CN113881796B - Rice qVE alpha-toco/total-toco molecular marker and application - Google Patents
Rice qVE alpha-toco/total-toco molecular marker and application Download PDFInfo
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Abstract
The invention discloses a main effect QTL for regulating and controlling the vitamin E content of rice, which belongs to the technical field of rice breeding and molecular biology, wherein the QTL is positioned on chromosome 11 of the rice, the genetic distance is 90.36-97.08cM, and the physical distance is 21077880-22646003bp; the invention also discloses a molecular marker closely linked with the QTL. By utilizing the QTL and the molecular marker thereof, rice with high vitamin E can be bred, and the screening efficiency can be improved.
Description
Technical Field
The invention belongs to the technical field of rice breeding and molecular biology, and particularly relates to a marker for regulating and controlling major QTL (quantitative trait locus) locus of rice vitamin E alpha-tocopherol accounting for total tocopherol content and application thereof.
Background
Rice (Oryza sativa L.) is one of the world important food crops, and with the development of society and the improvement of living standard, people pay more attention to the nutrition quality of rice. Rice kernels are one of the major sources of daily intake of micronutrients, where Vitamin E (VE) plays a vital antioxidant role in organisms [1-2] Not only is necessary for the metabolism of rice and the resistance to stress, but also is related to the nutrition and health of human beings [3-5] 。
The biological activities of the vitamin E isomers vary significantly, with the highest alpha-tocopherol activity being effective in lowering blood cholesterol levels and having been identified as a fat-soluble vitamin by federal regulations in the United statesSafe food additive [6] . In addition, the binding capacity of Tocopherol Binding Protein (TBP) to tocopherol isomers in human body is different, and under the same conditions, the tocopherol binding capacity to alpha-tocopherol is the strongest, and the alpha-tocopherol is preferentially absorbed [7] . Therefore, increasing the vitamin E content in rice, especially increasing the specific gravity of alpha-tocopherol, is one of the important ideas for cultivating high quality rice. The demand for high quality rice is also increasing. Cultivation of rice with both yield and quality has become the main direction of grain production in China [8] 。
In recent years, with the development of genome mapping technology and molecular marker technology, QTL localization of plant vitamin E has become a new research hotspot. Currently, in plants such as soybean, rape and arabidopsis thaliana, QTL related to vitamin E content or components is located, but for rice, QTL related to vitamin E content control is less studied and still in a starting stage. In the existing research results, the positioning result of the alpha-tocopherol related QTL of the main isomer of vitamin E in rice is as follows:
zhang Xiaona, etc [9] Recombinant Inbred Line (RIL) groups constructed by taking Zhenshan 97 (ZS 97) and Nanyang (NYZ) as parents are used as experimental materials, the vitamin E content is measured, and 4 QTLs related to the vitamin E are successfully positioned and are respectively positioned on chromosome 1, chromosome 2 and chromosome 6. Wherein, on chromosome 1 is an alpha-tocopherol related qVE-1 with additive effect of 0.8522 and contribution rate of 8.9%; meanwhile, the gene locus qVE-1 (RM 5-RM 488) and Sookwong et al [10] The located (RM 8144-RM 3642) has overlapping intervals, so that the two are possibly sourced from the same site. In addition, zhang Xiaona experiments were also performed on rice plants and F2 populations thereof, the parents of which were nanyang and zhenshan 97, 6 QTL sites associated with alpha-tocopherol, QTL alpha 2, qT alpha 4, qT alpha 5, qT alpha 7a, qT alpha 8, were detected during 2010 to 2011, with contribution rates in the range of 6.4% -10.3%, on chromosome 2, 4,5, 7, 8, respectively.
