阅读说明：本技术 用于构建半糯型粳稻品种dna指纹图谱库的成套引物对及其筛选方法和应用 (Complete primer pair for constructing semi-waxy japonica rice variety DNA fingerprint spectrum library and screening method and application thereof ) 是由 张亚东 赵春芳 赵庆勇 陈涛 姚姝 周丽慧 梁文化 路凯 赫磊 赵凌 朱镇 魏 于 2021-08-24 设计创作，主要内容包括：本发明提供了一种用于构建半糯型粳稻品种DNA指纹图谱库的成套引物对,包括18对SNP四引物对和36对InDel引物对,18对SNP四引物对分别特异性检测18个水稻SNP位点SNPjs0001～SNPjs0018,36对InDel引物对分别特异性检测36个水稻InDel位点InDeljs0001～InDeljs0036。还提供了相关筛选方法和应用。本发明的用于构建半糯型粳稻品种DNA指纹图谱库的成套引物对能够用于构建半糯型粳稻品种DNA指纹图谱库,能够用于半糯型粳稻品种的真伪和纯度鉴定,设计巧妙,使用简便快捷,成本低,不受环境影响,适于大规模推广应用。(The invention provides a set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library, which comprises 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs, wherein the 18 pairs of SNP four primer pairs respectively and specifically detect 18 rice SNP sites SNPjs 0001-SNPjs 0018, and the 36 pairs of InDel primer pairs respectively and specifically detect 36 rice InDel sites InDel 0001-InDeljs 0036. Related screening methods and uses are also provided. The primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library and identifying the authenticity and purity of the semi-waxy japonica rice variety, is ingenious in design, simple, convenient and quick to use, low in cost, free of environmental influence and suitable for large-scale popularization and application.)

1. The complete primer pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library is characterized by comprising 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs, wherein the 18 pairs of SNP four primer pairs respectively and specifically detect 18 rice SNP sites SNPjs 0001-SNPjs 0018, and the 36 pairs of InDel primer pairs respectively and specifically detect 36 rice InDel sites InDel 0001-InDel 0036.

2. The set of primer pairs for constructing the DNA fingerprint spectrum library of the semi-waxy japonica rice variety according to claim 1, wherein each pair of SNP four primer pairs respectively comprises an SNP forward inner primer, an SNP reverse inner primer, an SNP forward outer primer and an SNP reverse outer primer, and the sequences of 18 pairs of SNP four primer pairs are shown as SEQ ID NO:1 to SEQ ID NO: 72, each pair of InDel primer pairs respectively comprises an InDel forward primer and an InDel reverse primer, and the sequences of 36 pairs of InDel primer pairs are sequentially shown as SEQ ID NO: 73-SEQ ID NO: 144.

3. A screening method of a complete set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library is characterized by comprising the following steps:

(1) performing core sequence comparison on important trait genes of rice by using genome sequencing data of a plurality of rice varieties in a public database, screening SNP (Single nucleotide polymorphism) sites and InDel (InDel) sites, designing a candidate SNP four-primer pair aiming at the SNP sites, and designing a candidate InDel primer pair aiming at the InDel sites;

(2) respectively extracting leaf genome DNA of a plurality of semi-waxy japonica rice varieties, carrying out PCR amplification on the leaf genome DNA of each semi-waxy japonica rice variety by using the candidate SNP four-primer pair to obtain a PCR amplification product, carrying out electrophoresis strip detection on the PCR amplification product to obtain a PCR amplification band, carrying out primary screening on the specificity, polymorphism and band type easily-distinguishable degree of the PCR amplification band, screening out the SNP four-primer pair which has clear characteristic band, polymorphism and can be stably repeated among different semi-waxy japonica rice varieties, and carrying out cluster discrimination on the plurality of semi-waxy japonica rice varieties by using the characteristic band obtained by the amplification of the SNP four-primer pair to obtain a primary discrimination result;

(3) and further carrying out group distinguishing on the semi-waxy japonica rice varieties of each preliminary group obtained according to the preliminary distinguishing result by using the candidate InDel primer pairs until each semi-waxy japonica rice variety is divided into one group, wherein the number of the most similar difference primer pairs between the two semi-waxy japonica rice varieties accounts for more than 5% of the number of the total primer pairs, InDel primer pairs which are clear in characteristic bands, polymorphic and capable of being repeated stably among different semi-waxy japonica rice varieties are screened in the process, and the SNP four primer pairs and the InDel primer pairs form a set of primer pairs for constructing a DNA fingerprint spectrum library of the semi-waxy japonica rice varieties.

4. The screening method of set of primer pairs for constructing semi-waxy japonica Rice variety DNA fingerprinting database according to claim 3, characterized in that in the step (1), the public database is NCBI Genome database and Rice Genome Annotation Project database; the multiple rice varieties are Nipponbare, 9311, Suyunuo, Minghui 63 and Guichao 2; the important rice trait genes comprise a starch debranching enzyme gene II, a starch synthase gene II, a loose plant type gene LPA1, a heading stage gene Hd6, a rice stress-resistant gene OsLecRK1, a grain body type control gene GIF1, a rice starch regulating factor RSR1, a G protein gene RGA1, a heading stage gene Hd1, a thousand kernel weight gene TGW6, an endosperm protein gene RAG2, a heading stage control gene Ghd7, a heading stage gene Ghd8, a starch synthase gene III, a granule type starch synthase gene GBSSI, a heading stage gene OsFCA, a heading stage gene Ehd2, a low amylose content gene du3, a CO-like gene Ghd2, a starch branching enzyme gene I, a grain-setting gene OsBBY 2, casein kinase I, a MADS transcription factor, a rice stress-resistant gene OsLec 1, a glutamine synthetase gene Osc 2, a grain early rice blast resistance gene GW5, a rice blast resistance gene upright gene Pid 4642, a rice blast resistance gene, Histone methyltransferase SDG724, early panicle gene Ehd1, rice blast resistance gene Pib, granule type gene GW5, starch synthesis gene II, aroma gene BADH2, starch synthase gene I, starch synthase gene IIb, starch branching enzyme gene IV, ADP glucose pyrophosphorylase gene and starch branching enzyme gene III.

5. The method for screening the set of primer pairs for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety according to claim 3, wherein in the step (2), a plurality of the semi-waxy japonica rice varieties comprise Nanjing 46, Nanjing 5055, Nanjing 9108, Nanjing Jingjing, Nanjing 505, Nanjing 58, Nanjing 2728, Nanjing 5718, Nanjing 518, Nanjing 3908, Nanjing 56, Nanjing 5713, Zhongjiang Jing 9008, Nanjing 5818, Nanjing 9308, Nanjing 7718, Nanjing 9036, Jia 58, Suxiangjing No. 3, Ningjing No. 8, Suxiangjing 100, Songhuaxiang No. 1, Humianxiaxiang soft No. 1, Jianong early fragrance, Humian early fragrance, Hu rosin, Ningjing 1018, Jia 6, Jianong fragrance Wunong No. 9, Wunong fragrance No. 28, Miao fragrance No. 1606, Shanghai fragrance No. 1, Shanghai fragrant Yu No. 113, Yu No. 3, Yu No. 1, Yu No. 3; the number of the SNP four primer pairs is 18, each SNP four primer pair respectively comprises an SNP forward inner primer, an SNP reverse inner primer, an SNP forward outer primer and an SNP reverse outer primer, and the sequences of the 18 SNP four primer pairs are sequentially shown as SEQ ID NO:1 to SEQ ID NO: shown at 72.

6. The screening method of the set of primer pairs for constructing the DNA fingerprint spectrum library of the semi-waxy japonica rice variety according to claim 3, wherein in the step (3), the number of the InDel primer pairs is 36, each pair of InDel primer pairs respectively comprises an InDel forward primer and an InDel reverse primer, and the sequences of the 36 pairs of InDel primer pairs are sequentially shown as SEQ ID NO. 73-SEQ ID NO. 144.

