CN112501341B - Major QTL for regulating heading stage of rice, molecular marker and application - Google Patents
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Abstract
The invention discloses a main effect QTL for regulating and controlling the heading stage of rice, belonging to the technical field of rice breeding and molecular biology, wherein the QTL is positioned on the chromosome 4 of rice, the genetic distance is 146.5-148.3cM, and the physical distance is 34193251, 34596818 bp; and discloses molecular markers closely linked to the QTL. The QTL and the molecular marker thereof are utilized to breed the rice with the suitable heading stage, so that the screening efficiency can be improved, and the yield and income of the rice can be increased.
Description
Technical Field
The invention relates to the technical field of rice breeding and molecular biology, in particular to a major QTL for regulating and controlling the heading stage of rice, a molecular marker and application.
Background
Rice (Oryza sativa.L) is one of important grain crops in the world, and the heading stage is one of important agronomic traits of the rice, so that the rice has very important significance for ensuring the stable yield and the high yield of the rice. The proper heading period determines the regional adaptability and seasonal adaptability of a rice variety. Most of genes for controlling the heading stage are quantitative genes, the phenotype is greatly influenced by the environment, and the rice yield is reduced due to frequent occurrence of extreme weather in recent years, so that the research on the genes related to the heading stage of the rice plays an important role in rice breeding.
The molecular mechanism influencing the heading stage of rice is different in a plurality of reports, but more and more researches show that the regulation and control of the heading stage of rice is a complex process involving multiple genes. The molecular marker assisted breeding technology can effectively solve the problem of incomplete knowledge of related genes at the heading stage of rice, finds molecular markers closely linked with main effect QTL related to the heading stage by constructing a genetic linkage map and Quantitative Trait Loci (QTL) analysis, and can screen rice progeny by using the markers, thereby saving cost and improving breeding efficiency.
At present, researchers have limited research on the fine positioning of QTL sites at the heading stage of rice and related molecular markers; therefore, the QTL sites and related molecular markers of the rice heading stage need to be further and deeply excavated and analyzed, so as to provide a new choice for screening and identifying rice varieties with suitable heading stages.
Disclosure of Invention
In view of the above, the invention provides the main effect QTL for regulating and controlling the heading stage of the rice and the molecular marker tightly linked with the main effect QTL, which is used for breeding the rice with the suitable heading stage and can improve the screening efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method takes japonica rice variety heat-grinding No.2 as a female parent and indica rice variety Huazhan as a male parent for hybridization, takes a recombinant inbred line population obtained after continuous inbreeding of F1 generations as a material, carries out statistics and analysis on the heading stage of rice, simultaneously carries out QTL mapping analysis on data by utilizing an encrypted genetic map constructed by the population, finds out a major QTL with an LOD value as high as 5.75 on chromosome 4, and is named as HD-1, the genetic distance is 146.5-148.3c M, and the physical distance is 34193251-34818 bp.
The main effect QTL for regulating and controlling the heading stage of the rice can be applied to breeding of rice varieties, and by developing a molecular marker tightly linked with the main effect QTL, the QTL related to the heading stage in the detected rice varieties or strains in the rice varieties or strains can be detected, so that the breeding process of the rice with a proper heading stage can be accelerated.
Further, the major QTL is positioned between the molecular marker Indel hd-1 and the molecular marker Indel hd-2;
the primer pair of the molecular marker Indel hd-1 is as follows:
an upstream primer: 5'-GGTCAAACGTTGAGCATGAA-3', SEQ ID NO. 1;
a downstream primer: 5'-CACAAGTGGAGCGTGCTTTA-3', SEQ ID NO. 2;
the primer pair of the molecular marker Indel hd-2 is as follows:
an upstream primer: 5'-TGTAGCTGGTGAGGACTGGA-3', SEQ ID NO. 3;
a downstream primer: 5'-GGCGAAGAAAACGCTGGTA-3', SEQ ID NO. 4.
The molecular marker Indel hd-1 and the molecular marker Indel hd-2 are molecular markers closely linked with the main effect QTL of the heading stage of the rice, and the heading stage of rice plants can be predicted through the detection of the molecular markers, so that the breeding progress of breeding the rice with the proper heading stage is accelerated.
Further, the invention provides a rice breeding method, which comprises the following steps: extracting rice DNA, carrying out PCR amplification on the DNA by using the primer pair of the molecular marker, carrying out electrophoresis detection on an amplification product, and analyzing the heading stage of the rice by banding.
The method can be used for screening the rice with the suitable heading stage and identifying the molecular of the rice germplasm resources.
