CN117512188A - Molecular marker linked with major QTL for regulating and controlling rice seed setting rate and application - Google Patents
Molecular marker linked with major QTL for regulating and controlling rice seed setting rate and application Download PDFInfo
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Abstract
The invention discloses a molecular marker linked with a major QTL for regulating and controlling the rice setting rate, and relates to the technical field of rice setting rate molecular marker screening and application, wherein the molecular markers comprise Indel ssr-1 and Indel ss r-2; the substances for detecting the molecular marker Indel ssr-1 are shown in SEQ ID NO. 1-SEQ ID NO.2; the substances for detecting the molecular marker Indel ssr-2 are shown in SEQ ID NO. 3-SEQ ID NO. 4; the molecular marker is applied to an auxiliary breeding technology, can effectively solve the problem of incomplete knowledge of related genes, can efficiently find the molecular marker closely linked with the main effect QTL of the rice seed setting rate by constructing a genetic linkage map and analyzing quantitative character loci, can screen rice offspring by using the markers, and can effectively improve the breeding efficiency while saving the cost.
Description
Technical Field
The invention relates to the technical field of screening and application of molecular markers of rice setting percentage, in particular to a molecular marker linked with a major QTL for regulating and controlling the rice setting percentage and application thereof.
Background
Since rice domestication, yield-related traits have been the most interesting goal of breeders, and are one of the most important indexes for measuring whether rice varieties are good or not. The rice yield is mainly directly determined by three important constitutional factors: the number of ears per unit area, the number of solid grains per ear and grain weight, wherein the number of solid grains per ear is determined by the total grain number per ear and the fruiting rate, and the solid grains per ear are the characters with the largest variation amplitude in the yield constituent factors and are also most easily influenced by cultivation measures and environment. Therefore, the excavation of the main effective site of the rice seed setting rate and the development of related technologies have positive effects on the stability and improvement of rice yield per unit, can be used as one of the fundamental measures for relieving grain safety problems, and can assist in solving potential hidden hazards caused by global environmental problems.
The related research of the rice seed setting rate needs to take a molecular mechanism and a regulation mechanism formed by the yield traits as a basis, and is more based on the positioning and cloning of related QTL. By means of modern biological information technology, scientific researchers find out initial results in terms of molecular mechanism and regulation law of rice setting rate, and related researches mainly focus on transcription factors, protein kinase pathways and the like. Meina et al studied to develop the transcription factors OsPHR1 and OsPHR2 containing MYB domain, and if they are overexpressed, the phenomenon that the plant shows a decrease in fruiting rate under high phosphorus conditions; ruifang et al found that cyclin-dependent kinase inhibitor OsiICK6 can interact with cyclin OsCYCD and CDKA, and is induced by low temperature, ABA and osmotic stress, and pollen viability and fruiting rate of plants are reduced during overexpression. At present, most of rice setting rate related genes cloned by scientific researchers influence setting rate indirectly by influencing rice flower development, and the genes have low utilization value in rice production and limited application ways. In order to find the major genes with high maturing rate, the research is to carry out complementary verification and functional analysis on some important QTLs by means of map-based cloning and the like on the basis, optimize the breeding and matching of rice parent materials and further promote the rice breeding practice.
The research on the main effect QTL positioning and genetic mechanism of the rice seed setting rate in the scientific research field is relatively few, and the related QTL positioning and gene cloning research still needs further deep excavation and analysis.
Therefore, how to provide a major QTL related to the seed setting rate of rice and apply the major QTL to rice breeding is a technical problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a major QTL locus for regulating and controlling the rice seed setting rate, a molecular marker linked with the major QTL locus and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the main effect QTL locus for regulating and controlling the rice seed setting rate is positioned on a rice chromosome 2, the genetic distance is 128.62-147.53cM, and the physical distance is 30004559 ～ 34415035bp; the main effect QTL is located between a molecular marker Indel ssr-1 and a molecular marker Indel ssr-2;
the substances for detecting the molecular marker Indel ssr-1 are as follows:
an upstream primer: 5'-CCCGGGACTGATGAAGAAG-3', SEQ ID NO.1;
a downstream primer: 5'-CTCACCCTTGACTGCAGATTC-3', SEQ ID No.2;
the substances for detecting the molecular marker Indel ssr-2 are as follows:
an upstream primer: 5'-TGCCATAGATAACACCACTATGAAA-3', SEQ ID No.3;
a downstream primer: 5'-CGCCAAACCATCAAAAATGT-3', SEQ ID No.4.
