AU2019430940A1

AU2019430940A1 - Method for constructing rice molecular marker map based on Kompetitive Allele Specific PCR and application in breeding Using the same

Info

Publication number: AU2019430940A1
Application number: AU2019430940A
Authority: AU
Inventors: Chun Chen; Zhiqiang Chen; Tao Guo; Cuihong HUANG; Ming Huang; Yongzhu LIU; Kai Sun; Hui Wang; Jiafeng Wang; Wuming XIAO; Guili YANG; Danhua ZHOU
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-04-29
Filing date: 2019-10-25
Publication date: 2020-11-12
Also published as: AU2019101778A4; CN110029187A; WO2020220615A1

Abstract

The present invention provides a method for constructing a rice molecular marker map based on Kompetitive Allele Specific PCR (KASP) and application in breeding using the map, belonging to the field of agricultural technology. In the present invention, genome-wide recombination sequencing is conducted on rice and recombination sequencing data SNPs are mined for searching SNP sites; combined with existing SNP databases, 565 SNP sites required for breeding are obtained based on the SNP sites; and the rice molecular markers map is constructed by molecular marking with the selected SNP sites. The SNP markers of the constructed rice molecular marker map have the advantages of clear physical location and simple and convenient detection method, which can be directly used for molecular identification and genetic background analysis of some important agronomic traits, and has important application value for improving rice molecular breeding efficiency.

Description

METHOD FOR CONSTRUCTING RICE MOLECULAR MARKER MAP BASED ON KOMPETITIVE ALLELE SPECIFIC PCR AND APPLICATION IN BREEDING USING THE SAME TECHNICAL FIELD Method for constructing a rice molecular marker map based on Kompetitive Allele Specific PCR (KASP) and application in breeding using the same, belonging to the field of agricultural technology. BACKGROUND DNA molecular markers are polymorphic nucleotide sequences that are used to identify differences between two individuals or breeds and follow a simple Mendelian pattern of inheritance. The characteristics of ideal DNA markers include high polymorphism, co-dominant inheritance, frequent occurrence in the genome, selection of neutral behavior (i.e. no gene pleiotropy), simple operation and easy rapid determination, good repeatability and easy data exchange in the laboratory. In the past few decades, the use of molecular markers to reveal polymorphisms at the DNA level has played an increasingly important role in plant biotechnology and its genetic research. Since the single nucleotide polymorphism (SNP) was born in 1994, it has been quickly accepted by peer scholars. In 1996, Lander officially pointed out that SNP has opened a new era of molecular marking, which is the third-generation molecular marking technology developed on the basis of the second-generation molecular marking technology represented by SSR and ISSR. From 1998 to 2002, an international conference on "SNPs and complex genomes" was held every year, which aimed to discuss the prospects of SNPs in the research and application of complex genomes, and included the theory, methods and application of SNP markers. During this period each conference would add new contents on the original basis. SNP refers to a DNA sequence polymorphism at the genomic level due to single nucleotide variation with a polymorphism frequency greater than 1%. However, in practical research, single nucleotide variations located in cDNA with a frequency of less than 1% are often referred to as SNPs. As one of the most promising molecular markers, SNP markers are more suitable for a large number of detection and