CN117625832A - Molecular marker linked with major QTL for regulating thousand grain weight of rice and application - Google Patents

Molecular marker linked with major QTL for regulating thousand grain weight of rice and application Download PDF

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CN117625832A
CN117625832A CN202311680727.4A CN202311680727A CN117625832A CN 117625832 A CN117625832 A CN 117625832A CN 202311680727 A CN202311680727 A CN 202311680727A CN 117625832 A CN117625832 A CN 117625832A
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tgw
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饶玉春
朱超宇
朱哲楠
王跃星
杨惠敏
蔡依林
黄佳慧
刘之陶
汪理海
张芷宁
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Zhejiang Normal University CJNU
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Abstract

The invention discloses a molecular marker linked with a major QTL for regulating and controlling the thousand seed weight of rice, and relates to the technical fields of molecular marker screening and rice breeding of the major QTL linked with the thousand seed weight of rice, wherein the molecular markers comprise Indel tgw-1 and Indel tgw-2; wherein, the primer pair for amplifying Indel tgw-1 is shown in SEQ ID NO. 1-SEQ ID NO.2; the primer pair for amplifying Indel tgw-2 is shown as SEQ ID NO. 3-SEQ ID NO. 4; the molecular marker is applied to breeding high thousand grain rice, can screen rice offspring, and greatly improves the breeding efficiency of high thousand grain rice while saving cost.

