which can be used as an internal standard or positive control. Preferably, the primers are selected from one or more of the following groups: (1) 1 and 2 or a sequence having at least 90% identity thereto; (2) 8 and 9 or a sequence having at least 90% identity thereto; (3) 7 and 8 or a sequence having at least 90% identity thereto. Preferably, the probes have fluorescent labels, such as fluorescent by FAM, VIC, and CY5, respectively. The kit may further contain various reagents required for performing PCR, such as buffers, enzymes, dNTPs, and the like. In a preferred embodiment, the kit of the invention comprises: 1 and 2, or 4 and 5, or 7 and 8.

The SNP marker and the application thereof have the advantages that:

the invention utilizes a molecular biological method to detect molecular markers related to the plant type of the rice, can determine whether the rice progeny contains the genotype and the phenotype required by people or not through the molecular markers, and breeds the progeny containing excellent alleles according to the genotype and the phenotype. Therefore, the genotype and phenotype can be identified in the seedling stage, the rapid screening of the needed offspring is facilitated, and the breeding efficiency is improved.

The present invention will be illustrated below by way of specific examples. It is to be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The materials, reagents and methods not specifically described in the examples are not conventional in the art.

Examples

Example 1, molecular markers for the SD1 gene:

we sequenced 31 rice material SD1 genes. The molecular marker screening of the SD1 gene comprises the following steps:

1) extracting the genome DNA of the rice plant to be detected;

2) carrying out PCR amplification on the rice genome DNA by using the following primers;

SNP primer of SD 1:

primer name	Primer sequence (5'-3')
		SD1-SNP1F	GGAAATTTGTCACAAAGAGACAAG
SD1-SNP1R	AACAAATATACAGATCAACCTATG

Insertion/deletion primer for SD 1:

primer name	Primer sequence (5'-3')
		SD1-INDEL-2F	ACCTGAGGATGGAGCCCAAGATCCC
SD1-INDEL-2R	CTTGAATTACTTGTTCTGTTGCTTCG

3) For SNP molecular markers. And purifying the PCR product, then performing sequencing reaction, sequencing by a 3730 sequencer, and determining that the PCR product contains the corresponding gene. For Indel primers, the PCR products were sized by agarose gel electrophoresis.

4) The PCR reaction was calculated in 20. mu.l: 10 XPCR reaction buffer 2. mu.1, 25mM MgSO₄Mu.1, 2mM dNTP 2. mu.1, 5. mu.M primers F and R each 1.2. mu.1, genomic DNA 20ng, KOD-Plus polymerase 0.4. mu.1, ddH20 to 20. mu.1.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 15 sec, annealing at 55 ℃ for 30 sec, and extension at 68 ℃ for 1 min for 35 cycles; the temperature was maintained at 68 ℃ for 5 minutes.

The detection results of the SNP-containing sequence, the insertion/deletion sequence (deletion marker) and different varieties of rice are as follows, and the results show that the SNP and the insertion/deletion sequence can well distinguish high-stalk, semi-short-stalk and short-stalk in different varieties.

Sequence containing SNP:

GGAAATTTGTCACAAAGAGACAAGGTGACTCAACAGGCCCTCCAAACTGAAAATTTAATTACTTGCTCAAGATTTAAATATAACTACCCATCCAGTTTTTAATATATAAAGTTGTTAACTTTTAAACATATATATGTTTCACTGTTCTTATAATGTATTTTATCATTAAACATACTTTAAAACAT(A/G)TGGCTTATGTTTTTGAATATTTATATTAAAAATTTTAAATAAGATTAATGATCAAACGTATATTTACTAGTTAACGACATCATGTATTAAAAATCGGAGGAGGTATAGAAGTATGTTCTCCTTTCTTGTAAACATAGGTTGATCTGTATATTTGTT

insertion/deletion sequence, the difference sequence between the two amplification products (947bp/565bp)