The invention also provides a positioning method of the major QTL locus for regulating and controlling the rice setting rate, which comprises the following steps: 1) Hybridization is carried out by taking Huazhan and hot grinding as parents, 120 stable inheritance strains are obtained by a single-grain transmission method, and RILs are formed together; 2) The setting rate condition of the mature period of the recombinant inbred line group is inspected; 3) And analyzing the relation between the markers of the whole chromosome set and quantitative trait phenotype values by using a genetic map constructed by a large number of SNP and Indel markers developed in the early stage of a laboratory through R-QTL professional software, positioning the QTL to the corresponding positions of the linkage group one by one, and estimating the genetic effect of the QTL. If a molecular marker with LOD >2.5 is detected, 1 QTL exists between the 2 markers corresponding to the highest LOD value.
Molecular markers linked with a major QTL for regulating and controlling the rice seed setting rate, wherein the molecular markers comprise Indel ssr-1 and Indel ssr-2;
wherein, the substances for detecting the molecular marker Indel ssr-1 are as follows:
ssr-1-F：5’-CCCGGGACTGATGAAGAAG-3’，SEQ ID NO.1；
ssr-1-R：5’-CTCACCCTTGACTGCAGATTC-3’，SEQ ID NO.2；
the substances for detecting the molecular marker Indel ssr-2 are as follows:
ssr-2-F：5’-TGCCATAGATAACACCACTATGAAA-3’，SEQ ID NO.3；
ssr-2-R：5’-CGCCAAACCATCAAAAATGT-3’，SEQ ID NO.4。
the invention also claims the application of the molecular marker linked with the major QTL for regulating and controlling the rice seed setting rate in breeding rice with high seed setting rate.
The invention also claims a breeding method of the rice with high fruiting rate, which comprises the following steps: extracting rice DNA, carrying out PCR amplification on the DNA by using the detection molecular markers Indel ssr-1 and Indel ssr-2 according to claim 1, carrying out electrophoresis detection on amplified products, and analyzing the rice setting rate by a band type.
In the breeding method, the reaction system of PCR amplification is as follows: 0.5 mu L of upstream primer, 0.5 mu L of downstream primer, 1 mu L of DNA template and 8 mu L of Mix enzyme; the reaction procedure for PCR amplification was: pre-denaturation at 94℃for 10min, denaturation at 94℃for 20s, annealing at 55℃for 30s, extension at 72℃for 10s, amplification for 34 cycles, and final extension at 72℃for 1min.
The invention also claims a breeding kit for rice with high fruiting rate, which comprises the substances of the detection molecular markers Indel ssr-1 and Indel ssr-2.
Compared with the prior art, the invention uses the research means and method of increasingly developed molecular biology and genomics to heat and grind the japonica rice variety as male parent and the indica rice variety Huazhan as female parent to hybridize F 1 120 recombinant inbred line groups obtained after continuous selfing of the generation are used as materials, the encrypted genetic map constructed by the group is utilized to carry out QTL mapping analysis on the data, and a QTL with the LOD value up to 3.47 is detected on chromosome 2, so that a brand-new breakthrough is realized. The molecular marker assisted breeding technology can effectively solve the problem of incomplete knowledge of related genes, can efficiently find the molecular markers closely linked with the main effect QTL of the rice seed setting rate by constructing a genetic linkage map and quantitative trait locus (Quantitative Trait Locus, QTL) analysis, can screen rice offspring by using the markers, and can effectively improve the breeding efficiency while saving the cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing a genetic material construction flow chart used in the major QTL positioning process for regulating and controlling the seed setting rate of rice;
FIG. 2 is a graph showing frequency distribution of the setting percentage of RIL population;
FIG. 3 is a diagram showing the position of the major QTL qSSR2.1 on chromosome 2 for regulating the seed setting rate of rice;
FIG. 4 is a drawing showing the parent and F thereof of a primer pair of the molecular marker Indel ssr-1 1 Generation and RIL population amplification generated electropherograms; wherein 1 is Huazhan, 2 is hot grinding, and 3 is Huazhan/hot grinding hybrid offspring F 1 4-11 are RILs population of Huazhan/hot-grinding hybrid combination;
FIG. 5 is a drawing showing the primer pair of molecular marker Indel ssr-2 in parent and F thereof 1 Generation and RIL population amplification generated electropherograms; wherein 1 is Huazhan, 2 is hot grinding, and 3 is Huazhan/hot grinding hybrid offspring F 1 4-11 are RILs population of Huazhan/hot-grinding hybrid combination;
FIG. 6 is a diagram showing the use of primer pairs of the molecular Indel ssr-1 in the parent and BC thereof 3 F 1 Generating an electrophoresis pattern by amplification in generation; wherein 1 is Huazhan, 2 is Nippon, 3 is Huazhan/Nippon hybrid offspring F 1 BC with 4-10 being Huazhan/Nippon hybrid 3 F 1 A population;
FIG. 7 is a diagram showing the use of primer pairs of molecular Indel ssr-2 in the parent and BC thereof 3 F 1 Generating an electrophoresis pattern by amplification in generation; wherein 1 is Huazhan, 2 is Nippon, 3 is Huazhan/Nippon hybrid offspring F 1 BC with 4-10 being Huazhan/Nippon hybrid 3 F 1 A population.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 major QTL positioning for controlling seed setting Rate in Rice
1. Acquisition of Experimental materials
In terms of HuazhanAs donor parent, local rice variety is hot-ground to acceptor parent, hybrid is performed to construct RILs, and single grain transmission method (i.e., F 1 The bagging single plant is subjected to seed treatment until the phenotype of the offspring plant is not separated, and finally 120 stable inheritance plants (F) 12 All strain phenotypes stable), as in fig. 1.