Rice is taken as staple food of more than half of the world population, and the nutrition quality of the rice is closely related to the health of people. The improvement of the content of the vitamin E in the rice has very important social benefit and huge economic benefit, but the research on the positioning of the alpha-tocopherol QTL which is a main isomer of the vitamin E in the rice is limited at present. The method is mainly characterized in that the phenotype identification cost of the vitamin E content of the rice is high, the vitamin E content of the rice is low, and the accurate measurement difficulty is high. Therefore, the QTL locus and related molecular markers for controlling the vitamin E content in rice grains need to be further deeply excavated and analyzed, and a new choice is provided for high-quality rice breeding.
Reference to the literature
[1] Fitzgerald M.A., mcCouch S.R., and Hall R.D.,2009,Not just a grain of rice:the quest for quality,Trends Plant Sci, 14 (3) 133-139 (Merisha-FittgIjirad, susan-Michaux, robert-Hall, 2019, not just one meter: pursuit of quality [ J ]. Trend of plant science, 14 (3): 133-139);
[2] almeida J, quadra l, as r, setta n, goday f.d., berm u dez l, otaiza s.n., corr E a da Silva j.v., fernie a.r., carrari f, and Rossi m. 2011,Genetic dissection of vitamin E biosynthesis in tomato[J, journal of Experimental Botany,62 (11): 3781-3798 (Zhu Lian na·almida, lin Deluo ·quala, ramon, amari ya·saint, farbiana·goldoi, lu Yisha · Bei Mde si, san diego, otta, hesia, kov Lei Ya schirw, allis· Fei Ni, ferzedol karli, ma Geda na·rosi 2011, genetic analysis of vitamin E biosynthesis [ J ] (11): 3798);
[3] jing Liquan the environmental changes of Xuanshawu, mu Hairong, wang Yunxia and Yanglingxin lead to the overall deterioration of rice quality [ J ]. Chinese agricultural science, 2018,51 (13): 2462-2475; jing LQ, huS W, mu H R, wang Y X, yang L X. Change of atmospheric environment leads to deterioration of rice quality [ J ]. Scientia AgriculturaSinica,2018,51 (13): 2462-2475 (in Chinese withEnglish abstract);
[4] ross M W, robin D G.breeding for micronutrients instaple food crops from a human nutrition perspective [ J ]. Journal of Experimental Botany,2004,55 (396): 353-364 (Luo Si Welch, luo Bin Graham. Microelements of major food crops from a human nutrition point of view [ J ]. J.Experimental plant journal, 2004,55 (396): 353-364);
[5] uauy C, distelfeld A, fahima T, blech A, dubcovsky J.A NAC gene regulating senescence improves grainprotein, zinc, and iron content in wheat [ J ]. Science,2006,314:1298-1301 (Kristobel Wu Yi, alasf Derstfield, wengfa sigma, anbu Lei, george Du Bu kofski. An aging controlling NAC gene increases wheat grain protein, zinc and iron content [ J ]. Science,2006, 314:1298-1301);
[6] tigu F, zhang J L, liu G X, cai Z, li Y.A highly active pantothenate synthetase from Corynebacterium glutamicum enables the production of D-pantothenic acid with high productivity [ J ] Applied Microbiology and Biotechnology,2018,102:6039-6046 (Fei Tese M.grape Gu Zhangjun, liu Guoxia, cai Zhen, li Yin A highly active pantothenate synthase from Corynebacterium glutamicum can produce D-pantothenate [ J ] in high yield using microbiology and biotechnology,2018, 102:6039-6046);
[7] pan Weidong, li Xiaofeng, chen Shuangyan, liu Gongshe, 2006, cloning of plant vitamin E synthesis-related enzyme genes and development of functional studies in vivo, plant science report, 23 (1): 68-77;
[8] chen, zeng Yongjun, cheng Huihuang, tan Xueming, shang Qingyin, zeng Yanhua, dan Qinghua. Influence of different temperatures from ear alignment to milk maturation period on rice south japonica 9108 seed hormone and seed hormone content, starch accumulation and key enzyme activity synthesis [ J ]. Chinese rice science, 2019,33 (1): 57-67;
[9] zhang Xiaona positioning and analyzing the vitamin E content and composition related QTL in brown rice [ D ]. University of agriculture in China, 2013;
[10]Sookwong P.,Murata K.,Nakagawa K.,Shibata A.,Kimura T.,Yamaguchi M.,Kojima Y.,and Miyazawa T.,2009,Cross-fertilization for enhancing tocotrienol biosynthesis in rice plants and QTL analysis of their F2 progenies[J]J.Agric.food chem.,57 (11): 4620-4625 (Magnolia Zong Xiong, village and Zheng, zhongchuan Qing He, chai Tianzhang, mucun Jun, mountain tortoise, island)Yang Yilang, gong Zeguang male, 2009, outcrossing fertilization promotes rice tocotrienol synthesis and F thereof 2 QTL analysis of progeny [ J]J.agricultural and food chemistry 57 (11): 4620-4625).