7. A method for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety is characterized by comprising the following steps:

(A) respectively extracting leaf genome DNA of a plurality of semi-waxy japonica rice varieties;

(B) carrying out PCR amplification, electrophoresis imaging and effective characterization of amplification bands on the leaf genome DNA of each semi-waxy japonica rice variety by using the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library according to any one of claims 1-2, numbering the characterization bands in sequence according to the sequence of the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, and recording 0/1 on the characterization bands according to the numbering to obtain the DNA characteristic fingerprint spectrum of each semi-waxy japonica rice variety;

(C) and summarizing the DNA characteristic fingerprint spectrum to obtain the DNA fingerprint spectrum library of the semi-waxy japonica rice variety.

8. The method for constructing a DNA fingerprint library of a semi-waxy japonica rice variety according to claim 7, wherein in the step (a), the plurality of semi-waxy japonica rice varieties include nanjing 46, nanjing 5055, nanjing 9108, nanjing grain, nanjing 505, nanjing 58, nanjing 2728, nanjing 5718, nanjing 518, nanjing 3908, nanjing 56, nanjing 5713, zhongjiang japonica 9008, nanjing 5818, nanjing 9308, nanjing 7718, nanjing 9036, jia 58, suxiang No. 3, ninning No. 8, suxiang No. 100, shangxiaxiang No. 1, jianong early xiang, shangxiang ni 1212, kangsu 1018, jianong japonica No. 6, ningxiang jing No. 9, wunong xiang 28, wuxiang early xiang No. 1, shangyang No. 1606, shangxiang No. 113, xiang yun jing No. 3 and xu jing.

9. An identification method for identifying a semi-waxy japonica rice variety of a rice variety to be detected is characterized by comprising the following steps:

(I) extracting leaf genome DNA of the rice variety to be detected;

(II) carrying out PCR amplification, electrophoresis imaging and effective characterization of amplification bands on the leaf genome DNA of the rice variety to be detected by adopting the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library according to claim 1 or claim 2, numbering the characterization bands in sequence according to the sequence of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, recording the characterization bands by 0/1 according to the numbers, and establishing the DNA fingerprint spectrum library of the rice variety to be detected;

(III) merging the DNA fingerprint spectrum library of the rice variety to be detected with the DNA fingerprint spectrum library of the semi-waxy japonica rice variety constructed by the construction method of the DNA fingerprint spectrum library of the semi-waxy japonica rice variety according to claim 7 or claim 8, drawing a cluster map, and judging the target semi-waxy japonica rice variety with highest genetic similarity to the rice variety to be detected;

(IV) when the number of the difference primer pairs between the rice variety to be detected and the target semi-waxy japonica rice variety is more than 3, judging that the rice variety to be detected and the target semi-waxy japonica rice variety have obvious difference and belong to different varieties; and when the number of the difference primer pairs between the rice variety to be detected and the target semi-waxy japonica rice variety is less than 2, judging that the rice variety to be detected and the target semi-waxy japonica rice variety are the same variety or similar varieties.

10. Use of the primer set for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety according to any one of claims 1-2 in constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety, identifying a rice variety or molecular breeding.

Technical Field

The invention relates to the technical field of bioinformatics and molecular genetics, in particular to the technical field of primer pairs for constructing a rice DNA fingerprint spectrum library, and particularly relates to a complete set of primer pairs for constructing a semi-glutinous japonica rice variety DNA fingerprint spectrum library, and a screening method and application thereof.

Background

In recent years, breeders culture a series of excellent taste japonica rice varieties (Wangchailin, Zhangyadong, Chuanzhao and the like, inheritance and breeding research, inheritance 2021, 43 (5): 442 + 458) by reducing the amylose content of rice according to the dietary habits of people who love soft glutinous rice in Jianghuai regions, such as Nanjing japonica series japonica rice varieties, Ningjing 8, Suxiangjing 100, Fengjing 1606, Wuxiangjing 113 and the like in Jiangsu province, Shanghai Humian 1212, rosin japonica 1018 and the like in Shanghai, Jia 58 in Zhejiang, Jianong Zao Xiang and the like. The japonica rice with good taste is called semi-glutinous japonica rice because the amylose content of the rice is between that of ordinary japonica rice and glutinous rice, and the rice is soft in texture, high in viscosity, good in elasticity and not retrogradation when being cooked in cold. The popularization and application area of the semi-glutinous japonica rice varieties in Jianghuai areas of China is continuously increased in recent years, and according to incomplete statistics, the popularization area of only Jiangsu provinces in 2019 reaches more than 1300 mu of ten thousand; meanwhile, the rice is also a main provenance for creating high-quality high-grade rice brands.

Because the food quality is excellent, the rice brand is famous and good in marketing, under the temptation of huge economic benefits, the counterfeiting events of semi-glutinous japonica rice seeds and rice brands with excellent taste are endless, and some illegal vendors fake and inferior seeds or rice as high-quality varieties to form phenomena of false and false, true and false and indistinguishable and the like, so that the serious economic and benefit loss is brought to farmers, processing enterprises and consumers, and the serious negative influence is also caused to the brand image of high-grade rice. On the other hand, breeders also find that the superior taste rice has partial performance degradation and taste reduction phenomena in production, and the quality of the rice and the market competitiveness of brands are influenced. Therefore, the research and development of accurate and efficient methods for identifying the authenticity and purity of varieties and the realization of the accurate identification of the original species of the mainly-pushed semi-glutinous japonica rice variety with excellent taste become the problems of close attention of various parties such as breeding units, rice processing enterprises, quality monitoring parts, consumers and the like.

The field seed reproduction identification mode based on the plant pedigree phenotype cannot meet the requirement of accurately and quickly identifying different germplasms due to a plurality of limitations of long period, large variation of characters along with the environment and the like. The molecular marker technology based on DNA sequence variation is widely applied to the aspects of rice resource identification, variety purity detection and classification (Wangming lake, Zhangxiatian, Wu national forest, Jianqi, Shicourage bee, SSR marker application in rice variety DNA fingerprint map construction and purity identification in Ningbo areas, Chinese rice, 2009, 25(6), 50-54; Li banyan, stamen, old leaves, Zhengxing fei, Huzhou and Li lan sesame, rice germplasm resources are efficiently identified and fingerprint maps are constructed based on genome-wide SNP, molecular plant breeding is 2020, 18(18), 6050-doped 6057). The researches basically utilize SSR molecular markers, the existing industry standard NYT 1433-2014-rice variety identification technical regulation SSR marking method of rice variety authenticity molecule detection in China is DNA molecular identification carried out on the basis of 48 SSR markers, however, the standard selects indica type rice varieties with rich genetic variation as reference varieties, the distinguishing effect of more markers among the japonica type rice varieties is not ideal, only a few varieties present polymorphism, the main reason is that the japonica rice varieties are mainly pushed in production and are usually derived from the same backbone parent varieties or only a few derived varieties with even single character are improved on the basis of the same varieties, so that the genetic difference among the varieties is smaller and smaller, the researches show that the main pushed japonica rice varieties in the same rice area, particularly the derived varieties with the same character, such as Zhao-Chun-Fang, Yuanliang, Tianpan and northeast japonica rice, and Wx and OsSSIIa gene sequence analysis, the crop academic newspaper 2020, 46(06), 75-85). Therefore, the conventional SSR molecular markers cannot be used for identifying the authenticity and purity of the varieties.