analysis due to the large number and wide distribution in the genome and large-scale and high automation in the analysis process, and have been widely used in many fields, such as biology, agronomy, medicine, biological evolution. In rice research, since the completion of International Rice Genome Sequencing Project (International Rice Genome Sequencing Project 2005), rice functional genomics has been rapidly developed, and it also provides great information support for breeders to improve rice, an important crop through molecular breeding. Currently, 408,898 candidate DNA polymorphisms (SNPs/Indels) were identified based on the two subspecies of rice, Indica and Japonica. A total of 160,000 non-redundant SNPs were identified by determining 100 Mb of the specific part of the reference genome of 20 different varieties and local varieties through resequencing chips. Several resequencing projects from 2011 to 2012 also provided a large amount of information on the genomic structure and genetic diversity of rice. A large number of rice SNPs currently reported may be inquired through several related databases and a large number of rice SNP sites may be mined based on their information. (Gramme: http://ensembl.gramene.org/genomebrowser/index.html; Rice diversity: https://ricediversity.org/; Rice Genome Annotation Project: http://rice.plantbiology.msu.edu/; Rice SNP-seek Database: http://oryzasnp.org/iric-portal/). The above-mentioned large amount of rice SNP information provides beneficial information for molecular breeding. However, the currently published SNPs information has not been integrated in different databases, which is mainly reflected in the following aspects: 1. the specific physical locations of SNP markers are different in different databases, and even inconsistent; 2. SNP redundancy in different databases are unclear, and a large number of SNP markers have been repeatedly reported; 3. there is no systematic summary of relationship between currently published SNP markers and specific agronomic traits. These problems have greatly restricted rice breeders from discovering useful markers. Therefore, the SNPs information existing in rice germplasm resources with a wide genetic background is analyzed by using genome-wide sequencing to clarify their physical locations and construct a physical map; the physical map is utilized to further integrate and correlate the SNPs with the cloned agronomic trait gene so as to provide a clear and usable relationship between the SNPs and the agronomic trait and provide specific breeding guidance for rice breeding workers. Once a complete SNP physical map is constructed, how to efficiently detect SNPs is a key technical link that limits the application of SNPs. At present, SNPs detection technologies may be roughly classified based on the following principles: 1) a detection method based on the principle of allele PCR; 2) endonuclease and ligase technologies; 3) a conformation-based detection method; 4) mass spectrometry; 5) a detection method based on hybridization principle; 6) a sequencing method; 7) other methods: High Resolution Melting (HRM), Kompetitive Allele Specific PCR (KASP), etc. Where, KASP technology is based on fluorescence reading to detect different genotypes of SNPs. Compared with other detection technologies, the KASP technology has the advantages of high flexibility, supporting low, medium and high throughput research and single repeated experiment, high flexibility and high success rate of experimental design, lower cost and wide application prospect. Breeding application efficiency of the SNP markers is remarkably improved by converting SNPs information into reliable and efficient