Disclosure of Invention
The invention aims to solve the technical problem of providing a main effect QTL for regulating and controlling the ratio of vitamin E alpha-tocopherol in total tocopherol in rice grains and a molecular marker closely linked with the main effect QTL, which are used for breeding rice varieties with high vitamin E and can improve screening efficiency.
In order to solve the technical problems, the invention provides a main effect QTL for regulating and controlling the ratio of vitamin E alpha tocopherol to total tocopherol of rice, wherein the main effect QTL is positioned on a rice chromosome 11, named qVE alpha-toco/total-toco, the genetic distance between positioning intervals is 90.36-97.08cM, and the physical distance is 21077880-22646003bp.
The invention also provides a molecular marker of the main effect QTL (molecular marker closely linked to the main effect QTL) for regulating and controlling the vitamin E content of rice:
the main effect QTL is located between a molecular marker Indel Tococ-1 and a molecular marker Indel Tococ-2;
the primer pair of the molecular marker Indel Tococ-1 is as follows:
an upstream primer: 5'-TGGAAATCGCATGACCAGTA-3';
a downstream primer: 5'-CCCATGCTCCTAGCTGAGTC-3';
the primer pair of the molecular marker Indel Tococ-2 is as follows:
an upstream primer: 5'-TCGTTGTCCTTAAACTGGTCTG-3';
a downstream primer: 5'-TCAGGCATGGATGGCTTAG-3'.
The invention also provides application of the major QTL for regulating and controlling the vitamin E content of the rice in rice variety breeding:
and detecting QTL related to vitamin E content in rice varieties or lines by using molecular markers, so that rice with high ratio of alpha tocopherol to total tocopherol is bred through the molecular markers.
The invention also provides a breeding method of the rice with high vitamin E content, 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 vitamin E content of the rice by a band type (the ratio of vitamin E alpha-tocopherol in rice grains to total tocopherol by the band type analysis);
aiming at hybrid rice obtained by taking Huazhan as a parent;
when the strip tends to be occupied by parent, the rice grain of the strain has a high ratio of vitamin E alpha tocopherol to total tocopherol; conversely, it is indicated that the ratio of vitamin E alpha tocopherol to total tocopherol is low.
Description: another parent is rice varieties with less vitamin E alpha tocopherol than total tocopherol, such as hot ground No.2, nippon sunny, etc.
As the improvement of the breeding method of the rice with high vitamin E content, the invention:
the reaction system (11. Mu.L) for PCR amplification was: 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 2. Mu.L of DNA template, 6. Mu.L of mix enzyme, ddH 2 O 1μL；
Mix enzyme was purchased from Optimago (2×Taq Master Mix);
the reaction procedure for PCR amplification was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 30s, amplification for 38 cycles; finally, the extension is carried out for 10min at 72 ℃.