With the rise of functional genomes of rice, a large number of important character regulation genes such as rice quality, resistance, high yield, plant type and the like are cloned successively, and the genes have various allelic variation forms in rice germplasm resources (Zhang Changquan, Von Linhao, Gu Ming Xue Feng, Liu Qiao quan, Jiangsu province rice quality character heredity and important gene cloning research progress, heredity, 2021, 43(5), 17; Wanmin, important gene cloning and breeding utilization of rice, academic annual meeting of Chinese crop society, 2019). Therefore, in order to realize the accurate identification of the authenticity of the main cultivated semi-waxy type good-taste japonica rice variety in the middle and lower reaches of Yangtze river, construct a DNA fingerprint database of a single variety, better keep the stable and heredity of the variety, maintain the legal rights and interests of growers and rice processing enterprises, and create a good-taste rice variety brand, a set of rapid and accurate detection system based on the functional marker of the rice core gene is necessary to be established.

Therefore, it is desirable to provide a set of primer pairs for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety, which can be used for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety and can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library, which can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety and is suitable for large-scale popularization and application.

The invention also aims to provide a complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, which has the advantages of ingenious design, simple and convenient and quick use, low cost, no environmental influence and suitability for large-scale popularization and application.

The invention also aims to provide a screening method of the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, the obtained primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety, and is suitable for large-scale popularization and application.

The invention also aims to provide a screening method of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, which has the advantages of ingenious design, simple and quick operation, low cost and suitability for large-scale popularization and application.

The invention also aims to provide application of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library in constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, identifying rice varieties or molecular breeding, so that the primer set can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety, can be used for molecular breeding and is suitable for large-scale popularization and application.

The invention also aims to provide application of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library in constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, identifying rice varieties or molecular breeding, and the primer set is ingenious in design, simple, convenient and quick to use, low in cost, free of environmental influence and suitable for large-scale popularization and application.

In order to achieve the above object, in a first aspect of the present invention, a set of primer pairs for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety is provided, wherein the set of primer pairs for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety comprises 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs, 18 pairs of SNP four primer pairs respectively and specifically detect 18 rice SNP sites SNPjs0001 to SNPjs0018, and 36 pairs of InDel primer pairs respectively and specifically detect 36 rice InDel sites indejs 0001 to indejs 0036.

Preferably, each pair of the SNP four primer pairs respectively comprises an SNP forward inner primer, an SNP reverse inner primer, an SNP forward outer primer and an SNP reverse outer primer, and the sequences of the 18 pairs of the SNP four primer pairs are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 72, each pair of InDel primer pairs respectively comprises an InDel forward primer and an InDel reverse primer, and the sequences of 36 pairs of InDel primer pairs are sequentially shown as SEQ ID NO: 73-SEQ ID NO: 144.

In a second aspect of the invention, the invention provides a screening method of a set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library, which is characterized by comprising the following steps:

(1) performing core sequence comparison on important trait genes of rice by using genome sequencing data of a plurality of rice varieties in a public database, screening SNP (Single nucleotide polymorphism) sites and InDel (InDel) sites, designing a candidate SNP four-primer pair aiming at the SNP sites, and designing a candidate InDel primer pair aiming at the InDel sites;

(2) respectively extracting leaf genome DNA of a plurality of semi-waxy japonica rice varieties, carrying out PCR amplification on the leaf genome DNA of each semi-waxy japonica rice variety by using the candidate SNP four-primer pair to obtain a PCR amplification product, carrying out electrophoresis strip detection on the PCR amplification product to obtain a PCR amplification band, carrying out primary screening on the specificity, polymorphism and band type easily-distinguishable degree of the PCR amplification band, screening out the SNP four-primer pair which has clear characteristic band, polymorphism and can be stably repeated among different semi-waxy japonica rice varieties, and carrying out cluster discrimination on the plurality of semi-waxy japonica rice varieties by using the characteristic band obtained by the amplification of the SNP four-primer pair to obtain a primary discrimination result;

(3) and further carrying out group distinguishing on the semi-waxy japonica rice varieties of each preliminary group obtained according to the preliminary distinguishing result by using the candidate InDel primer pairs until each semi-waxy japonica rice variety is divided into one group, wherein the number of the most similar difference primer pairs between the two semi-waxy japonica rice varieties accounts for more than 5% of the number of the total primer pairs, InDel primer pairs which are clear in characteristic bands, polymorphic and capable of being repeated stably among different semi-waxy japonica rice varieties are screened in the process, and the SNP four primer pairs and the InDel primer pairs form a set of primer pairs for constructing a DNA fingerprint spectrum library of the semi-waxy japonica rice varieties.

Preferably, in the step (1), the public databases are the NCBI Genome database and the Rice Genome Annotation Project database; the multiple rice varieties are Nipponbare, 9311, Suyunuo, Minghui 63 and Guichao 2; the important rice trait genes comprise a starch debranching enzyme gene II, a starch synthase gene II, a loose plant type gene LPA1, a heading stage gene Hd6, a rice stress-resistant gene OsLecRK1, a grain body type control gene GIF1, a rice starch regulating factor RSR1, a G protein gene RGA1, a heading stage gene Hd1, a thousand kernel weight gene TGW6, an endosperm protein gene RAG2, a heading stage control gene Ghd7, a heading stage gene Ghd8, a starch synthase gene III, a granule type starch synthase gene GBSSI, a heading stage gene OsFCA, a heading stage gene Ehd2, a low amylose content gene du3, a CO-like gene Ghd2, a starch branching enzyme gene I, a grain-setting gene OsBBY 2, casein kinase I, a MADS transcription factor, a rice stress-resistant gene OsLec 1, a glutamine synthetase gene Osc 2, a grain early rice blast resistance gene GW5, a rice blast resistance gene upright gene Pid 4642, a rice blast resistance gene, Histone methyltransferase SDG724, early panicle gene Ehd1, rice blast resistance gene Pib, granule type gene GW5, starch synthesis gene II, aroma gene BADH2, starch synthase gene I, starch synthase gene IIb, starch branching enzyme gene IV, ADP glucose pyrophosphorylase gene and starch branching enzyme gene III.

Preferably, in the step (2), the plurality of semi-glutinous japonica rice varieties include nanjing 46, nanjing 5055, nanjing 9108, nanjing crystal grain, nanjing 505, nanjing 58, nanjing 2728, nanjing 5718, nanjing 518, nanjing 3908, nanjing 56, nanjing 5713, zhongjiang japonica 9008, nanjing 5818, nanjing 9308, nanjing 7718, nanjing 9036, jia 58, su xiangjing No. 3, ningjing No. 8, su xiangjing 100, shang zaixiang No. 1, shang zaoxiang No. 1, jianong shang zaixiang, shang shangguan 1212, rosin japonica 1018, jianong nong jing No. 6, yangxiang xiangjing No. 9, yangxianxiang 28, shangxiang 1, jinxiangyu No. 1, jinxiangfeng, wuxiangxiangyu 1606, wuxiang xiangjing 1813, wuxiangjing 181113 and xu jing; the number of the SNP four primer pairs is 18, each SNP four primer pair respectively comprises an SNP forward inner primer, an SNP reverse inner primer, an SNP forward outer primer and an SNP reverse outer primer, and the sequences of the 18 SNP four primer pairs are sequentially shown as SEQ ID NO:1 to SEQ ID NO: shown at 72.

Preferably, in the step (3), the number of the InDel primer pairs is 36, each pair of the InDel primer pairs respectively comprises an InDel forward primer and an InDel reverse primer, and the sequences of the 36 pairs of the InDel primer pairs are shown as SEQ ID NO: 73-SEQ ID NO:144 in sequence.

In a third aspect of the invention, a method for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library is provided, which is characterized by comprising the following steps:

(A) respectively extracting leaf genome DNA of a plurality of semi-waxy japonica rice varieties;

(B) carrying out PCR amplification, electrophoresis imaging and effective characterization of amplification bands on the leaf genome DNA of each semi-waxy japonica rice variety by using the complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, numbering the characterization bands in sequence according to the sequence of the complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, and carrying out 0/1 recording on the characterization bands according to the numbers to obtain the DNA characteristic fingerprint spectrum of each semi-waxy japonica rice variety;

(C) and summarizing the DNA characteristic fingerprint spectrum to obtain the DNA fingerprint spectrum library of the semi-waxy japonica rice variety.