Selecting parent and seed of each strain (F) 12 ) 100 seeds each, soaking seeds for 2 days after surface sterilization, wrapping with wet towels, and after germination accelerating in a 37 ℃ incubator for 48 hours, selecting seeds with consistent white exposure for sowing. After 30 days, selecting parents with similar growth conditions and 30 seedlings of each strain, transplanting each strain, and planting all rice materials in a Zhejiang university college laboratory test field in Jinhua city of Zhejiang province for routine management.
2. Rice setting rate determination
After the rice is mature, the actual setting rate of each strain of HZ and Nekken2 is measured. After the seeds are ripe, the seeds of each single spike of the population are harvested, the total grain number (containing blighted grains) and blighted grains (without rice empty shells) are counted, the total grain number is reduced by the empty shell number, the total grain number is equal to the solid grain number, and the fruiting rate is calculated by dividing the solid grain number by the total grain number.
As shown in FIG. 2, the rice seed setting rate data are normally distributed and have a wide range, and a plurality of super-parent individuals exist, so that the genetic characteristics of quantitative traits are shown.
3. QTL positioning analysis
And (3) carrying out Quantitative Trait Locus (QTL) interval mapping on the rice seed setting rate by utilizing a genetic map constructed by a large number of SNP (single nucleotide polymorphism) and Indel markers developed in the early stage of a laboratory, analyzing the relation between the markers of the whole chromosome set and quantitative trait phenotype values by using R-QTL professional software, positioning the QTL to the corresponding positions of the linkage group one by one, and estimating the genetic effect of the QTL. If a molecular marker with LOD >2.5 is detected, 1 QTL exists between the 2 markers corresponding to the highest LOD value.
Finally, we found a major QTL located between the Indel ssr-1 marker and the Indel ssr-2 marker on chromosome 2 throughout the whole chromosome set, with a fecundity LOD value as high as 3.47. The genetic distance is 128.62-147.53cM, the physical distance is 30004559 ～ 34415035bp, and the genetic distance is qSSR2.1, as shown in FIG. 3.
Example 2 molecular marker assisted selection
Setting a molecular marker Indel ssr-1 and a molecular marker In del ssr-2 respectively at the upstream and downstream of a QTL locus qSSR2.1, and designing a primer;
the primer pair of the molecular marker Indel ssr-1 is as follows:
the upstream primer 5'-CCCGGGACTGATGAAGAAG-3', SEQ ID NO.1;
the downstream primer 5'-CTCACCCTTGACTGCAGATTC-3', SEQ ID NO.2;
the primer pair of the molecular marker Indel ssr-2 is as follows:
the upstream primer 5'-TGCCATAGATAACACCACTATGAAA-3', SEQ ID NO.3;
the downstream primer 5'-CGCCAAACCATCAAAAATGT-3', SEQ ID No.4.
Taking parent Huazhan, heat grinding and F 1 Extracting genome DNA from rice leaves of the generation and RIL population, and carrying out PCR amplification on the genome DNA by using the molecular markers;
PCR reaction system: 0.5 mu L of upstream primer, 0.5 mu L of downstream primer, 1 mu L of DNA template and 8 mu L of Mix enzyme;
the reaction procedure is: pre-denaturation at 94℃for 10min, denaturation at 94℃for 20s, annealing at 55℃for 30s, extension at 72℃for 10s, amplification for 34 cycles, and final extension at 72℃for 1min.