SNP markers based on KASP. In conclusion, the present invention combines the integrated SNP map KASP, which can effectively overcome the current technical difficulties of unclear SNPs information and difficult detection and application, and has strong application value. SUMMARY In response to the current disconnection between rice SNPs and breeding practice, the present invention combines the integrated SNP map with KASP, which can effectively overcome the current technical difficulties of unclear SNPs information, unknown relationship between SNP markers and traits, and difficult detection and application, and has strong application value. The present invention is implemented by the following technical solution: A KASP-based rice molecular marker map and application in breeding using the same, which includes the following steps of: SI. conducting genome-wide sequencing on 530 rice germplasm materials from home and abroad, and analyzing SNPs of 530 materials in the genome-wide range; S2. selecting 565 SNP sites closely related to breeding practice from the above-mentioned genome-wide SNPs based on the following rules: 1) markers associated with subgroup grouping or quality control of seeds/samples, mainly used for the classification of germplasm materials; 2) markers associated with Indica/Indica polymorphism; 3) high polymorphism markers; 4) functional diagnostic markers; 5) cloned gene interval markers; 6) key gene targeted site markers; 7) markers that have been reported to be significantly linked to agronomic traits; 8) SNP markers used to fill in the gaps; S3. mapping a genome map of the 565 SNP sites according to the physical locations of the SNP sites; and S4. constructing a rice molecular marker map by molecular marking of the mapped genome map of the 565 SNP sites on the basis of KASP. Preferably, the genomic map described in S3 is mapped using Map Gene 2 Chromosome v2 in a mapping website. Preferably, three primer sequences are designed for the molecular marker at each site in S4, and two allele-specific forward primer sequences and one reverse primer sequence are designed for each marker; DNA templates are respectively added during PCR reaction, three corresponding primers are marked, and two fluorescent markers which are respectively complementary with 14-15 bases at the tail part of the forward primer and respectively carry red and blue fluorescent groups are added. More preferably, the 14-15 bases at the end of the allele-specific forward primer sequence are universal, with one allele-specific forward primer sequence end being a linker sequence complementary to a red fluorescent marker and the other allele-specific forward primer sequence end being a linker sequence complementary to a blue fluorescent marker. More preferably, the linker sequence at the end of one allele-specific forward primer sequence complementary to the red fluorescent marker is AGTCGGATTACGAAT, and the linker sequence at the end of the other allele-specific forward primer sequence complementary to the blue fluorescent marker is CTTAGGATACTAGG. Application of the rice molecular marker map constructed in claim 1 in breeding, in particular, the map can be used for identifying quality and disease resistance genes of rice materials from different sources. Application of the rice molecular marker map constructed in claim 1 in breeding, in particular, the map can be used for identifying genetic distance of rice materials from different sources. The present invention has the following beneficial effects as compared with the prior art: 1. The SNP map provided by the present invention is a physical map, which reflects the actual distance between genes or markers in the biological genome, so that the location range of the target gene on the chromosome may be determined as long as the linkage relationship between the target gene and the genetic markers is verified through experiments, while the genetic map distance determined by the traditional genetic map based on the recombination rate is more relative; 2. All SNP sites provided by the present invention are integrated on one map, there is no duplication among each other, and the problem of high redundancy of the existing genetic map is effectively overcome; 3. The SNP sites provided by the present invention are clear in use, such as for functional diagnostic markers, cloned gene interval markers, key gene targeted site markers and the like, while the traditional genetic map cannot provide the specific relationship between the SNP sites and genes; 4. The KASP-based SNP markers detection method provided by the present invention has the advantages of being simple and convenient, high in accuracy, strong in flexibility, lower in cost and easy to realize automation. Therefore, compared with the reported genetic map, the present invention can effectively improve the application efficiency of the SNP markers in rice breeding. BRIEF DESCRIPTION OF THE DRAWING In order to illustrate the examples of the present invention or the technical solutions of the prior art, the accompanying drawings to be used will be described briefly below. Notably, the following accompanying drawing merely illustrates some examples of the present invention, but other accompanying drawings can also be obtained for those of ordinary skill in the art based on the accompanying drawing without any creative efforts. FIG. 1 shows information for 565 SNPs with successfully designed primers in the present invention; FIG. 2 shows an exemplary graph of the results of detecting a portion of markers in the present invention; FIG. 3 shows a phylogenetic tree based on Nei's genetic distance. DETAILED DESCRIPTION The following describes the present invention in more detail with reference to accompanying drawings and examples. The test methods used in the examples are conventional, if not otherwise specified; the materials and reagents used are commercially available if not otherwise specified. 1. A collection of 530 rice materials from different sources at home and abroad There were a total of 530 germplasm materials from a wide range of sources, and the sources of different materials were shown in Table 1. Where, two types of foreign germplasm (KY and KIR) were obtained from International Rice Research Institute (IRRI), and the rest materials were provided by South China Agricultural University (SCAU). Table 1 The collected 530 rice materials