ACCTGAGGATGGAGCCCAAGATCCCGGAGCCATTCGTGTGGCCGAACGGCGACGCGAGGCCGGCGTCGGCGGCGGAGCTGGACATGCCCGTGGTCGACGTGGGCGTGCTCCGCGACGGCGACGCCGAGGGGCTGCGCCGCGCCGCGGCGCAGGTGGCCGCCGCGTGCGCCACGCACGGGTTCTTCCAGGTGTCCGAGCACGGCGTCGACGCCGCTCTGGC GCGCGCCGCGCTCGACGGCGCCAGCGACTTCTTCCGCCTCCCGCTCGCCGAGAAGCGCCGCGCGCGCCGCGTCCCG GGCACCGTGTCCGGCTACACCAGCGCCCACGCCGACCGCTTCGCCTCCAAGCTCCCATGGAAGGAGACCCTCTCCT TCGGCTTCCACGACCGCGCCGCCGCCCCCGTCGTCGCCGACTACTTCTCCAGCACCCTCGGCCCCGACTTCGCGCC AATGGGGTAATTAAAACGATGGTGGACGACATTGCATTTCAAATTCAAAACAAATTCAAAACACACCGACCGAGAT TATGCTGAATTCAAACGCGTTTGTGCGCGCAGGAGGGTGTACCAGAAGTACTGCGAGGAGATGAAGGAGCTGTCGCTGACGATCATGGAACTCCTGGAGCTGAGCCTGGGCGTGGAGCGAGGCTACTACAGGGAGTTCTTCGCGGACAGCAGCTCAATCATGCGGTGCAACTACTACCCGCCATGCCCGGAGCCGGAGCGGACGCTCGGCACGGGCCCGCACTGCGACCCCACCGCCCTCACCATCCTCCTCCAGGACGACGTCGGCGGCCTCGAGGTCCTCGTCGACGGCGAATGGCGCCCCGTCAGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGGTAAACCATCTCCTATTCTCCTCTCCTCTGTTCTCCTCTGCTTCGAAGCAACAGAACAAGTAATTCAAG(947bp)

ACCTGAGGATGGAGCCCAAGATCCCGGAGCCATTCGTGTGGCCGAACGGCGACGCGAGGCCGGCGTCGGCGGCGGAGCTGGACATGCCCGTGGTCGACGTGGGCGTGCTCCGCGACGGCGACGCCGAGGGGCTGCGCCGCGCCGCGGCGCAGGTGGCCGCCGCGTGCGCCACGCACGGGTTCTTCCAGGTGTCAGCGAGGAGATGAAGGAGCTGTCGCTGACGATCATGGAACTCCTGGAGCTGAGCCTGGGCGTGGAGCGAGGCTACTACAGGGAGTTCTTCGCGGACAGCAGCTCAATCATGCGGTGCAACTACTACCCGCCATGCCCGGAGCCGGAGCGGACGCTCGGCACGGGCCCGCACTGCGACCCCACCGCCCTCACCATCCTCCTCCAGGACGACGTCGGCGGCCTCGAGGTCCTCGTCGACGGCGAATGGCGCCCCGTCAGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGGTAAACCATCTCCTATTCTCCTCTCCTCTGTTCTCCTCTGCTTCGAAGCAACAGAACAAGTAATTCAAG(565bp)

Table 1: SD1 type and SNP and deletion marker status of 31 parts of material

Example 2 molecular marker of IPA1 Gene

We sequenced 31 rice material IPA1 genes, which were divided into two genotypes. The molecular marker screening of the IPA1 gene comprises the following steps:

1) extracting the genome DNA of the rice plant to be detected;

2) performing PCR amplification on rice genomic DNA by using primers selected from the following table;

the primers selected were:

name of primer	Primer sequence (5'-3')
		IPA-SNP1F	TTCGCCGGTGCTGCCGAGCTCCG
IPA-SNP1R	TCATTGGTTATCTTCTTATAGCAGG

3) For SNP molecular markers. The PCR product was purified and then subjected to a sequencing reaction, and sequenced by a 3730 sequencer. Determining to contain the corresponding gene.

4) The PCR reaction was performed in 20. mu.l with 10 XPCR reaction buffer 2. mu.1, 25mM MgSO40.8. mu.1, 2mM dNTP 2. mu.1, 5. mu.M primers F and R each 1.2. mu.1, genomic DNA 20ng, KOD-Plus polymerase 0.4. mu.1, and ddH20 added to make up to 20. mu.1.

The PCR reaction conditions were: pre-denaturation at 94 ℃ for 2 min, denaturation at 94 ℃ for 15 sec, annealing at 58 ℃ for 30 sec, and extension at 68 ℃ for 1 min for 35 cycles; the temperature was maintained at 68 ℃ for 5 minutes.