Preferably, the reaction system for PCR amplification is: 1 uL of upstream primer, 1 uL of downstream primer, 2 uL of DNA template, 6 uL of mix enzyme, ddH2O 1μL；
The reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, and amplification for 38 cycles; final extension at 72 ℃ for 10 min.
Furthermore, the primer pair of the molecular marker can be used for preparing a rice breeding kit.
In conclusion, the invention positions the main effect QTL HD-1 for regulating the heading stage of the rice, 2 pairs of molecular markers which are closely linked with the QTL locus are obtained by applying the QTL locus, the heading stage of the rice material can be predicted by utilizing the molecular markers, and the breeding of the ideal plant type of the rice is accelerated.
Drawings
FIG. 1 is a flow chart showing the construction of genetic materials used in the process of regulating the location of a major QTL at the heading stage of rice;
FIG. 2 is a graph showing the frequency profile of the heading date of the RIL population;
wherein RY represents the rice variety Hongshan No.2, and HZ represents the rice variety Huazhan;
FIG. 3 shows the position of major QTL HD-1 on chromosome 4 for regulation of heading date of rice;
FIG. 4 shows the electrophoretogram generated by the amplification of primer pair of molecular marker Indel hd-1 in the parent and its F1 generation and RIL population;
wherein, 1 is Huazhan, 2 is Rehan No.2, 3 is the filial generation F1 of Rehan No. 2/Huazhan (heading time is biased to Rehan No. 2), and 4-12 is the rice line material with earlier heading in RIL population of the combination of the Rehan No. 2/Huazhan.
FIG. 5 shows the electrophoretogram generated by the amplification of the primer pair of the molecular marker Indel hd-2 in the parent and its F1 generation and RIL population;
wherein, 1 is Huazhan, 2 is Hot Zhen No.2, 3 is the hot Zhen No. 2/Huazhan filial generation F1, and 4-12 is the rice line material with earlier heading in the RIL group of the hot Zhen No. 2/Huazhan hybridization combination.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Regulation and control of major QTL location at heading date of Rice
1. Acquisition of test materials
Huazhan is taken as a donor parent, a rice variety Hongshan No.2 is taken as a receptor parent for hybridization, and a single event transmission method (namely, F1 is subjected to bagging single plant seed receiving treatment until phenotype of a progeny strain is not separated) is utilized to finally obtain 134 strains with stable inheritance (F13, all strains have stable phenotypes) to form a recombinant inbred line RIL group, as shown in figure 1.
Selecting 60 seeds of each parent and each strain (F13), soaking the seeds for 2 days after surface disinfection, changing water every other day, wrapping the seeds with a wet towel, putting the wrapped seeds in a constant temperature box at 37 ℃ for accelerating germination for 2 days, ensuring that the towel is in a wet state midway, and selecting the seeds with consistent exposure and whiteness for sowing. After 30 days, 24 seedlings of each plant line and parents with similar growth conditions are selected and transplanted, and all the rice materials are planted in test fields of biochemical academy colleges of Zhejiang university in Jinhua city, Zhejiang province and are managed conventionally.
2. Heading date statistics
Investigating the heading time of each individual plant of the parent, the F1 generation plant and the F2 generation population, and recording the result; the heading time is the days from sowing to 1cm heading of the rice.
As shown in FIG. 2, the heading date data showed a continuous normal distribution and a wide range, wherein individuals with an early heading of Homophorus H.sub.2, a late heading of Huazhang, and more heading dates earlier than Homophorus H.sub.2 or later than Huazhang existed.
QTL localization analysis
The Quantitative Trait Locus (QTL) interval mapping is carried out on the rice heading stage by utilizing a genetic map constructed by a large number of SNP and Indel markers developed in the early stage of a laboratory, the relation between the markers of the whole chromosome set and the quantitative trait phenotype values is analyzed through R-QTL professional software, the QTL is positioned to the corresponding positions of the linkage group one by one, and the genetic effect of the linkage group is estimated. If the detected LOD >2.5 molecular marker, then the 2 markers corresponding to the highest LOD value are considered to have 1 QTL.
Finally, a major QTL between the Indel HD-1 marker and the Indel HD-2 marker on chromosome 4 was found in the entire genome of rice hot-research No.2 with LOD values as high as 5.75, genetic distances of 146.5-148.3cM, physical distances of 34193251-.