Preferably, in the step (a), the plurality of semi-waxy japonica rice varieties include nanjing 46, nanjing 5055, nanjing 9108, nanjing crystal grain, nanjing 505, nanjing 58, nanjing 2728, nanjing 5718, nanjing 518, nanjing 3908, nanjing 56, nanjing 5713, zhongjiang japonica 9008, nanjing 5818, nanjing 9308, nanjing 7718, nanjing 9036, jia 58, su xiangjing No. 3, ningjing No. 8, su xiangjing 100, shang zaixiang No. 1, shang zaoxiang No. 1, jianong shang zaixiang, shang shangguan 1212, rosin japonica 1018, jianong nong japonica No. 6, ningxiang jing No. 9, yangxianxiang 28, shangxianjing No. 1, jinxiangyu No. 1, jinxiangfeng, wuxiangxiangyu 1606, wuxiangyu 1813, wuchang xiangjing 1819 and xu.

In a fourth aspect of the present invention, there is provided an identification method for identifying a semi-waxy japonica rice variety of a rice variety to be tested, comprising the steps of:

(I) extracting leaf genome DNA of the rice variety to be detected;

(II) carrying out PCR amplification, electrophoresis imaging and effective characterization of amplification bands on the leaf genome DNA of the rice variety to be detected by adopting the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, numbering the characterization bands in sequence according to the sequence of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, carrying out 0/1 recording on the characterization bands according to the numbers, and establishing the DNA fingerprint spectrum library of the rice variety to be detected;

(III) merging the DNA fingerprint spectrum library of the rice variety to be detected with the DNA fingerprint spectrum library of the semi-waxy japonica rice variety constructed by the construction method of the DNA fingerprint spectrum library of the semi-waxy japonica rice variety, drawing a cluster map, and judging the target semi-waxy japonica rice variety with highest genetic similarity with the rice variety to be detected;

(IV) when the number of the difference primer pairs between the rice variety to be detected and the target semi-waxy japonica rice variety is more than 3, judging that the rice variety to be detected and the target semi-waxy japonica rice variety have obvious difference and belong to different varieties; and when the number of the difference primer pairs between the rice variety to be detected and the target semi-waxy japonica rice variety is less than 2, judging that the rice variety to be detected and the target semi-waxy japonica rice variety are the same variety or similar varieties.

In the fifth aspect of the invention, the application of the primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library in constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, identifying rice varieties or molecular breeding is provided.

The invention has the beneficial effects that:

a. the complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library comprises 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs, wherein the 18 pairs of SNP four primer pairs respectively and specifically detect 18 rice SNP sites SNPjs 0001-SNPjs 0018, and the 36 pairs of InDel primer pairs respectively and specifically detect 36 rice InDel sites InDel 0001-InDel 0036, so that the primer pairs can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety, and is suitable for large-scale popularization and application.

b. The primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library comprises 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs, wherein the 18 pairs of SNP four primer pairs respectively and specifically detect 18 rice SNP sites SNPjs 0001-SNPjs 0018, and the 36 pairs of InDel primer pairs respectively and specifically detect 36 rice InDel sites InDel 0001-InDel 0036, so the primer set is ingenious in design, simple, convenient and quick to use, low in cost, free of environmental influence and suitable for large-scale popularization and application.

c. The screening method of the complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library utilizes the genome sequencing data of a plurality of rice varieties in a public database, carries out core sequence comparison aiming at important character genes of rice, screens SNP sites and InDel sites, and respectively designs candidate SNP four primer pairs and candidate InDel primer pairs; carrying out PCR amplification on semi-waxy japonica rice varieties by using a candidate SNP four-primer pair, carrying out primary screening on specificity, polymorphism and band type distinguishing easiness of a PCR amplification band, screening out the SNP four-primer pair which has clear characteristic bands among different semi-waxy japonica rice varieties, has polymorphism and can be stably repeated, and carrying out group distinguishing on a plurality of semi-waxy japonica rice varieties by using the characteristic bands obtained by amplification of the SNP four-primer pair; the candidate InDel primer pairs are utilized to further carry out group distinguishing until each semi-waxy japonica rice variety is divided into a group, the number of the difference primer pairs between the two most similar semi-waxy japonica rice varieties accounts for more than 5% of the number of the total primer pairs, InDel primer pairs which are clear in characteristic bands, polymorphic and capable of being repeated stably between different semi-waxy japonica rice varieties are screened out in the process, and the SNP four primer pairs and the InDel primer pairs form a set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library, so that the set of primer pairs obtained by the method for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and the purity of the semi-waxy japonica rice variety, and is suitable for large-scale popularization and application.

d. The screening method of the complete set of primer pairs for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library utilizes the genome sequencing data of a plurality of rice varieties in a public database, carries out core sequence comparison aiming at important character genes of rice, screens SNP sites and InDel sites, and respectively designs candidate SNP four primer pairs and candidate InDel primer pairs; carrying out PCR amplification on semi-waxy japonica rice varieties by using a candidate SNP four-primer pair, carrying out primary screening on specificity, polymorphism and band type distinguishing easiness of a PCR amplification band, screening out the SNP four-primer pair which has clear characteristic bands among different semi-waxy japonica rice varieties, has polymorphism and can be stably repeated, and carrying out group distinguishing on a plurality of semi-waxy japonica rice varieties by using the characteristic bands obtained by amplification of the SNP four-primer pair; the candidate InDel primer pairs are utilized to further carry out group distinguishing until each semi-waxy japonica rice variety is divided into a group, the number of the difference primer pairs between the two most similar semi-waxy japonica rice varieties accounts for more than 5% of the number of the total primer pairs, InDel primer pairs which are clear in characteristic bands, polymorphic and capable of being stably repeated between different semi-waxy japonica rice varieties are screened out in the process, and the SNP four primer pairs and the InDel primer pairs form a set of primer pairs for constructing a DNA fingerprint spectrum library of the semi-waxy japonica rice variety, so that the primer pair is ingenious in design, simple, convenient and fast to operate, low in cost and suitable for large-scale popularization and application.

e. The primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library is applied to constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, identifying rice varieties or molecular breeding, so that the primer set can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice varieties, can be used for molecular breeding and is suitable for large-scale popularization and application.

f. The primer set for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library is applied to constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, identifying rice varieties or molecular breeding, and is ingenious in design, simple, convenient and quick to use, low in cost, free of environmental influence and suitable for large-scale popularization and application.

Drawings

FIG. 1 shows the amplification map and effective characterization sites of 3 SNP four primer pairs in a set of primer pairs for constructing a DNA fingerprint spectrum library of a semi-waxy japonica rice variety, wherein M is a DNA Marker, D1-D34 correspond to different semi-waxy japonica rice varieties respectively, and x is an effective characterization site and an ineffective site.

FIG. 2 shows the amplification map and effective characterization sites of 4 pairs of InDel primer pairs in the semi-waxy japonica rice variety for constructing the complete set of primer pairs of the semi-waxy japonica rice variety DNA fingerprint map library, wherein M is a DNA Marker, D1-D34 correspond to different semi-waxy japonica rice varieties respectively, and x is an effective characterization site and an ineffective site.

FIG. 3 is a cluster analysis diagram of a DNA fingerprint spectrum library of a rice variety to be tested and a DNA fingerprint spectrum library of a semi-waxy japonica rice variety, which are constructed by using the primer set for constructing the DNA fingerprint spectrum library of the semi-waxy japonica rice variety, wherein a square frame shows that the rice variety to be tested and the Nanjing 9108 belong to the same group and belong to the same variety, and the rice variety to be tested is a Nanjing 9108 suspected variety.