The PCR amplified products were detected by electrophoresis on a 4% agarose gel, and the partial results are shown in FIGS. 4 and 5.
And analyzing the band type of the electrophoresis detection band, wherein the band tends to the parent Huazhan, so that the rice seed setting rate of the strain is good, and the seed setting rate is poor when the band tends to the hot grinding.
Comparing the seed setting rate of the rice of the tested strain with the result predicted by the band type analysis, and displaying that the predicted result is identical with the actual detection result.
Example 3 application of molecular markers linked to QTL related to rice seed setting Rate in rice breeding
The molecular marker set in the example 2 can be applied to rice molecular assisted breeding, and the specific implementation modes are as follows: mixing other rice varieties with smaller fruiting rate with HuazhanCrossing to obtain corresponding F 1 Backcrossing to BC by taking Japanese sunny as recurrent parent 3 F 1 And (3) replacing. Extraction of BC 3 F 1 And (3) carrying out PCR amplification on the single plant DNA of the generation part, and then using the primers of Indel ssr-1 and Indel ssr-2, and determining whether corresponding marks exist or not through band type analysis, wherein the existence of the marks indicates that the plant has strong capability of regulating and controlling the fruiting rate. By utilizing the method for screening and directional selection, the rice with stronger regulation and control rate and retaining the fine properties of Japanese sun can be obtained, and the breeding efficiency is greatly improved.
The laboratory backcrosses the rice variety Nippon with smaller fruiting rate with Huazhan through the molecular marker, and screens and directionally selects by the method to obtain the rice filial generation with stronger regulating and controlling fruiting rate and retaining the good property of Nippon, and the BC can be seen from the figures 6 and 7 3 F 1 The tendency of the generations to bloom indicates that the superior characters with stronger setting rate are reserved.
In conclusion, the main effect QTL for regulating and controlling the rice seed setting rate can effectively increase the rice regulating and controlling capability, effectively improve the quality and yield of rice in the breeding process, and accelerate the progress of optimizing rice varieties. Meanwhile, rice varieties with higher yield can be cultivated in the rice molecular assisted breeding process, and the rice quality and yield are optimized. The method is simple, convenient, easy, safe and effective, is beneficial to improving the economic value of rice varieties, gives consideration to economic and ecological benefits, and is suitable for large-scale popularization and application
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. The molecular marker is linked with a major QTL for regulating and controlling the rice seed setting rate, and is characterized by comprising Indel ssr-1 and Indel ssr-2;
wherein, the substances for detecting the molecular marker Indel ssr-1 are as follows:
ssr-1-F：5’-CCCGGGACTGATGAAGAAG-3’，SEQ ID NO.1；
ssr-1-R：5’-CTCACCCTTGACTGCAGATTC-3’，SEQ ID NO.2；
the substances for detecting the molecular marker Indel ssr-2 are as follows:
ssr-2-F：5’-TGCCATAGATAACACCACTATGAAA-3’，SEQ ID NO.3；
ssr-2-R：5’-CGCCAAACCATCAAAAATGT-3’，SEQ ID NO.4。
2. the use of the molecular marker linked with the major QTL for regulating and controlling the rice seed setting rate in breeding rice with high seed setting rate according to claim 1.
3. A breeding method of rice with high fruiting rate is characterized in that the process comprises the following steps: extracting rice DNA, carrying out PCR amplification on the DNA by using the detection molecular markers Indel ssr-1 and Indel ssr-2 according to claim 1, carrying out electrophoresis detection on amplified products, and analyzing the rice setting rate by a band type.
4. A method for breeding rice with high fruiting rate according to claim 3, wherein,
the PCR amplification reaction system is as follows: 0.5 mu L of upstream primer, 0.5 mu L of downstream primer, 1 mu L of DNA template and 8 mu L of Mix enzyme;
the reaction procedure for PCR amplification was: pre-denaturation at 94℃for 10min, denaturation at 94℃for 20s, annealing at 55℃for 30s, extension at 72℃for 10s, amplification for 34 cycles, and final extension at 72℃for 1min.
5. A breeding kit for rice with high fruiting rate is characterized by comprising the substances of the detection molecular markers Indel ssr-1 and Indel ssr-2 in claim 1.
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| CN202311680892.XA CN117512188A (en) | 2023-12-08 | 2023-12-08 | Molecular marker linked with major QTL for regulating and controlling rice seed setting rate and application |
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