The detection results of the SNP-containing sequence and rice of different varieties are shown in the following, and the results show that the SNP can well distinguish the genotypes of the IPA1 genes in different varieties.

Containing the SNP sequences:

TTCGCCGGTGCTGCCGAGCTCCGCCGCGCCTGCCGGAGCACGCTGCCATGGCCGCCCTGGAGAAGACACGAGAGAATTAGGTGGAGGGTGGGGGAAGGGTGAGATTTTTTATATTATCTATGGG(T/C)TCCATTATAAATTTTCTAAACCACACTTATACTGTGGGTGCAGTGTCATTTAGAGTTCCCAAACCACCTATGTTGCAGCTGTGGTATAACAATTTGCTAGGACGCATTGCTACTGCCCTTGTACCCTGCTATAAGAAGATAACCAATGA

table 2: SNP of IPA1 of 31 parts of Material

Sequence listing

<110> China academy of sciences molecular plant science remarkable innovation center

<120> molecular marker of rice plant type and genotype and application thereof

<130> 210087

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 1

ggaaatttgt cacaaagaga caag 24

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (125)..(125)

<223> n is t or c

<400> 2

aacaaatata cagatcaacc tatg 24

<210> 3

<211> 342

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (186)..(186)

<223> n is t or c

<400> 3

ggaaatttgt cacaaagaga caaggtgact caacaggccc tccaaactga aaatttaatt 60

acttgctcaa gatttaaata taactaccca tccagttttt aatatataaa gttgttaact 120

tttaaacata tatatgtttc actgttctta taatgtattt tatcattaaa catactttaa 180

aacatntggc ttatgttttt gaatatttat attaaaaatt ttaaataaga ttaatgatca 240

aacgtatatt tactagttaa cgacatcatg tattaaaaat cggaggaggt atagaagtat 300

gttctccttt cttgtaaaca taggttgatc tgtatatttg tt 342

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

acctgaggat ggagcccaag atccc 25

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 5

cttgaattac ttgttctgtt gcttcg 26

<210> 6

<211> 565

<212> DNA

<213> Artificial Sequence

<400> 6

acctgaggat ggagcccaag atcccggagc cattcgtgtg gccgaacggc gacgcgaggc 60

cggcgtcggc ggcggagctg gacatgcccg tggtcgacgt gggcgtgctc cgcgacggcg 120

acgccgaggg gctgcgccgc gccgcggcgc aggtggccgc cgcgtgcgcc acgcacgggt 180

tcttccaggt gtcagcgagg agatgaagga gctgtcgctg acgatcatgg aactcctgga 240

gctgagcctg ggcgtggagc gaggctacta cagggagttc ttcgcggaca gcagctcaat 300

catgcggtgc aactactacc cgccatgccc ggagccggag cggacgctcg gcacgggccc 360

gcactgcgac cccaccgccc tcaccatcct cctccaggac gacgtcggcg gcctcgaggt 420

cctcgtcgac ggcgaatggc gccccgtcag ccccgtcccc ggcgccatgg tcatcaacat 480

cggcgacacc ttcatggtaa accatctcct attctcctct cctctgttct cctctgcttc 540

gaagcaacag aacaagtaat tcaag 565

<210> 7

<211> 947

<212> DNA

<213> Artificial Sequence

<400> 7

acctgaggat ggagcccaag atcccggagc cattcgtgtg gccgaacggc gacgcgaggc 60

cggcgtcggc ggcggagctg gacatgcccg tggtcgacgt gggcgtgctc cgcgacggcg 120

acgccgaggg gctgcgccgc gccgcggcgc aggtggccgc cgcgtgcgcc acgcacgggt 180

tcttccaggt gtccgagcac ggcgtcgacg ccgctctggc gcgcgccgcg ctcgacggcg 240

ccagcgactt cttccgcctc ccgctcgccg agaagcgccg cgcgcgccgc gtcccgggca 300

ccgtgtccgg ctacaccagc gcccacgccg accgcttcgc ctccaagctc ccatggaagg 360

agaccctctc cttcggcttc cacgaccgcg ccgccgcccc cgtcgtcgcc gactacttct 420

ccagcaccct cggccccgac ttcgcgccaa tggggtaatt aaaacgatgg tggacgacat 480

tgcatttcaa attcaaaaca aattcaaaac acaccgaccg agattatgct gaattcaaac 540

gcgtttgtgc gcgcaggagg gtgtaccaga agtactgcga ggagatgaag gagctgtcgc 600

tgacgatcat ggaactcctg gagctgagcc tgggcgtgga gcgaggctac tacagggagt 660

tcttcgcgga cagcagctca atcatgcggt gcaactacta cccgccatgc ccggagccgg 720

agcggacgct cggcacgggc ccgcactgcg accccaccgc cctcaccatc ctcctccagg 780