Detailed Description

In order to clearly understand the technical contents of the present invention, the following examples are given in detail.

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. In the examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 screening of primer set for constructing DNA fingerprint library of semi-waxy japonica rice variety and construction of DNA fingerprint library of semi-waxy japonica rice variety

First, test materials

The invention selects 34 semi-glutinous japonica rice varieties which are from the main rice varieties produced in Shanghai region of Jiangzhe at present, the taste quality is excellent, the varieties are widely accepted by the market, and the name and the information of the breeding unit are detailed in Table 1. All the materials are known public materials, and the agricultural germplasm resource middle-term library in Jiangsu province can be provided free of charge.

TABLE 1 34 semi-waxy japonica rice varieties and breeding units of the present invention

Second, primer pair design and screening

(1) Primer pair sequence source and design

Based on genome sequencing data of rice varieties of Nissan, 9311, Suyunuo, Minghui 63 and Guichao 2 in a public database (https:// www.ncbi.nlm.nih.gov/, http:// rice, plant biology.msu.edu /), anchoring important character control genes of rice quality, yield, resistance, heading date and the like, performing multi-sequence comparison on a core region (coding region) of the genes, and screening sequence variation of nucleotide insertion deletion and single nucleotide substitution; designing primer pairs at proper positions on two sides of a variation site respectively, designing SNP four-primer pairs aiming at a single nucleotide substitution variation type, specifically designing an InDel primer pair aiming at an insertion deletion variation type by utilizing the SNP four-primer pair designed by a four-primer amplification hindered mutation system PCR technology, the primer pair design of part of the starch synthase genes also refers to literature data (Chentao, Luomiri, Zhang Yao and the like, a four-primer amplification hindered mutation system PCR technology is utilized to detect the low amylose content gene Wx-mq of rice, Chinese rice science, 2013, 27(5), plus 534, field happiness, Seigy Jie, Qian and the like, the molecular marker development information of the amylase genes in the rice starch synthesis related genes is established and reported scientifically, 2010, 55(26) and 2591 plus 2601), and the primer pair sequences are submitted to Nanjing Optimaokae biological Limited company for primer synthesis.

(2) Primer pair screening

Firstly, initially screening the specificity and polymorphism of PCR amplification bands and the easily distinguishable degree of band types of the SNP four primer pairs in each semi-waxy japonica rice variety, screening out the SNP four primer pairs which have clear characteristic bands, polymorphism and stable repetition among different semi-waxy japonica rice varieties, and counting 18 SNP four primer pairs related to important characteristic gene function variation sites of rice in total; then, taking the 18 SNP four-primer pairs as core primers constructed by a fingerprint spectrum library, and carrying out group distinguishing on each semi-waxy japonica rice variety according to a characteristic band of the core primers; taking the distinguishing result of the core primers as a reference, adding InDel primer pairs in each group for further distinguishing, and by analogy, dividing 34 semi-glutinous japonica rice varieties into more groups until each group is divided into only one semi-glutinous japonica rice variety, and ensuring that the number of the difference primer pairs between the two most similar semi-glutinous japonica rice varieties (the difference primer pairs refer to the difference of the characteristic bands amplified by the primer pairs between the two) accounts for more than 5% of the number of the total primer pairs. Finally, 18 pairs of SNP four primer pairs and 36 pairs of InDel primer pairs which have good amplification effect and high polymorphism and are derived from 41 important rice trait regulatory genes are screened to form a complete set of primer pairs for constructing a DNA fingerprint spectrum library of the semi-glutinous japonica rice variety, and the sequence information of 54 pairs of primer pairs containing 54 primer pairs is shown in Table 2.

Sequence and Source Gene information of primer pairs in Table 254

The 54 sites corresponding to 54 pairs of primers are shown in Table 3.

Table 354 corresponds to the primer pair 54 bit information

Construction of DNA fingerprint spectrum library of three-half waxy japonica rice variety

(1) Extraction of leaf genomic DNA

Respectively sampling 34 semi-glutinous japonica rice varieties by using leaves, putting 0.1g of leaves into a 2ml centrifuge tube, freeze-drying, grinding into powder, adding 500ul of CTAB buffer solution preheated in advance, incubating in a thermostat at 65 ℃ for 30min, taking out the centrifuge tube in the middle, turning upside down, fully shaking up, taking out the centrifuge tube, adding 500ul of chloroform and isoamyl alcohol solution, uniformly mixing according to a ratio of 24:1, then centrifuging at 12000rpm for 10min at room temperature, discarding the precipitate, transferring the supernatant into a new centrifuge tube, adding 2/3 volumes of isopropanol, uniformly mixing, and standing at-20 ℃ for 1 hour; finally centrifuging at 4 deg.C and 10000rpm for 5min, discarding supernatant, retaining precipitate, adding 500ul 75% ethanol, washing precipitate for 2 times,removing supernatant to obtain DNA precipitate, air drying at room temperature, adding 100ul deionized H₂And O, dissolving the DNA, and storing in a refrigerator at the temperature of 20 ℃ below zero for later use.

(2) PCR amplification

A set of primer pairs consisting of 34 leaf genome DNAs of semi-waxy japonica rice varieties serving as templates and 54 primer pairs serving as amplification primer pairs are respectively subjected to PCR amplification according to the amplification conditions of the primer pairs. The PCR reaction adopts a 15ul amplification system, and specifically comprises the following steps: 2 × Taq Master Mix 7.5 ul; mix primers 2.0 ul; total DNA 1ul (50-200 ng); ddH₂O4.5 ul. The PCR amplification procedure was: carrying out pre-denaturation at 94 ℃ for 2min, then circulating, carrying out denaturation at 94 ℃ for 30s, annealing at 52-57 ℃ for 45s, extending at 72 ℃ for 1min, circulating for 33 times, extending at 72 ℃ for 5min, and storing at 10 ℃ for 10min to obtain PCR amplification products of leaf genome DNA of each semi-waxy japonica rice variety on different primer pairs.

(3) Electrophoretic detection of PCR products

And (3) carrying out electrophoresis on the amplification products of the 18 pairs of SNP four primer pairs on agarose gel with the mass ratio concentration of 2.0%, dyeing by using nucleic acid dye, observing under a gel imaging system, and shooting images. Selecting different types of gels from the amplification products of 336 pairs of InDel primer pairs according to the length and type of the characteristic band, carrying out electrophoresis on 2% agarose gel when the number of the differential bases is more than 10bp, carrying out electrophoresis on 9% polyacrylamide gel when the number of the differential bases is less than 10bp, and shooting images after the polyacrylamide gel electrophoresis is finished and the gel is subjected to silver staining, washing and formaldehyde color development.

(4) Result analysis and construction of semi-waxy japonica rice variety DNA fingerprint spectrum library

According to a detection image after agarose gel electrophoresis or polyacrylamide gel electrophoresis, selecting a characteristic band type which accords with the size of a primer design target product to carry out gel recovery and sequence analysis so as to confirm the correctness of the characteristic band. Then, using DNA Marker as control, PCR bands on the electrophoretogram are effectively characterized, and a schematic diagram of effectively characterizing PCR amplification bands of a part of primer pairs is shown in FIG. 1 and FIG. 2. And finally, numbering the characteristic strips in sequence according to the sequence of SNP 1-18-InDel 1-36, recording the characteristic strips obtained by each semi-glutinous japonica rice variety on each pair of primer pairs in sequence according to the number 0/1, wherein 0 represents that the characteristic strip is not amplified, 1 represents that the characteristic strip is amplified, obtaining the DNA characteristic fingerprint spectrum of each semi-glutinous japonica rice variety, and establishing a DNA fingerprint spectrum library (table 3) of the semi-glutinous japonica rice variety, which comprises 155 data.