Types of materials Quantity Example Materials

Diversity Germplasm Material "9311" numbered KBen-122 and material "Minghui 63" numbered 234 and Breeding Parents KBen-031

Foreign Germplasm Refrigerator material "BR 7232-6-2-3" numbered KIR-005, from "6th Rice Heat 38 (IRRI) Tolerance (IRHTN 2011)"

Foreign Germplasm 50 Indica material "ZO09NEAMAT" from Vietnam numbered KY006 (Other)

Huahang Youzhan and Huangruanzhan hybrid advanced strain "(Huahang

Advanced breeding line 114 Youzhan/Huangruanzhan) F2-22-1-1-1-2-2-1" numbered KDOO1, namely a breeding

line 1

IRRI Introduced 24 Progeny recovery material "MingHui63/IR03A550" numbered KP-001 Three-line Parents

Sterile line 28 Sterile line material "Y58S" numbered KC014

F1 material numbered KF002, namely "Huangruan Xiuzhanx Huahang 31" Fl 42 "Ben-4xHuahang No. 31"

2. Genome resequencing analysis of 530 rice materials The DNA of 530 rice materials was extracted to construct shotgun library according to a standard process of Illumina platform, all of which were Paired-end sequencing libraries, with an insert length between 200-500 bp. After quality test met the requirements, the library was sequenced on an Illumina Hiseq 2000/2500 platform for conducting base recognition, original data arrangement and data quality assessment according to an Illumina standard process. The SNPs information among 530 materials was compared against the reference genome. A total of 652457 SNP sites were detected by SAMTOOLS software. After filtering under conditions of dp2, Miss.7, and Maf0.01, a total of 60144 high-quality SNP sites were obtained for subsequent analysis. 3. A collection of SNP sites for constructing a map In order to develop rice genome-wide markers more effectively, this research combined the published data and available databases to select the following 8 types of SNP data from the SNPs obtained in step 2 as SNP markers for the development of the genome-wide (Table 2): 1) the first type of markers were associated with subgroup grouping or quality control of seeds/samples, mainly used for classification of germplasm materials; 2) the second type and third type of markers were mainly polymorphic markers, which may be better used for materials classification and genome background diversity assessment; since the materials used in this research were mostly indica, so the markers associated with the Indica/Indica polymorphism were classified into one type; 3) rice functional diagnostic markers that have been reported or self-developed and verified by this project; 4) the fifth type and the sixth type of markers were the reported key functional gene targeted sites and related SNP sites in cloned gene interval; 5) the reported markers that were significantly linked to target traits found through genome-wide association analysis; and 6) the eighth type of markers were SNP markers self-developed by this research, which could fill in the gap so that the markers were relatively evenly distributed on 12 chromosomes. Table 2 The screened SNP sites information SN SNP Classification Characteristic of markers SNP (s)

Associated with subgroup grouping or 1 Subgrouping 61 quality control

Associated with Indica/Indica specific 2 Indica/Indica-variation 64 polymorphism

3 High PIC Hight PIC value 53

4 FNP Functional diagnosis 21

5 Key gene targeted Key functional gene targeted sites 95

6 Identified gene overlapped Cloned gene interval 173

7 GWAS MS SNP GWAS highly significant linkage 33

8 Supplement Filling in the gap 96

4. Primer design and markers transformation of 596 SNP sites With the screened SNP sequences information, primer sequences of the SNP site were generated by Kraken software. As can be seen from Table 3 and FIG. 1, 59 pairs of primers were designed for 61 SNPs associated with subgroup grouping or quality control in the first type, with a success rate of 96.7%; 62 pairs of primers were designed for 64 SNPs associated with Indica/Indica specific polymorphism in the second type, with a success rate of 96.8%; 52 pairs of primers were designed for 53 SNPs with high PIC polymorphism in the third type, with a success rate of 98.1%; 18 pairs of primers were designed for 21 functional diagnostic SNPs in the fourth type, with a success rate of 85.7%; 90 pairs of primers were designed for 95 key functional genes targeted SNP sites in the fifth type, with a success rate of 94.7%; 156 pairs of primers were designed for 173 cloned gene interval SNPs in the sixth type, with a success rate of 90.1%; 32 pairs of primers were designed for 33 GWAS highly significant linked SNPs in the seventh type, with a success rate of 97.0%; and 100 pairs of primers were designed for 96 SNPs in the eight type, with a success rate of 100%. Except for the fourth type of sites, the success rate of primers at the remaining sites may reach more than 90%. 565 pairs of primers were designed for the candidate 596 SNPs, with a success rate of 94.8%. According to the data, a conversion rate of the KASP marker reached 94.8%, the primer design and the markers transformation had higher success rate, and a plurality of valuable candidate SNP markers were provided for experimental development. Table 3 Proportions of SNPs with successfully designed primers Candidate SNP SNP (s) with successfully designed SN SNP Classification Success rate(%) (s) primers

