CN117512188A - Molecular marker linked with major QTL for regulating and controlling rice seed setting rate and application - Google Patents

Abstract

The invention discloses a molecular marker linked to the main QTL for regulating rice seed setting rate, and relates to the technical field of screening and application of rice seed setting rate molecular markers. The molecular markers include Indel ssr-1 and Indel ss r-2; the detection molecular marker Indel ssr The substances of ‑1 are shown in SEQ ID NO.1～SEQ ID NO.2; the substances of detection molecular marker Indel ssr‑2 are shown in SEQ ID NO.3～SEQ ID NO.4; the molecular markers of the present invention are used in Assisted breeding technology can effectively solve the problem of incomplete understanding of related genes. By constructing genetic linkage maps and analyzing quantitative trait loci, molecular markers that are closely linked to the main QTL of rice seed setting rate can be efficiently found. Using these markers can Screening rice progeny can effectively improve breeding efficiency while saving costs.

Description

Molecular markers linked to QTL that regulates rice seed setting rate and their applications

Technical field

The present invention relates to the technical field of screening and application of molecular markers for rice seed setting rate, and more specifically to molecular markers linked to the main QTL for regulating rice seed setting rate and their applications.

Background technique

Since the domestication of rice, yield-related traits have been the most important target for breeders and one of the most important indicators to measure the excellence of rice varieties. Rice yield is mainly directly determined by three important factors: the number of panicles per unit area, the number of solid grains per panicle and the grain weight. The number of solid grains per panicle is jointly determined by the total number of grains per panicle and the seed setting rate, and is one of the yield components. The traits with the greatest variation and the most easily affected by cultivation practices and the environment. Therefore, the excavation of the main effect locus of rice seed setting rate and the development of related technologies will have a positive effect on the stability and improvement of rice yield. At the same time, it can be used as one of the fundamental measures to alleviate food security problems and help solve potential problems caused by global environmental problems. Hidden danger.

Relevant research on rice seed setting rate needs to be based on the molecular mechanism and regulatory mechanism of yield trait formation, and is more based on the mapping and cloning of related QTL. With the help of modern bioinformatics technology, scientific researchers are currently exploring the molecular mechanism and regulatory rules of rice seed setting rate and have achieved preliminary results. Related research mainly focuses on transcription factors and protein kinase pathways. Meina et al. studied the MYB domain-containing transcription factors OsPHR1 and OsPHR2. If they are overexpressed, plants will show a decrease in seed setting rate under high phosphorus conditions; Ruifang et al. found that the cyclin-dependent kinase inhibitor OsiICK6 can Interacts with cell cycle proteins OsCYCD and CDKA and is induced by low temperature, ABA and osmotic stress. When overexpressed, the pollen viability and seed setting rate of plants are reduced. At present, most of the rice seed-setting rate-related genes cloned by scientific researchers indirectly affect the seed-setting rate by affecting rice flower development. Such genes have little value in rice production and have limited application pathways. In order to find the main genes that maintain high seed setting rate, the research will conduct complementary verification and functional analysis of some important QTL through map-based cloning and other methods, optimize the selection and combination of rice parent materials, and further promote rice breeding. practice.

In the current scientific research field, there are relatively few studies on the main QTL mapping and genetic mechanism of rice seed setting rate, and related QTL mapping and gene cloning research still requires further in-depth excavation and analysis.

Therefore, how to provide a major QTL related to rice seed setting rate and apply it in rice breeding is an urgent technical problem that those skilled in the art need to solve.