TABLE 3 DNA fingerprint spectrum library of semi-glutinous japonica rice variety (0/1 database)

(5) 155-data-based clustering analysis among semi-waxy japonica rice varieties

According to 155 0/1 data detected by 54 pairs of primer pairs, the genetic similarity of 34 semi-glutinous japonica rice varieties is calculated by using a similarity program in SLT _ NT-sys _2.10 software, UPGMA (non-weighted group mean method) clustering analysis is carried out by using SHAN in a clustering program, and a genetic relationship clustering tree-like graph among the varieties is drawn. The detected variation range of the genetic similarity between the semi-waxy japonica rice varieties is 0.471-0.897, the minimum value of the genetic similarity coefficient is 0.471 detected between Nanjing 7718 and Suxiangjing 100, and the relationship between the Nanjing 7718 and the Suxiangjing is the farthest; and the highest genetic similarity value (0.897) is detected between the Nanjing 9308 and the Nanjing 9036, 5 primer pair differences exist between the Nanjing 9308 and the Nanjing 9036, and the percentage of the number of the difference primer pairs to the number of the total primer pairs is more than 5 percent and is higher than the judgment value of similar varieties. As can be seen from the cluster map (FIG. 3), there are large genetic variations among 34 semi-waxy japonica rice varieties, and 54 pairs of primer pairs can clearly distinguish and distinguish the 34 semi-waxy japonica rice varieties of the invention.

Example 2 detection of whether a certain Rice variety to be tested is one of the 34 semi-waxy japonica Rice varieties of the present invention

First, test materials

The rice variety DX to be detected is one or more suspected varieties of the 34 semi-waxy japonica rice varieties.

Second, identification procedure

(1) Leaf genome DNA extraction of rice variety DX to be detected

According to the method for extracting the leaf genome DNA of the third step (1) in the example 1, the leaf genome DNA of the rice variety DX to be tested is obtained by replacing 34 leaves of the semi-waxy japonica rice variety with the leaf of the rice variety DX to be tested, and the other steps are not changed.

(2) PCR amplification

According to the PCR amplification method of the third step (2) in the example 1, the leaf genome DNA of the 34 semi-waxy japonica rice varieties is replaced by the leaf genome DNA of the rice variety DX to be detected, and other steps are not changed, so that PCR amplification products of the rice variety DX to be detected on 54 primer pairs are obtained.

(3) Amplification band analysis, fingerprint library construction

According to the methods of the third step (3) and the third step (4) in the embodiment 1, the PCR amplification result of the rice variety DX to be detected is subjected to amplification strip electrophoresis detection, effective characterization and 0/1 recording, and a DNA fingerprint spectrum library of the rice variety to be detected is obtained.

(4) Clustering analysis between rice variety to be detected and 34 semi-waxy japonica rice varieties

According to the method of the third step (5) in the embodiment 1, the '34 semi-waxy japonica rice varieties' are replaced by the 'rice variety DX to be detected and the 34 semi-waxy japonica rice varieties', other steps are not changed, the cluster map of the semi-waxy japonica rice variety containing the rice variety DX to be detected is obtained, and the semi-waxy japonica rice variety with the highest genetic similarity to DX is found out according to the cluster map.

(5) Determination of judgment standard for judging whether rice variety to be detected is true semi-waxy japonica rice variety

According to the agricultural industry standard (NYT1433-2014) SSR marking method of rice variety identification technical regulation, varieties with differences of more than or equal to 2 markers in 48 SSR markers are considered as different varieties, and the differences are calculated to be about 5% according to the marking ratio. The percentage of the difference mark of 5 percent is also used as a judgment standard for variety authenticity detection results in the fields of corn and other researches. According to the invention, 54 pairs of primer pairs are selected, and the number of the difference primer pairs is 3, which is used as a judgment standard for judging whether the rice variety to be detected is the same as the semi-waxy japonica rice variety with the highest genetic similarity.

(a) If the number of the difference primer pairs between the rice variety DX to be detected and the semi-waxy japonica rice variety with the highest genetic similarity is more than 3 (including 3), the conclusion is that the rice variety DX to be detected does not belong to any one of the 34 semi-waxy japonica rice varieties, or the rice variety DX to be detected has obvious difference with the semi-waxy japonica rice variety with the highest genetic similarity and belongs to different varieties;

(b) if the number of the difference primer pairs between the rice variety DX to be detected and the semi-waxy type japonica rice variety with the highest genetic similarity is less than 2 pairs (including 2 pairs), the conclusion is that the rice variety DX to be detected and the semi-waxy type japonica rice variety with the highest genetic similarity are the same variety, or the rice variety DX to be detected and the semi-waxy type japonica rice variety with the highest genetic similarity do not detect obvious difference and belong to the same variety.

Referring to fig. 3, the rice variety DX and the semi-waxy japonica rice variety nanjing 9108 have the highest genetic similarity, and the number of the differential primer pairs is less than 2, which indicates that the rice variety DX and the semi-waxy japonica rice 9108 have no obvious difference and belong to the same variety.

Compared with the prior art, the invention has the following beneficial effects:

1. at present, molecular identification of authenticity of rice varieties is mostly based on 48 SSR markers in an industry standard NYT 1433-2014-rice variety identification technical regulation SSR marking method issued in 2014 of China, but the SSR markers are suitable for indica rice, japonica rice subspecies or indica rice varieties, and have the defects of poor polymorphism, more interference bands, low discrimination, low repeatability and the like in japonica rice varieties with closer genetic relationship. Therefore, a new way needs to be found for accurate molecular identification and fingerprint map construction of different japonica rice cultivars.

2. The 54 pairs of primer pairs obtained by screening in the invention are from core sequences of 41 important rice trait control genes, and the 41 genes are related to diversified rice key traits such as quality (starch synthesis related genes, branching enzyme genes and debranching enzyme genes), fragrance, stress resistance (disease resistance and stress resistance), plant type, grain property and the like. Therefore, the DNA fingerprint atlas database is constructed, and the phenotype characteristics of important rice traits are linked. And the primer pair sequence mainly comes from the coding region of the gene and has low homology with other position sequences of the genome, so the primer pair sequence has the characteristics of low mismatching rate, low cost, high stability, high repeatability, high distinguishing capability, high throughput, simple operation and the like, and not only meets the construction requirements of a DNA fingerprint spectrum library of a semi-glutinous japonica rice variety, but also meets the requirements of the construction of fingerprint spectrum data of other types of rice varieties.

3. The semi-waxy japonica rice varieties used for constructing the DNA fingerprint spectrum library are mainly good food flavor varieties in the production of rice in the middle and lower reaches of Yangtze river at present, so the constructed DNA fingerprint spectrum library can be directly used for guiding the authenticity identification of the rice varieties in production and the brand rice of the varieties in the market.

4. The complete set of primer pairs consisting of 54 pairs of primer pairs provided by the invention can be applied to the construction of DNA fingerprint spectra of rice varieties, the identification of variety authenticity, the differentiation of rice germplasm resources and the breeding of rice molecules, provides scientific and technological support for rice variety management, seed and rice market monitoring, enterprise right maintenance and the like, and ensures the safety of grain production; meanwhile, a technical basis is laid for analyzing important gene resources of parents in rice molecular breeding, and the breeding process of a new rice variety with high quality, high yield and high water resistance is accelerated by marker-assisted selection.