The invention also provides a rice breeding kit with high vitamin E content: comprising a pair of molecularly marked primers as described above. Namely, the primer pair of the molecular marker can be used for preparing a Gao-tocopherol to total tocopherol ratio rice breeding kit. The kit for rapidly and simply screening the ratio of the vitamin E alpha tocopherol in the grains to the total tocopherol can utilize molecular markers to assist breeding and improve rice quality.
The invention can be used for screening rice fine varieties and identifying rice germplasm resource molecules.
The invention uses japonica rice variety Huatan 2 as female parent and indica rice variety Huazhan as male parent to hybridize, uses recombined inbred line group obtained after F1 generation continuous selfing as material, adopts high performance liquid chromatograph to measure vitamin E alpha-tocopherol and total tocopherol, and uses the encryption genetic map constructed by the group to carry out QTL mapping analysis on data, detects a QTL with LOD value up to 4.92, is named qVE alpha-toco/total-toco, the locus is positioned on rice chromosome 11, the genetic distance is 90.36-97.08cM, and the physical distance is 21077880-22646003bp.
The main effect QTL for regulating and controlling the ratio of the vitamin E alpha-tocopherol to the total tocopherol of the rice can be applied to rice variety breeding, and the breeding process of the rice variety with the high vitamin E alpha-tocopherol to the total tocopherol ratio can be accelerated by developing molecular markers closely linked with the main effect QTL and detecting related QTLs for controlling the ratio of the vitamin E alpha-tocopherol to the total tocopherol in the rice variety or the rice strain.
The molecular markers Indel Tococ-1 and Indel Tococ-2 are molecular markers closely linked with the main effect QTL of the ratio of the vitamin E alpha-tocopherol to the total tocopherol of the rice, and the content of the vitamin E in the rice grains can be predicted by detecting the molecular markers, so that the breeding progress of the rice varieties with the ratio of the vitamin E alpha-tocopherol to the total tocopherol is accelerated.
In conclusion, the main effect QTL qVE alpha-toco/total-toco for regulating and controlling the ratio of vitamin E alpha-tocopherol in rice grains is positioned, 2 molecular markers closely linked with the QTL are obtained by applying the QTL locus, the ratio of vitamin E alpha tocopherol in total tocopherol in rice varieties can be predicted by using the molecular markers, and the breeding of ideal rice plant types is accelerated.
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The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a flow chart of genetic material construction used in the major QTL localization process for regulating the ratio of vitamin E alpha-tocopherol to total tocopherol in rice kernels;
FIG. 2 is a RIL frequency distribution plot;
wherein Nekken represents the rice variety of hot-grinding No.2, and HZ represents the rice variety Huazhan;
FIG. 3 is a position of a major QTL qVE alpha-toco/total-toco on chromosome 11 for regulating the ratio of vitamin E alpha-tocopherol to total tocopherol in rice kernels;
FIG. 4 shows an electropherogram generated by amplification of a primer pair of the molecular marker Indel Tococ-1 in the parent and F1 generation and RIL populations;
wherein, 1 is Huazhan, 2 is hot-ground No.2, 3 is hot-ground No. 2/Huazhan filial generation F1, and 4-12 is rice plant material with higher content in RIL group (F12) of hot-ground No. 2/Huazhan filial combination.
FIG. 5 shows an electropherogram generated by amplification of a primer pair of the molecular marker Indel Tococ-2 in the parent and F1 generation and RIL populations;
wherein, 1 is Huazhan, 2 is hot-ground No.2, 3 is hot-ground No. 2/Huazhan filial generation F1, and 4-12 is hot-ground No. 2/Huazhan filial generation RIL group with higher content of rice plant materials.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1 major QTL positioning for controlling the ratio of vitamin E alpha-tocopherol to Total tocopherol in Rice grains
1. Acquisition of Experimental materials
Hybridization is carried out by taking hot grinding No.2 as a receptor parent and rice variety Huazhan as a donor parent, and a single seed transmission method (namely, F1 is subjected to bagging single plant seed receiving treatment until offspring strain phenotype is not separated) is utilized, so that 120 stably inherited strains (F12, all strain phenotypes are stable) are finally obtained, and a recombinant inbred line RIL population is formed, as shown in figure 1.