Contents of the invention

In view of this, the present invention provides major QTL sites that regulate rice seed setting rate, molecular markers linked to them, and their applications.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The main QTL site that regulates rice seed setting rate is located on rice chromosome 2, with a genetic distance of 128.62-147.53cM and a physical distance of 30004559-34415035bp; the main QTL is located at the molecular marker Indel ssr-1 and the molecular marker Indel ssr -2;

The substance for detecting the molecular marker Indel ssr-1 is:

Upstream primer: 5’-CCCGGGACTGATGAAGAAG-3’, SEQ ID NO.1;

Downstream primer: 5’-CTCACCCTTGACTGCAGATTC-3’, SEQ ID NO.2;

The substances used to detect the molecular marker Indel ssr-2 are:

Upstream primer: 5’-TGCCATGATAACACCACTATGAAA-3’, SEQ ID NO.3;

Downstream primer: 5’-CGCCAAACCATCAAAAATGT-3’, SEQ ID NO.4.

The present invention also provides a method for locating major QTL sites that regulate rice seed setting rate, including the following steps: 1) Crossing Huazhan and Reyan as parents, and obtaining 120 stable genetic lines through single-grain transmission. , together form the RILs population; 2) Examine the seed setting rate of the recombinant inbred line population at maturity; 3) Use the genetic map constructed by a large number of SNPs and Indel markers previously developed in the laboratory to analyze the genetic pattern of the entire chromosome set through R-QTL professional software Based on the relationship between markers and phenotypic values of quantitative traits, QTLs were mapped to the corresponding positions in the linkage group one by one, and their genetic effects were estimated. If a molecular marker with LOD>2.5 is detected, it is considered that there is a QTL between the two markers corresponding to the highest LOD value.

Molecular markers linked to the main QTL that regulates rice seed setting rate, the molecular markers include Indel ssr-1 and Indel ssr-2;

Among them, the substance for detecting the molecular marker Indel ssr-1 is:

ssr-1-F: 5’-CCCGGGACTGATGAAGAAG-3’, SEQ ID NO.1;

ssr-1-R: 5’-CTCACCCTTGACTGCAGATTC-3’, SEQ ID NO.2;

The substances used to detect the molecular marker Indel ssr-2 are:

ssr-2-F: 5’-TGCCATGATAACACCACTATGAAA-3’, SEQ ID NO.3;

ssr-2-R: 5’-CGCCAAACCATCAAAAATGT-3’, SEQ ID NO.4.

The present invention also claims the application of the molecular markers linked to the main QTL for regulating rice seed setting rate in the breeding of rice with high seed setting rate.

The present invention also claims a method for breeding rice with high seed setting rate. The process includes: extracting rice DNA, and using the detection molecular markers Indel ssr-1 and Indel ssr-2 as claimed in claim 1 to amplify the DNA by PCR. The amplified product was detected by electrophoresis, and the rice seed setting rate was analyzed by banding pattern.

In the above breeding method, the reaction system of PCR amplification is: 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of DNA template, and 8 μL of Mix enzyme; the reaction program of PCR amplification is: pre-denaturation at 94°C for 10 min, denaturation at 94°C 20 s, annealing at 55°C for 30 s, extension at 72°C for 10 s, amplification for 34 cycles, and final extension at 72°C for 1 min.

The present invention also claims a breeding kit for rice with high seed setting rate, including the substances for detecting molecular markers Indel ssr-1 and Indel ssr-2.

It can be seen from the above technical solutions that compared with the existing technology, the present invention relies on the increasingly developed research means and methods of molecular biology and genomics to hybridize the japonica rice variety Reyan as the male parent and the indica rice variety Huazhan as the female parent. A population of 120 recombinant inbred lines obtained after continuous self-crossing of the F ₁ generation was used as the material. The encrypted genetic map constructed by the population was used to conduct QTL mapping analysis on the data. A QTL with an LOD value as high as 3.47 was detected on chromosome 2. , achieving new breakthroughs. The molecular marker-assisted breeding technology of the present invention can effectively solve the problem of incomplete understanding of related genes. By constructing a genetic linkage map and analyzing quantitative trait loci (Quantitative Trait Locus, QTL), it can efficiently find the main QTL related to rice seed setting rate. Tightly linked molecular markers can be used to screen rice offspring, which can effectively improve breeding efficiency while saving costs.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without exerting creative efforts.