Therefore, the invention utilizes the genome sequencing data of a plurality of rice varieties in a public database to carry out core sequence comparison aiming at important character genes of rice, screens SNP sites and InDel sites, and respectively designs a candidate SNP four-primer pair and a candidate InDel primer pair; carrying out PCR amplification on semi-waxy japonica rice varieties by using a candidate SNP four-primer pair, carrying out primary screening on specificity, polymorphism and band type distinguishing easiness of a PCR amplification band, screening out the SNP four-primer pair which has clear characteristic bands among different semi-waxy japonica rice varieties, has polymorphism and can be stably repeated, and carrying out group distinguishing on a plurality of semi-waxy japonica rice varieties by using the characteristic bands obtained by amplification of the SNP four-primer pair; the candidate InDel primer pairs are utilized to further carry out group discrimination until each semi-waxy japonica rice variety is divided into a group, the number of the difference primer pairs between the two most similar semi-waxy japonica rice varieties accounts for more than 5% of the number of the total primer pairs, InDel primer pairs which are clear in characteristic bands, polymorphic and capable of being stably repeated between different semi-waxy japonica rice varieties are screened out in the process, and the SNP four primer pairs and the InDel primer pairs form a complete set of primer pairs for constructing a semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for constructing the rice variety DNA fingerprint spectrum library, identifying variety authenticity, dividing germplasm resources, breeding rice molecular breeding and other related researches, and provide technical support for rice germplasm resources and new variety protection.

In conclusion, the primer set pair for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library can be used for constructing the semi-waxy japonica rice variety DNA fingerprint spectrum library, can be used for identifying the authenticity and purity of the semi-waxy japonica rice variety, is ingenious in design, simple, convenient and quick to use, low in cost, free of environmental influence and suitable for large-scale popularization and application.

In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

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<221> misc_feature

<222> (1)...(22)

<223> Forward primer IF of SNP-7 in Rice

<400> 25

ccgcgggagg cggcggcgga ga 22

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(30)

<223> reverse primer IR of SNP-7 of rice SNP four primer pairs

<400> 26

cggaagaggg gtcgagctcg acgccgcagc 30

<210> 27

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-7 for rice

<400> 27

agctgctggt gttggtggtg cggcggcg 28

<210> 28

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-7 of rice SNP four primer pair

<400> 28

gagacggcgg cgaggagcga ctccggca 28

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer IF of SNP four primer pairs SNP-8 for rice

<400> 29

gggaaaagct caacggcggc ga 22

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(26)

<223> reverse primer IR of SNP-8 of rice SNP four primer pairs

<400> 30

cattaggagc cacgtcagct ggctgc 26

<210> 31

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-8 OF rice

<400> 31

cacgtggaac ccacaagcca actagcaa 28

<210> 32

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-8 of rice SNP four primer pairs

<400> 32

gggtggggag aaggcgagca gttgtagt 28

<210> 33

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer IF of SNP four primer pairs SNP-9 for rice

<400> 33

cgtggcgtgc gacgtgcagg ggt 23

<210> 34

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> reverse primer IR of SNP-9 of rice SNP four primer pairs

<400> 34

gccgggagcg ggttcgcgga ttg 23

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(25)

<223> Forward primer OF OF SNP four primer pairs SNP-9 for rice

<400> 35

gtacctgtgc gcgtcgtgcg acgcg 25

<210> 36

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-9 of rice SNP four primer pairs

<400> 36

tccacctcct cgtccttgtc gccgagga 28

<210> 37

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(35)

<223> Forward primer IF of SNP four primer pairs SNP-10 for rice

<400> 37

ttattaatct acgtttaata ttttgaatgt gtttc 35

<210> 38

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(29)

<223> reverse primer IR of SNP-10 of rice SNP four primer pairs

<400> 38

agttttggtg tgtaacatca gatatagga 29

<210> 39

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-10 OF rice

<400> 39

aatcttttaa gcctaattgc tccataat 28

<210> 40

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-10 of four primer pairs of rice SNP

<400> 40

aattgttcgt tttacatctt tatacgga 28

<210> 41

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer IF of SNP four primer pairs SNP-11 for rice

<400> 41

ccgccgcgcg ctccaagtct cc 22

<210> 42

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(26)

<223> reverse primer IR of SNP-11 of rice SNP four primer pair

<400> 42

cgaggtgttc cccggctgcc ggaaat 26

<210> 43

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(32)

<223> Forward primer OF OF SNP four primer pairs SNP-11 for rice

<400> 43

ccagtagcag acaccacctg tcccattggg ga 32

<210> 44

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-11 of rice SNP four primer pair

<400> 44

caggtgctcg gcgctcaacc acatggtt 28

<210> 45

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(30)

<223> Forward primer IF of SNP four primer pairs SNP-12 for rice

<400> 45

tttgacgaat ctagataagg agcctgtatg 30

<210> 46

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(32)

<223> reverse primer IR of SNP-12 of rice SNP four primer pairs

<400> 46

cctataatat aagggatttt ttagggaggg ct 32

<210> 47

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(30)

<223> Forward primer OF OF SNP four primer pairs SNP-12 for rice

<400> 47

cgctataaag gttacgtaaa catatcgcag 30

<210> 48

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-12 of rice SNP four primer pairs

<400> 48

ttctttttgg aacggaggga gtaagtat 28

<210> 49

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer IF of SNP four primer pairs SNP-13 of rice

<400> 49

atggcggacc aatctccggt gtgcaccc 28

<210> 50

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(25)

<223> reverse primer IR of SNP-13 of rice SNP four primer pairs

<400> 50

cgctcccagc agacctgcgg tgccc 25

<210> 51

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-13 OF rice

<400> 51

ccgctcatca caaccaaacc gccggctc 28

<210> 52

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-13 of rice SNP four primer pairs

<400> 52

gcagggcact gaccacctgc ctggtgga 28

<210> 53

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(33)

<223> Forward primer IF of SNP four primer pairs SNP-14 for rice

<400> 53

aaaaaaataa ttggagttac aatctaacat tac 33

<210> 54

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(29)

<223> reverse primer IR of SNP-14 of rice SNP four primer pairs

<400> 54

aagttctgca atttacagtg taaatacgt 29

<210> 55

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-14 for rice

<400> 55

tacaaaatat ctggtatttg ttgaatgg 28

<210> 56

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-14 of four primer pairs of rice SNP

<400> 56

cagcataaaa tgactgtaaa acgtaaac 28

<210> 57

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer IF of SNP four primer pairs SNP-15 for rice

<400> 57

agtgccagcg gtggtggccg ca 22

<210> 58

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(34)

<223> reverse primer IR of SNP-15 of rice SNP four primer pair

<400> 58

tcatgaactt cttgaaactg aatcatacct gagc 34

<210> 59

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-15 for rice

<400> 59

aggaggcacc gctaggtggt gtgttcag 28

<210> 60

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-15 of rice SNP four-primer pair

<400> 60

aatcttgcat gccccttcag aaggacga 28

<210> 61

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer IF of SNP four primer pairs SNP-16 of rice

<400> 61

atgttgtgtt cttgtgttct ttgcaggc 28

<210> 62

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer IR of SNP-16 of rice SNP four primer pairs

<400> 62

gtagatcttc tcaccggtct ttccccaa 28

<210> 63

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-16 OF rice

<400> 63

gggtgaggtt tttccattgc tacaatcg 28

<210> 64

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(26)

<223> reverse primer OR of SNP-16 of rice SNP four-primer pair

<400> 64

gtcgatgaac acacggtcga ctcaat 26

<210> 65

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(29)

<223> Forward primer IF of SNP four primer pairs SNP-17 of rice

<400> 65

ttccctgttc aattgcagaa taatcaact 29

<210> 66

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> reverse primer IR of SNP-17 of rice SNP four primer pairs

<400> 66

aggaccctgc aagggttgtc tct 23

<210> 67

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> Forward primer OF OF SNP four primer pairs SNP-17 OF rice

<400> 67

accttttact ttgtagggca tgaaccct 28

<210> 68

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-17 of four primer pairs of rice SNP

<400> 68

gaccagataa cctattctgg ccaaaaga 28

<210> 69

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer IF of SNP four primer pairs SNP-18 for rice

<400> 69

cgcagcaggc ggcggtggtt gc 22

<210> 70

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(27)

<223> reverse primer IR of SNP-18 of rice SNP four primer pairs

<400> 70

cctgccaggg ttgcaacttc gccgaca 27

<210> 71

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(29)