1 Subgrouping 61 59 96.7

Indica/Indica-variatio 2 64 62 96.8 n

3 Hight PIC 53 52 98.1

4 FNP 21 18 85.7

Key gene targeted 95 90 94.7

Identified gene 6 173 156 90.1 overlapped

7 GWAS MS SNP 33 32 97.0

8 Supplement 96 96 100.0

Total 596 565 94.8

5. Typing and verification of 565 SNP markers The candidate markers designed above were detected according to a standard KASP amplification system. Taking the detected results of marker SK07.16957375 as an example, the detected results of different sample plates varied according to their samples. FIG. 2-a shown a non-polymorphism sample plate for marker SK07.16957375, where the samples were gathered according to their fluorescence signal values with only one typing result, without classification; FIG. 2-b shown that marker SK07.16957375 gathered in the upper left corner, the lower right comer and the middle according to its fluorescence signal sample distribution and classified as three typing results. The marker SK07.16957375 was polymorphic in the sample plate. The randomly selected 480 experimental materials were detected and typed by using the successfully transformed markers in the fourth type. 558 in 565 markers that could be successfully transformed displayed fluorescent signals in the detection, where 91 markers shown only one effective fluorescent signal value, which were non-polymorphic markers; 467 markers could display at least 2 effective fluorescent signal values, which were polymorphic markers. These markers could be typed on at least 2 experimental materials. Therefore, 565 SNP molecular markers developed in this research were available. 6. Mapping of Physical Map of 565 KASP-SNP Markers A physical map of 565 markers was constructed using Map Gene 2 Chromosome v2, a mapping website, and the distribution of the markers on the chromosomes was shown in Table 4. Table 4 Distribution of markers with the detected results and polymorphic markers on rice chromosomes Number Number of Chromosome Polymorphic marker Marker density Polymorphic marker Chr. of marker Polymorphic marker length (Mb) proportion (%) (SNP/Mb) density (SNP/Mb) (s) (s)

1 58 47 43.27 81.03 1.34 1.09

2 52 44 35.94 84.62 1.45 1.22

3 63 51 36.41 80.95 1.73 1.40

4 43 35 35.50 81.40 1.21 0.99

43 40 29.96 93.02 1.44 1.34

6 89 82 31.25 92.13 2.85 2.62

7 31 21 29.70 67.74 1.04 0.71

8 36 34 28.44 94.44 1.27 1.20

9 22 16 23.01 72.73 0.96 0.70

10 25 22 23.21 88.00 1.08 0.95

11 58 44 29.02 75.86 2.00 1.52

12 38 31 27.53 81.58 1.38 1.13

Ave. 46.5 39 31.10 83.69 1.48 1.24

Total 558 467 373.25

Note: the rice chromosome length data were referenced to the Rice Genome Annotation Project website. 7. Identification of target genes in rice germplasm using functional SNP sites 18 in 565 KASP-SNP markers were marked as functional sites of cloned genes. In this research, 5 markers were selected for the identification of target genes of germplasm resources. Where, SK06.1765761, SK06.1768006, SK06.176899, and SK06.6752887 were associated with the quality of rice, the first three markers (SK06.1765761, SK06.1768006 and SK06.176899) were mainly related to the synthesis of amylose in rice, and SK06.6752887 was mainly related to the gelatinization temperature, quality of rice during cooking; marker SK12.10607554 was associated with rice blast resistance, with genotype G/G corresponding to rice blast resistance. According to experimental verification, genotypes T/T, A/A, C/C marked by SK06.1765761, SK06.1768006, and SK06.176899 were associated with low amylose, and genotype TT/TT marked by SK06.6752887 corresponded to a lower gelatinization temperature, facilitating the cooking of rice (Table 5). Table 5 Functional Marker Information Associated With Quality and Disease Resistance Marking Gene Phenotypic trait Allele Favorable gene type