Figure 1 is a flowchart showing the genetic material construction process used in the process of mapping the main QTL that regulates rice seed setting rate;

Figure 2 is a frequency distribution diagram of the seed setting rate of the RIL population;

Figure 3 attached shows the location of the main QTL qSSR2.1 that regulates rice seed setting rate on chromosome 2;

Figure 4. The accompanying picture shows the electrophoresis pattern produced by the primer pair of the molecular marker Indel ssr-1 amplified in the parents, their F ₁ generation and the RIL population; among them, 1 is Huazhan, 2 is Reyan, and 3 is Huazhan/Reyan. Yan hybrid progeny F ₁ and 4-11 are the RILs population of the Huazhan/Reyan hybrid combination;

Figure 5. The attached picture shows the electrophoresis pattern produced by the primer pair of the molecular marker Indel ssr-2 amplified in the parents, their F ₁ generation and the RIL population; among them, 1 is Huazhan, 2 is Reyan, and 3 is Huazhan/Reyan. Yan hybrid progeny F ₁ and 4-11 are the RILs population of the Huazhan/Reyan hybrid combination;

Figure 6. The attached picture shows the electrophoresis pattern produced by using the primer pair of the molecule Indel ssr-1 to amplify the parent and its BC ₃ F ₁ generation; 1 is Huazhan, 2 is Nipponbare, and 3 is the Huazhan/Nipponbare hybrid progeny F. ₁ and 4-10 are the BC ₃ F ₁ population of Huazhan/Nipponbare hybrid;

Figure 7 The attached picture shows the electrophoresis pattern produced by using the primer pair of the molecule Indel ssr-2 to amplify the parent and its BC ₃ F ₁ generation; 1 is Huazhan, 2 is Nipponbare, and 3 is the F hybrid progeny of Huazhan/Nipponbare. ₁ and 4-10 are the BC ₃ F ₁ population of Huazhan/Nipponbare hybrid.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Example 1 Mapping of major QTL regulating rice seed setting rate

1. Acquisition of experimental materials

Using Huazhan as the donor parent and the local rice variety Reyan as the recipient parent, hybridization was carried out to construct RILs, and the single-grain transmission method was used (i.e., F ₁ was bagged and single plants were inoculated until the phenotype of the offspring lines was no longer the same). segregation occurred), and finally 120 stable genetic strains (F ₁₂ , all strains were phenotypically stable) were obtained, as shown in Figure 1.

Select 100 seeds each from the parent and each strain (F ₁₂ ). After surface disinfection, soak the seeds for 2 days, then wrap them in a moist towel, place them in a 37°C incubator for 48 hours to germinate, and then select seeds with consistent white color for sowing. After 30 days, parents with similar growth conditions and 30 seedlings of each strain were selected and transplanted. All rice materials were planted in the experimental field of the College of Chemistry, Zhejiang Normal University, Jinhua City, Zhejiang Province, and were managed routinely.

2. Determination of rice seed setting rate

After the rice matured, HZ, Nekken2 and the seed setting rate of each line were measured. After maturity, harvest the seeds of each single ear of the group, count the total number of grains (including grains) and grains (empty shells without rice), subtract the number of empty shells from the total number of grains, equal to the number of solid grains, divide the number of solid grains The seed setting rate was calculated from the total number of grains.

The results can be seen in Figure 2. The rice seed setting rate data is normally distributed and has a wide range. There are many super-parent individuals, showing the genetic characteristics of quantitative traits.

3. QTL positioning analysis

Using the genetic map constructed by a large number of SNP and Indel markers previously developed in the laboratory, quantitative trait loci (QTL) interval mapping was performed on rice seed setting rate, and the markers and quantitative trait phenotypic values of the entire chromosome set were analyzed through R-QTL professional software. relationship, locate QTL one by one to the corresponding position of the linkage group, and estimate its genetic effect. If a molecular marker with LOD>2.5 is detected, it is considered that there is a QTL between the two markers corresponding to the highest LOD value.