Description

Molecular marker linked with major QTL for regulating thousand grain weight of rice and application
Technical Field
The invention relates to the technical field of molecular marker screening and rice breeding of rice thousand-grain weight major QTL linkage, in particular to a molecular marker linked with a rice thousand-grain weight major QTL and application thereof.
Background
The rice yield is composed of the effective spike number, the solid grain number and thousand grain weight of each plant, wherein the thousand grain weight has direct influence on the yield. Therefore, the genetic control mechanism of thousand-grain weight characters is deeply analyzed, and high-grain-weight crops are cultivated, so that a solid theoretical basis and available gene resources are laid for rice high-yield breeding.
The effective spike number, the spike grain number and the grain weight jointly determine the yield of the rice. Thousand grain weight is one of the key factors affecting rice yield and quality, is closely related to grain type, and is a quantitative trait controlled by multiple genes. And the frequent hazards of drought, soil salinization, heavy metal pollution and the like and the gradual increase of the demand of people on rice form contradictions. Therefore, the cultivation and popularization of rice varieties with higher thousand grain weight have important significance for ensuring the rice to keep high yield under the current contradictory background, are one of the fundamental measures for solving the problem of rice yield reduction caused by the problems of available land area reduction and the like, can improve the economic benefit of planting and protect the grain safety of China.
To date, a great deal of research on genetic control mechanisms of thousand-grain weight traits of rice has been carried out by the former, and researchers have found 315 major QTLs affecting thousand-grain weight on 12 chromosomes of rice, and have successfully cloned some genes involved in thousand-grain weight control, such as qTGW2, TGW6, xia and the like. Although many thousand kernel weight QTLs are located, complex quantitative traits of rice, which are controlled by multiple genes, are now discovered, and many studies have found genes that can alter thousand kernel weight, it is not necessarily possible to directly increase yield by thousand kernel weight modulation by these genes alone. Therefore, in improving thousand seed weight of rice and increasing yield, it is necessary to find more thousand seed weight related QTLs and to understand their functions and corresponding regulatory mechanisms in depth. Lays a foundation for further improving thousand seed weight characteristics, increasing single yield and cultivating high-yield and high-quality varieties.
At present, the research on thousand seed weight genetic mechanism of rice is relatively few. Therefore, thousand-grain weight QTL localization and gene cloning research still need further deep excavation and analysis, and new breakthrough is expected to be realized by means of research means and methods of increasingly developed molecular biology and genomics.
Therefore, how to attach and regulate the major QTL of thousand grain weight of rice to locate and screen the molecular markers linked with the major QTL is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a molecular marker linked with a major QTL for regulating and controlling thousand seed weight of rice and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a positioning method of a major QTL of thousand grain weight of rice, which comprises the following steps:
1) Hybridization is carried out by taking hot grinding and Huazhan as parents, 120 stable inheritance strains are obtained by a single-grain transmission method, and RILs are formed together;
2) The thousand seed weight 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 the molecular marker with LOD >2.5 is detected, 1 QTL exists among the 2 markers corresponding to the highest LOD value, the corresponding marker is named qTGW2.1, the main effect QTL is positioned on chromosome 2, the genetic distance is 40.42-90.16cM, the physical distance is 9429264-21032630 bp, and the LOD value reaches 5.28. By developing the molecular marker closely linked with the major QTL, whether the rice variety or strain has the thousand grain weight related QTL or not is detected, and the breeding process of the rice excellent seed can be accelerated.
Based on the positioned major QTL, the invention also requests to protect a molecular marker linked with the major QTL for regulating and controlling the thousand grain weight of the rice, wherein the molecular marker comprises Indel tgw-1 and Indel tgw-2; wherein,
the primer pair of the molecular marker Indel tgw-1 is as follows:
the upstream primer tgw-1-F:5'-GTACGTCACTTGCTGCATGG-3', SEQ ID NO.1;
the downstream primer tgw-1-R:5'-TGGCTCACCACTTGTTGGTA-3', SEQ ID No.2;
the primer pair of the molecular marker Indel tgw-2 is as follows:
the upstream primer tgw-2-F:5'-TCCATGCTTATGGCTCCAGT-3', SEQ ID No.3;
the downstream primer tgw-2-R:5'-GGGCCAAATTTCGATTTTCT-3', SEQ ID No.4.
As the invention concept same as the technical scheme, the invention also claims the application of the molecular marker in breeding high thousand grain weight rice.
As the invention conception same as the technical scheme, the invention also claims a breeding method of high thousand grain weight rice, which comprises the following steps:
extracting rice DNA, performing P CR amplification on the DNA by using the primer pair with molecular markers as set forth in claim 4, performing electrophoresis detection on the amplified product, and analyzing the thousand grain weight of the rice by using a band type.
Preferably, the reaction system for PCR amplification is: 0.75 mu L of upstream primer, 0.75 mu L of downstream primer, 1 mu L of DNA template, 12.5 mu L of Mix enzyme and ddH 2 O10μL；
The reaction procedure for PCR amplification was: pre-denaturation at 94℃for 3min, denaturation at 94℃for 20s, annealing at 57℃for 30s, extension at 72℃for 5s, amplification for 35 cycles, and final extension at 72℃for 1min.
The invention also claims a high thousand grain weight rice breeding kit, which comprises the primer pair of the molecular marker.
Compared with the prior art, the invention adopts the hybridization F which is the indica rice variety Huazhan male parent and the japonica rice variety which is hot ground as female parent 1 120 recombinant inbred line groups obtained after continuous selfing of the generation are used as materials, the data are subjected to QTL mapping analysis by utilizing the established encrypted genetic map of the groups, and a QTL with the LOD value as high as 5.28 is detected. The molecular marker assisted breeding technology can effectively solve the problem of incomplete knowledge of related genes, can effectively find out the molecular markers closely linked with the major QTL related to thousand seed weight by constructing a genetic linkage map and quantitative trait locus (Quantitative Trait Locus, QTL) analysis, can screen rice offspring by using the markers, and greatly improves the breeding efficiency while saving the cost.
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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 a major QTL positioning process for regulating thousand kernel weight of rice;
FIG. 2 is a graph showing the frequency distribution of thousand kernel weight of RIL population;
FIG. 3 is a diagram showing the position of the major QTL qTGW2.1 for regulating thousand kernel weight of rice on chromosome 2;