<223> Forward primer OF OF SNP four primer pairs SNP-18 for rice

<400> 71

tgacacatgc gatcgatgtg gcattggca 29

<210> 72

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(28)

<223> reverse primer OR of SNP-18 of rice SNP four primer pair

<400> 72

ggcgccggct cactagggac ttcctcgg 28

<210> 73

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-1

<400> 73

cgcggggtgg cgacgacaac 20

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-1

<400> 74

accgcctata gaccaacgac 20

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> Forward primer F of rice InDel primer pair InDel-2

<400> 75

ggaagggatc cgtaatacaa a 21

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-2

<400> 76

cccatatcta catgacggtt 20

<210> 77

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(16)

<223> Forward primer F of rice InDel primer pair InDel-3

<400> 77

ggaaatggga gtcgcc 16

<210> 78

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(17)

<223> reverse primer R of rice InDel primer pair InDel-3

<400> 78

cgaagaaacc acgctca 17

<210> 79

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-4

<400> 79

caatatgcct gaagtccac 19

<210> 80

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> reverse primer R of rice InDel primer pair InDel-4

<400> 80

tgtgctcaat tgctagctt 19

<210> 81

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-5

<400> 81

ccaaaacgac ttactttaga gc 22

<210> 82

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> reverse primer R of rice InDel primer pair InDel-5

<400> 82

ttcctccgta ctcataagag c 21

<210> 83

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(18)

<223> Forward primer F of rice InDel primer pair InDel-6

<400> 83

ctcgatcccc tagctctc 18

<210> 84

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(18)

<223> reverse primer R of rice InDel primer pair InDel-6

<400> 84

tcacctcgtt ctcgatcc 18

<210> 85

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-7

<400> 85

ccagtgtcgc cttctccggc tt 22

<210> 86

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-7

<400> 86

ggggaaacga gacggcggtc ca 22

<210> 87

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-8

<400> 87

tatgtgcagc gttcattgac ct 22

<210> 88

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> reverse primer R of rice InDel primer pair InDel-8

<400> 88

agggtcagtc ataacctcag t 21

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> Forward primer F of rice InDel primer pair InDel-9

<400> 89

agcagcaaac atccaaaggc a 21

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> reverse primer R of rice InDel primer pair InDel-9

<400> 90

gggtgtcaag tcgaatccag c 21

<210> 91

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-10

<400> 91

gataaccatc ggtaattgct 20

<210> 92

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> reverse primer R of rice InDel primer pair InDel-10

<400> 92

ggcaatcatg aagattcgaa g 21

<210> 93

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-11

<400> 93

ggaacaattg gttaaatact tca 23

<210> 94

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-11

<400> 94

agaaaaaatt ggatctttgt ca 22

<210> 95

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-12

<400> 95

tccgaatata tttctggatt gtg 23

<210> 96

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-12

<400> 96

ggaaaaataa taatggcttc 20

<210> 97

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-13

<400> 97

agcaggttat aagctaggcc 20

<210> 98

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-13

<400> 98

ctaccaacaa gttcatcaaa 20

<210> 99

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-14

<400> 99

gtgcgtgacc ctttgctgtt 20

<210> 100

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-14

<400> 100

atcttccagg ttccaattct tc 22

<210> 101

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-15

<400> 101

cggggcataa acttcacct 19

<210> 102

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> reverse primer R of rice InDel primer pair InDel-15

<400> 102

attactccca aatgtttgtc gat 23

<210> 103

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-16

<400> 103

taactgactc ctttggcta 19

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> reverse primer R of rice InDel primer pair InDel-16

<400> 104

tatatgcatg aagaacatgt c 21

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(21)

<223> Forward primer F of rice InDel primer pair InDel-17

<400> 105

gaacaatgcc caaacttgag a 21

<210> 106

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-17

<400> 106

gggtccacat gtcagtgagc 20

<210> 107

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-18

<400> 107

cgtcttgcaa ccaacgccga 20

<210> 108

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-18

<400> 108

gagcgtgtgt agggaaggag ct 22

<210> 109

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-19

<400> 109

aagcaatgta agttcaagta gc 22

<210> 110

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-19

<400> 110

gattagggat gatggttttc 20

<210> 111

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(18)

<223> Forward primer F of rice InDel primer pair InDel-20

<400> 111

gggaggcgct gaagagga 18

<210> 112

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-20

<400> 112

gggtagtcac caccctacct tg 22

<210> 113

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-21

<400> 113

ataccccatc aatcgaaat 19

<210> 114

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-21

<400> 114

gaaaaggaca acattgagaa 20

<210> 115

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-22

<400> 115

cttctatcca ttccttaatc cca 23

<210> 116

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-22

<400> 116

atgctattga tgttaagagg gc 22

<210> 117

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(24)

<223> Forward primer F of rice InDel primer pair InDel-23

<400> 117

tgctacataa cacgcataca aagt 24

<210> 118

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> reverse primer R of rice InDel primer pair InDel-23

<400> 118

agacaaaagc gaaaggtaat gag 23

<210> 119

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-24

<400> 119

gtggggaaaa caagtaagtc tg 22

<210> 120

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-24

<400> 120

agttccatca gaagaatcag gg 22

<210> 121

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-25

<400> 121

gtcgccgccg tcgtcagcac 20

<210> 122

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> reverse primer R of rice InDel primer pair InDel-25

<400> 122

agctctgcct ccgtccctt 19

<210> 123

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-26

<400> 123

ccatcacctc aaatacatca ctc 23

<210> 124

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-26

<400> 124

agactggaat gccccttagg 20

<210> 125

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(25)

<223> Forward primer F of rice InDel primer pair InDel-27

<400> 125

ccaataccgt aaactagcga ctatg 25

<210> 126

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-27

<400> 126

tacaggtaga atggcagtgg tg 22

<210> 127

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(24)

<223> Forward primer F of rice InDel primer pair InDel-28

<400> 127

gcactcctgc ctgtttatct gaag 24

<210> 128

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(24)

<223> reverse primer R of rice InDel primer pair InDel-28

<400> 128

gtcgtacagc ttgaagtgat ccag 24

<210> 129

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-29

<400> 129

ggttctcggt gaagatggc 19

<210> 130

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> reverse primer R of rice InDel primer pair InDel-29

<400> 130

gtggtcccag ctgaggtcc 19

<210> 131

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-30

<400> 131

ctttgatagt tcgaatggtt 20

<210> 132

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-30

<400> 132

caatgtttct ccgtgatgat 20

<210> 133

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-31

<400> 133

gaacttgtgc cttaagctga ctg 23

<210> 134

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-31

<400> 134

ggaatagtaa gccgaaggac tt 22

<210> 135

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-32

<400> 135

aagtccttcg gcttactatt cc 22

<210> 136

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> reverse primer R of rice InDel primer pair InDel-32

<400> 136

ggagaaggaa cataacaggg ac 22

<210> 137

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> Forward primer F of rice InDel primer pair InDel-33

<400> 137

gaccaaccga ttaccttctt 20

<210> 138

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-33

<400> 138

ttgctctttt ctcaacctgt 20

<210> 139

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(19)

<223> Forward primer F of rice InDel primer pair InDel-34

<400> 139

tacgctatgc tcttgaaac 19

<210> 140

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-34

<400> 140

tatcttccca gtaaccatca 20

<210> 141

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(22)

<223> Forward primer F of rice InDel primer pair InDel-35

<400> 141

aaggttagca ttggttggtg ag 22

<210> 142

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-35

<400> 142

tctccttgaa cagcgacagc 20

<210> 143

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(23)

<223> Forward primer F of rice InDel primer pair InDel-36

<400> 143

caccaattat attagcgtgc tcc 23

<210> 144

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (1)...(20)

<223> reverse primer R of rice InDel primer pair InDel-36

<400> 144

cgtggctctt ggctctcttg 20