Finally, we found a major QTL between the Indel ssr-1 marker and the Indelssr-2 marker on chromosome 2 in the entire genome, with an LOD value of 3.47 for seed setting rate. Its genetic distance is 128.62-147.53cM, and its physical distance is 30004559~34415035bp, and it is named qSSR2.1, as shown in Figure 3.

Example 2 Molecular marker assisted selection

Set the molecular marker Indel ssr-1 and the molecular marker In delssr-2 respectively upstream and downstream of the QTL site qSSR2.1, and design primers;

The primer pair of molecular marker Indel ssr-1 is:

Upstream primer 5’-CCCGGGACTGATGAAGAAG-3’, SEQ ID NO.1;

Downstream primer 5’-CTCACCCTTGACTGCAGATTC-3’, SEQ ID NO.2;

The primer pair of molecular marker Indel ssr-2 is:

Upstream primer 5’-TGCCATGATAACACCACTATGAAA-3’, SEQ ID NO.3;

Downstream primer 5'-CGCCAAACCATCAAAAATGT-3', SEQ ID NO.4.

Take rice leaves from the parents Huazhan and Reyan and their F ₁ generation and RIL populations, extract genomic DNA, and use the above molecular markers to perform PCR amplification of their genomic DNA;

PCR reaction system: 0.5 μL upstream primer, 0.5 μL downstream primer, 1 μL DNA template, 8 μL Mix enzyme;

The reaction program was: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 10 s, 34 cycles of amplification, and final extension at 72°C for 1 min.

The PCR amplification products were detected by 4% agarose gel electrophoresis, and some results are shown in Figures 4 and 5.

Analyze the band pattern of the electrophoresis detection band. If the band tends to be the parent Huazhan, it means that the rice seed setting rate of this strain is better. If the band tends to be hot grinding, it means the seed setting rate is poor.

The rice seed setting rate of the tested lines was compared with the results predicted by banding analysis, and it was shown that the predicted results were consistent with the actual test results.

Example 3 Application of molecular markers linked to QTL related to rice seed setting rate in rice breeding

The molecular markers set in Example 2 can be applied to molecular-assisted breeding of rice. The specific implementation method is as follows: cross other rice varieties Nipponbare, which has a smaller seed setting rate, with Huazhan to obtain the corresponding F ₁ , using Nipponbare as the recurrent parent. Backcross was performed to BC ₃ F ₁ generation. Extract the DNA of some individual strains of BC ₃ F _1st generation, and then use the primers of Indel ssr-1 and Indel ssr-2 for PCR amplification. Band pattern analysis is used to determine whether there are corresponding markers. The presence of markers indicates the regulation of the strain. The seed yield is strong. By using this method for screening and directional selection, rice can be obtained that has strong regulation of seed setting rate and retains the excellent traits of Nipponbare, which greatly improves breeding efficiency.

Our laboratory used this molecular marker to backcross the rice variety Nipponbare, which has a low seed-setting rate, with Huazhan, and used the above method to conduct directional selection to obtain rice offspring with strong control over the seed-setting rate and retain the excellent traits of Nipponbare. It can be seen from Figure 6 and Figure 7 that the BC ₃ F ₁ generation is biased towards Huazhan, indicating that the excellent trait of strong seed setting rate is retained.

In summary, the main QTL for regulating rice seed setting rate of the present invention can effectively increase the regulatory ability of rice, effectively improve rice quality and yield during the breeding process, and at the same time accelerate the process of optimizing rice varieties. At the same time, in the process of molecular-assisted rice breeding, rice varieties with higher yields can be cultivated and rice quality and yield can be optimized. This method is simple, safe and effective, beneficial to improving the economic value of rice varieties, taking into account both economic and ecological benefits, and is suitable for large-scale promotion and application

Each embodiment in this specification is described in a progressive manner. Each embodiment focuses on its differences from other embodiments. The same and similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables those 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 practiced 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.

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.