FIG. 4 is a diagram showing the parent and F thereof of a primer pair of the molecular marker Indel tgw-1 1 Generation and RIL populations. Wherein 1 is Huazhan, 2 is hot grinding, and 3 is Huazhan/hot grinding hybrid offspring F 1 4-12 are RILs population of Huazhan/hot-grinding hybrid combination;
FIG. 5 is a diagram showing the parent and F thereof of a primer pair of the molecular marker Indel tgw2 1 Generation and RIL populations. Wherein, 1 is Huazhan, 2 is heat grinding, and 3 is Huazhan/heat grinding hybridizationOffspring F 1 4-12 are RILs populations of Huazhan/Hot-ground hybridization combinations.
FIG. 6 is a diagram showing the use of primer pairs of the molecule Indel tgw-1 in the parent and BC thereof 3 F 1 Generating an electrophoresis pattern by amplification in generation; wherein 1 is hot grinding, 2 is 9311,3 is hot grinding/9311 filial generation F 1 BC with hot-milling/9311 hybridization at 4-10 3 F 1 A population;
FIG. 7 is a diagram showing the use of primer pairs of the molecule Indel tgw-2 in the parent and BC thereof 3 F 1 Generating an electrophoresis pattern by amplification in generation; wherein 1 is hot grinding, 2 is 9311,3 is hot grinding/9311 filial generation F 1 BC with hot-milling/9311 hybridization at 4-10 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 regulating thousand seed weight of Rice
1. Acquisition of Experimental materials
Hybrid construction of RILs using a Huazhan donor parent and a local rice variety hot-ground as an acceptor parent, using a 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 thousand grain weight measurement
After the rice is mature, the thousand grain weights of HZ, nekken2 and each strain are measured. And after the seeds of each individual plant of the colony are harvested after the seeds are ripe, selecting 1000 full seeds of each individual plant for each material, mixing, and counting the thousand seed weight of the seeds by an electronic particle counter and an electronic balance.
As shown in FIG. 2, the thousand grain weight data of rice 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 thousand kernel weight of the rice by utilizing a genetic map constructed by a large number of SNP 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 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.
Finally we found a major QTL located between the Indel tgw-1 and Indel tgw-2 markers on chromosome 3 in the whole chromosome set, with thousand-grain-weight LOD values as high as 5.28. The genetic distance is 40.42-90.16cM, the physical distance is 9429264 ～ 21032980bp, and the genetic distance is qTGW2.1, as shown in FIG. 3.
Example 2 molecular marker assisted selection
Setting a molecular marker Indel tgw-1 and a molecular marker Indel tgw-2 respectively at the upstream and downstream of the QTL locus qTGW2.1, and designing a primer;
the primer pair of the molecular marker Indel tgw-1 is as follows:
the upstream primer 5'-GTACGTCACTTGCTGCATGG-3', SEQ ID NO.1;
the downstream primer 5'-TGGCTCACCACTTGTTGGTA-3', SEQ ID NO.2;
the primer pair of the molecular marker Indel tgw-2 is as follows:
the upstream primer 5'-TCCATGCTTATGGCTCCAGT-3', SEQ ID NO.3;
the downstream primer 5'-GGGCCAAATTTCGATTTTCT-3', SEQ ID No.4.
Parent hot grinding, hua Zhan 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: 0.75. Mu.L of upstream primer, 0.75. Mu.L of downstream primer, 1. Mu.L of DNA template, 12.5. Mu.L of Mix enzyme, ddH 2 O10μL；
The reaction procedure is: pre-denaturation at 94℃for 3min, denaturation at 94℃for 20s, annealing at 57℃for 30s, extension at 72℃for 5s, amplification for 35 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 thousand grain weight of the rice of the strain is poor, and if the band tends to the hot grinding, the thousand grain weight is good.
Comparing thousand grain weight of the rice of the tested strain with the result predicted by band type analysis, and displaying that the predicted result is identical with the actual detection result.
Example 3 application of Rice thousand seed weight related QTL 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: hybridization of other thousand-grain rice varieties with smaller weight, such as 9311, with hot grinding to obtain corresponding F 1 Backcrossing to BC by taking 9311 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 tgw-1 and Indel tgw-2 to determine whether corresponding markers exist or not through band type analysis, wherein the presence of the markers indicates that the thousand seed weight regulation capability of the plant is stronger. By utilizing the method for screening and directional selection, the rice with strong thousand grain weight regulation and control and kept 9311 excellent properties can be obtained, and the breeding efficiency is greatly improved.
The laboratory backcrosses the rice variety 9311 with small thousand grain weight with hot grinding through the molecular marker, and uses the method to screen and directionally select to obtain the rice filial generation with strong thousand grain weight and kept 9311 excellent character, and the method is used for the breeding of the riceAs can be seen from fig. 6 and 7, BC 3 F 1 The generation of the Chinese herbal medicine is biased to be hot-ground, which shows that the excellent characters of strong thousand grain weight are reserved.
In conclusion, the main effect QTL for regulating and controlling the thousand grain weight of the rice can effectively increase the regulation capability of the rice, effectively improve the quality and yield of the rice in the breeding process, and accelerate the progress of optimizing the rice variety. 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 to implement, safe and effective, is beneficial to improving the economic value of rice varieties, has both 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 thousand grain weight of rice, and is characterized by comprising Indel tgw-1 and Indel tgw-2; wherein,
the primer pair of the molecular marker Indel tgw-1 is as follows:
the upstream primer tgw-1-F:5'-GTACGTCACTTGCTGCATGG-3', SEQ ID NO.1;
the downstream primer tgw-1-R:5'-TGGCTCACCACTTGTTGGTA-3', SEQ ID No.2;
the primer pair of the molecular marker Indel tgw-2 is as follows:
the upstream primer tgw-2-F:5'-TCCATGCTTATGGCTCCAGT-3', SEQ ID No.3;
the downstream primer tgw-2-R:5'-GGGCCAAATTTCGATTTTCT-3', SEQ ID No.4.
2. The use of the molecular marker of claim 1 in breeding high thousand seed weight rice.
3. A breeding method of high thousand grain weight rice is characterized by comprising the following steps:
extracting rice DNA, carrying out PCR amplification on the DNA by using the primer pair of the molecular marker as claimed in claim 4, carrying out electrophoresis detection on an amplified product, and analyzing the thousand grain weight of the rice by a band type.
4. A method for breeding high thousand seed weight rice according to claim 3, wherein,
the PCR amplification reaction system is as follows: 0.75 mu L of upstream primer, 0.75 mu L of downstream primer, 1 mu L of DNA template, 12.5 mu L of Mix enzyme and ddH 2 O10μL；
The reaction procedure for PCR amplification was: pre-denaturation at 94℃for 3min, denaturation at 94℃for 20s, annealing at 57℃for 30s, extension at 72℃for 5s, amplification for 35 cycles, and final extension at 72℃for 1min.
5. A high thousand grain weight rice breeding kit, comprising the molecular marker primer pair of claim 1.
CN202311680727.4A 2023-12-08 2023-12-08 Molecular marker linked with major QTL for regulating thousand grain weight of rice and application Pending CN117625832A (en)

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