acgacgtcgg cggcctcgag gtcctcgtcg acggcgaatg gcgccccgtc agccccgtcc 840

ccggcgccat ggtcatcaac atcggcgaca ccttcatggt aaaccatctc ctattctcct 900

ctcctctgtt ctcctctgct tcgaagcaac agaacaagta attcaag 947

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 8

ttcgccggtg ctgccgagct ccg 23

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 9

tcattggtta tcttcttata gcagg 25

<210> 10

<211> 274

<212> DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (125)..(125)

<223> n is t or c

<400> 10

ttcgccggtg ctgccgagct ccgccgcgcc tgccggagca cgctgccatg gccgccctgg 60

agaagacacg agagaattag gtggagggtg ggggaagggt gagatttttt atattatcta 120

tgggntccat tataaatttt ctaaaccaca cttatactgt gggtgcagtg tcatttagag 180

ttcccaaacc acctatgttg cagctgtggt ataacaattt gctaggacgc attgctactg 240

cccttgtacc ctgctataag aagataacca atga 274

Claims

1. An isolated nucleic acid molecule from rice, which

(1) Contains a first SNP marker, and/or

(2) Contains a deletion marker, and/or

(3) Contains a second SNP marker, wherein,

the first SNP marker was: taking rice genome DNA as a template, and taking SEQ ID NO 1 and 2 as primers to carry out PCR amplification to obtain 186 th nucleotide from the 5' end of an amplification product, wherein the nucleotide is A or G;

the deletion marker is: the 194 nd and 195 th nucleotide sequences of SEQ ID NO. 6 or the 194 nd and 577 th nucleotide sequences of SEQ ID NO. 7,

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is 10bp to 1000bp in length; preferably, the nucleotide sequence of the nucleic acid molecule comprises the sequence shown in SEQ ID NO 3, 6, 7 or 10.

3. Primers for detecting (1) a first SNP marker and optionally a deletion marker, and/or (2) a second SNP marker in the genome of rice, wherein,

the first SNP marker was: taking rice genome DNA as a template, and taking SEQ ID NO 1 and SEQ ID NO 2 as primers to carry out PCR amplification to obtain 186 th nucleotide from the 5' end of an amplification product, wherein the nucleotide is A or G;

the second SNP marker was: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by taking the rice genome DNA as a template and SEQ ID NO 8 and 9 as primers is T or C,

preferably, the first and second electrodes are formed of a metal,

the amplification product of the primer comprises SEQ ID NO. 3 or a fragment thereof comprising nucleotide 186 from the 5' end of SEQ ID NO. 3, and/or,

the amplification product of the primer comprises SEQ ID NO. 6 or a fragment thereof, wherein the fragment comprises the 194 nd and 195 th nucleotide sequences of SEQ ID NO. 6; or the amplification product of the primer comprises SEQ ID NO 7 or a fragment thereof comprising the nucleotide sequence at position 194 and 577 of SEQ ID NO 7, and/or,

the amplification product of the primer comprises SEQ ID NO. 10 or a fragment thereof comprising nucleotide number 125 from the 5' end of SEQ ID NO. 10.

4. The primer of claim 3, wherein the primer is selected from one or more of the group consisting of: (1) the sequences shown in SEQ ID NOS: 1 and 2 or a sequence that hybridizes to SEQ ID NO:3 under stringent conditions, (2) the sequences shown in SEQ ID NOS: 4 and 5 or a sequence that hybridizes to SEQ ID NO:6 or 7 under stringent conditions, and (3) the sequences shown in SEQ ID NOS: 8 and 9 or a sequence that hybridizes to SEQ ID NO:10 under stringent conditions.