Example 2 molecular marker assisted selection
Setting a molecular marker Indel HD-1 and a molecular marker Indel HD-2 respectively at the upstream and downstream of the QTL locus HD-1, and designing primers;
the primer pair of the molecular marker Indel hd-1 is as follows:
an upstream primer: 5'-GGTCAAACGTTGAGCATGAA-3', SEQ ID NO. 1;
a downstream primer: 5'-CACAAGTGGAGCGTGCTTTA-3', SEQ ID NO. 2;
the primer pair of the molecular marker Indel hd-2 is as follows:
an upstream primer: 5'-TGTAGCTGGTGAGGACTGGA-3', SEQ ID NO. 3;
a downstream primer: 5'-GGCGAAGAAAACGCTGGTA-3', SEQ ID NO. 4.
Taking parental hot grinding No.2, Huazhan and F1 generation thereof and rice leaves of RIL group, extracting genome DNA, and carrying out PCR amplification on the genome DNA by using the molecular marker;
and (3) PCR reaction system: 1 μ L of upstream primer (10 μmol), 1 μ L of downstream primer (10 μmol), 2 μ L of DNA template (> 100ng/μ L), 6 μ L of Mix enzyme (2 XTAQA Master Mix), ddH2O 1μL；
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, and amplification for 38 cycles; final extension at 72 ℃ for 10 min.
The PCR amplification products were detected by electrophoresis on a 5% agarose gel, and the results are shown in FIGS. 4 to 5.
And analyzing the electrophoresis detection strip type, wherein the strip tends to the parental hot grinding No.2, which indicates that the heading stage of the rice of the line is earlier, and if the strip tends to Huazhan, the heading stage is later.
And comparing the heading time of the tested strain rice with the result predicted by the banding pattern analysis, and displaying that the predicted result is matched with the actual statistical result.
Example 3 application of rice heading stage-related QTL in rice breeding
Hybridizing the rice variety 9311 with later heading stage as male parent and the hot grinding No.2 as female parent to obtain corresponding F1, and hybridizing the corresponding F1 with 9311 backcrossing to BC for recurrent parent3F1And (4) generation. Extraction of BC3F1The DNA of a part of the individual strain is generated, and then PCR amplification is carried out by using primers of Indel hd-1 and Indel hd-2, and electrophoresis detection is carried out.
And analyzing the band type of the electrophoresis detection, wherein the band tends to the parental hot grinding No.2, which indicates that the heading period of the rice of the line is earlier. The method is used for screening and directional selection, so that the rice with early heading stage and 9311 excellent characters can be obtained, and the breeding efficiency is greatly improved.
In conclusion, the main effect QTL for regulating and controlling the heading stage of the rice can effectively accelerate the process of optimizing rice varieties. The rice with proper heading stage can be cultivated in the rice molecular assisted breeding process. The method is simple, convenient, feasible, safe and effective, is beneficial to improving the economic value of the rice variety, gives consideration to economic and ecological benefits, and is suitable for large-scale popularization and application.
The general principles defined herein may be implemented in 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.
Sequence listing
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Claims (5)
1. A molecular marker closely linked with a main effect QTL for regulating and controlling the heading stage of rice, which is characterized in that,
comprises a molecular marker Indel hd-1 and a molecular marker Indel hd-2;
the primer pair of the molecular marker Indel hd-1 is as follows:
an upstream primer: 5'-GGTCAAACGTTGAGCATGAA-3', SEQ ID NO. 1;
a downstream primer: 5'-CACAAGTGGAGCGTGCTTTA-3', SEQ ID NO. 2;
the primer pair of the molecular marker Indel hd-2 is as follows:
an upstream primer: 5'-TGTAGCTGGTGAGGACTGGA-3', SEQ ID NO. 3;
a downstream primer: 5'-GGCGAAGAAAACGCTGGTA-3', SEQ ID NO. 4.
2. The use of the molecular marker of claim 1 in rice variety breeding,
and breeding the rice with the proper heading stage by the molecular marker.
3. A method for breeding rice is characterized in that,
extracting rice DNA, carrying out PCR amplification on the DNA by using the primer pair with the molecular marker according to claim 1, carrying out electrophoresis detection on an amplification product, and analyzing the heading stage of the rice through banding.
4. The method for breeding rice as claimed in claim 3,
the reaction system of PCR amplification is: 1 uL of upstream primer, 1 uL of downstream primer, 2 uL of DNA template, 6 uL of mix enzyme, ddH2O 1μL；
The reaction procedure for PCR amplification was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, and amplification for 38 cycles; final extension for 10min at 72 ℃.
5. A kit for breeding rice is characterized in that,
a primer pair comprising the molecular marker of claim 1.
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