SK06.1765761 GBSSI/Wx Amylose starch content T/G T

SK06.1768006 GBSSI/Wx Amylose starch content A/C A

SK06.1768997 GBSSI/Wx Amylose starch content C/T C

SK06.6752887 SSIIa/ALK Gelatinization temperature GC/TT TT

SK12.10607554 Pita Rice blast resistance G/T G

481 rice germplasms were screened using the above 5 markers, 129 rice germplasms containing 4 rice quality-associated favorable genotypes and 1 rice blast resistance-associated genotype were obtained. Of the 129 rice germplasms, most were domestic rice germplasms and few were foreign germplasms, which may be related to the source of the population. These germplasm resources carrying 5 favorable genes may be used as excellent gene donors for molecular breeding practice. 8. Genetic diversity analysis on the germplasm resources by using genome-wide KASP-SNP markers Genotypes of 481 rice germplasms were analyzed using 565 KASP-SNP markers, and an NJ cluster map was constructed based on Nei's genetic distance according to the genotype data (FIG. 3). The 481 germplasms may be divided into two subspecies Japonica and Indica, most of which were Indica germplasms, only 32 Japonica germplasms. The materials belonging to the subspecies of Indica may be divided into two types: one type mainly contained diversity germplasms and breeding parents (experiment ID KBen) and advanced breeding lines (experiment ID KD); the other type mainly contained foreign germplasms with experiment IDs KIR, KY and KP, sterile line germplasm with experiment ID KC, and some of the diversity germplasms and breeding parents. The above-mentioned genetic diversity analysis results may provide guidance for the selective breeding of parents. The selection of materials with certain genetic differences for hybrid combination may create more favorable gene recombination in the generation of genetic segregation. Several examples are used for illustration of the principles and implementation methods of the present invention. The description of the examples is used to help illustrate the method and its core principles of the present invention. In addition, those skilled in the art can make various modifications in terms of specific examples and scope of application in accordance with the teachings of the present invention. In conclusion, the content of this specification shall not be construed as a limitation to the present invention.

Claims

What is claimed is: 1. A method for constructing a rice molecular marker map based on Kompetitive Allele Specific PCR (KASP), wherein the method comprises the following steps of: Si. conducting genome-wide sequencing on 530 rice germplasm materials from home and abroad, and analyzing SNPs of 530 materials in the genome-wide range; S2. selecting 565 SNP sites closely related to breeding practice from the above-mentioned genome-wide SNPs; S3. mapping a genome map of the 565 SNP sites according to physical locations of the SNP sites; and S4. constructing a rice molecular marker map by molecular marking of the mapped genome map of the 565 SNP sites on the basis of KASP.
2. The method for constructing a rice molecular marker map based on KASP according to claim 1, wherein the genomic map in S3 is mapped using Map Gene 2 Chromosome v2 in a mapping website.
3. The method for constructing a rice molecular marker map based on KASP according to claim 1, wherein three primer sequences are designed for the molecular marker at each site in S4, and two allele-specific forward primer sequences and one reverse primer sequence are designed for each marker; DNA templates are respectively added during PCR reaction, three corresponding primers are marked, and two fluorescent markers which are respectively complementary with 14-15 bases at the tail part of the forward primer and respectively carry red and blue fluorescent groups are added.
4. The method for constructing a rice molecular marker map based on KASP according to claim 3, wherein the 14-15 bases at the end of the allele-specific forward primer sequence are universal, with one allele-specific forward primer sequence end being a linker sequence complementary to a red fluorescent marker and the other allele-specific forward primer sequence end being a linker sequence complementary to a blue fluorescent marker.
5. The method for constructing a rice molecular marker map based on KASP according to claim 4, wherein the linker sequence at the end of one allele-specific forward primer sequence complementary to the red fluorescent marker is AGTCGGATTACGAAT, and the linker sequence at the end of the other allele-specific forward primer sequence complementary to the blue fluorescent marker is CTTAGGATACTAGG.
6. Application of the rice molecular marker map constructed according to claim 1 in breeding, wherein the map can be used for identifying quality and disease resistance genes of rice materials from different sources.
7. Application of the rice molecular marker map constructed according to claim 1 in breeding, wherein the map can be used for identifying genetic distance of rice materials from different sources.