5. A probe for detecting (1) a first SNP marker and optionally a deletion marker, and/or (2) a second SNP marker in a rice genome, wherein,

preferably, the probe comprises:

(1.1) a probe recognizing SEQ ID NO 3 or a fragment thereof comprising the 186 th base from the 5' end of SEQ ID NO 3 and/or the complementary sequence of (1.2) (1.1); and/or

(2.1) a probe recognizing SEQ ID NO:6 or a fragment thereof comprising bases 194 and 195 of SEQ ID NO:6 and/or a probe recognizing SEQ ID NO:7 or a fragment thereof comprising bases 194 and 577 of SEQ ID NO:7 and/or the complementary sequence of (2.2) (2.1); and/or

(3.1) a probe recognizing SEQ ID NO:10 or a fragment thereof comprising the 125 th base from the 5' end of SEQ ID NO:10 and/or the complementary sequence of (3.2) (3.1).

6. A kit comprising reagents for detecting (1) a first SNP marker and optionally a deletion marker, and/or (2) a second SNP marker in a rice genome, wherein,

the deletion markers are: 6 th 194 nd 195 th nucleotide sequence or 7 th 194 th 577 th nucleotide sequence;

7. The kit of claim 6, wherein the kit comprises

(1) A primer for detecting (1.1) the first SNP marker and optionally the deletion marker, and/or (1.2) the second SNP marker, and

optionally (2) a probe for detecting (2.1) the first SNP marker and optionally the deletion marker, and/or (2.2) the second SNP marker, and

optionally (3) a nucleic acid molecule having (3.1) a first SNP marker and optionally a deletion marker, and/or (3.2) a second SNP marker,

preferably, the kit comprises: the primer of claim 3 or 4, optionally the probe of claim 5 and optionally the nucleic acid molecule of claim 1 or 2.

8. A method for identifying the plant type and/or genotype of rice, said method comprising,

(1) detecting in the genome of the rice (a) a first SNP marker and optionally a deletion marker, and/or (b) a second SNP marker, wherein,

the first SNP marker is: taking rice genome DNA as a template, and taking SEQ ID NO 1 and SEQ ID NO 2 as primers to carry out PCR amplification to obtain 186 th nucleotide from the 5' end of an amplification product, wherein the nucleotide is A or G;

the deletion markers are: the nucleotide sequence at the 194 th-195 th site of SEQ ID NO. 6 or the nucleotide sequence at the 194 th-577 th site of SEQ ID NO. 7;

(2) identifying the plant type and/or genotype of the rice according to the first SNP marker, the deletion marker and the second SNP marker, wherein,

the first SNP marker is GG, the deletion marker is the nucleotide sequence of No. 194 and 577 of SEQ ID NO. 7 and is identified as a highstalk, the first SNP marker is AA, the deletion marker is the nucleotide sequence of No. 194 and 577 of SEQ ID NO. 7 and is identified as a semi-dwarfstalk, the first SNP marker is GG, the deletion marker is the nucleotide sequence of No. 194 and 195 of SEQ ID NO. 6 and is identified as a dwarfstalk;

the second SNP marker CC is identified as rice with C-type homozygous IPA1 genotype, and the second SNP marker TT is identified as rice with T-type homozygous IPA1 genotype.

9. The application of the reagent for detecting the first SNP marker and the optional deletion marker and/or (2) the second SNP marker in the rice genome in the identification of the plant type of the rice, the identification of the genotype or the breeding of the rice or the preparation of a kit for the identification of the plant type of the rice, the identification of the genotype or the breeding of the rice is provided, wherein,

the first SNP marker was: taking rice genome DNA as a template, and taking SEQ ID NO 1 and SEQ ID NO 2 as primers to carry out PCR amplification to obtain 186 th nucleotide from the 5' end of an amplification product;

the second SNP marker was: the 125 th nucleotide from the 5' end of an amplification product obtained by PCR amplification by using rice genome DNA as a template and SEQ ID NO 8 and 9 as primers.

10. The use according to claim 9, wherein the plant type of rice, the genotype or the rice breeding is identified based on the SNP marker detected, wherein,

the first SNP marker is GG, the deletion marker is the nucleotide sequence of the 194 nd through 577 th of SEQ ID NO. 7 and is identified as a highstalk, the first SNP marker is AA, the deletion marker is the nucleotide sequence of the 194 th through 577 th of SEQ ID NO. 7 and is identified as a semi-dwarfstalk, the first SNP marker is GG, the deletion marker is the nucleotide sequence of the 194 th through 195 th of SEQ ID NO. 6 and is identified as a dwarfstalk,