60 seeds of the parent and each strain of seeds (F12) are selected, the seeds are soaked for 2 days after surface sterilization, then the seeds are wrapped by wet towels, and the seeds are selected and sowed after germination in a 37 ℃ incubator for 48 hours. After 30 days, the parents with similar growth conditions and 24 seedlings of each strain are selected and transplanted, and all rice materials are planted in Zhejiang university college laboratory test fields in Jinhua city of Zhejiang province for routine management.
2. Rice grain vitamin E content data determination
Measuring the vitamin E alpha-tocopherol and tocopherol content of rice grains by using high performance liquid chromatography (high performance liquid chromatogra, HPLC);
the results are shown in figure 2, the vitamin E alpha-tocopherol ratio data represent continuous normal distribution and wide range, more super-parent individuals exist, and the genetic characteristics of quantitative characters are represented.
The abscissa in fig. 2 represents the ratio of alpha-tocopherol to total tocopherol, and the ordinate represents the RIL population number.
3. QTL positioning analysis
And (3) carrying out Quantitative Trait Locus (QTL) interval mapping on the vitamin E content of the rice 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 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 of LOD >3 is detected, 1 QTL exists between the 2 markers corresponding to the highest LOD value.
Finally, a major QTL located between the Indel Tococ-1 marker and the Indel Tococ-2 marker on chromosome 11 was found in the whole chromosome group of rice, with a LOD value of up to 4.92 for vitamin E content in the rice seeds, a genetic distance of 90.36-97.08cM, a physical distance of 21077880-22646003bp, and a designation qVE. Alpha. -toco/total-toco (FIG. 3).
Example 2 molecular marker assisted selection
Setting a molecular marker Indel Tococ-1 and a molecular marker Indel Tococ-2 on the upstream and downstream of a QTL locus qVE alpha-toco/total-toco respectively, and designing a primer;
the primer pair of the molecular marker Indel Tococ-1 is as follows:
an upstream primer: 5'-TGGAAATCGCATGACCAGTA-3' (SEQ ID NO. 1);
a downstream primer: 5'-CCCATGCTCCTAGCTGAGTC-3' (SEQ ID NO. 2);
the primer pair of the molecular marker Indel Tococ-2 is as follows:
an upstream primer: 5'-TCGTTGTCCTTAAACTGGTCTG-3' (SEQ ID NO. 3);
a downstream primer: 5'-TCAGGCATGGATGGCTTAG-3' (SEQ ID NO. 4).
Taking rice leaves of parent hot-grinding No.2 and Hua Zhan and F1 generation and RIL population (obtained in step 1 of example 1), extracting genome DNA, and carrying out PCR amplification on the genome DNA by using the molecular markers;
PCR reaction system: 1. Mu.L of the upstream primer (10. Mu. Mol), 1. Mu.L of the downstream primer (10. Mu. Mol), 2. Mu.L of the DNA template (> 100 ng/. Mu.L), 6. Mu.L of the Mix enzyme (2 XTaq Master Mix, prinsepia, inc.), ddH 2 O 1μL；
The reaction procedure is: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 30s, amplification for 38 cycles; finally, the extension is carried out for 10min at 72 ℃.
The PCR amplified products were detected by electrophoresis on a 4% agarose gel,
the primer pair of the molecular marker Indel Tococ-1 is adopted, and the electrophoresis detection result is shown in figure 4;
the result of the electrophoresis test using the primer pair of the molecular marker Indel Tococ-2 is shown in FIG. 5.
And analyzing the band type of the electrophoresis detection band, wherein the band tends to the parent Huazhan, so that the rice grain of the strain has high vitamin E alpha-tocopherol to total tocopherol ratio, and the band tends to the hot grinding No.2, so that the rice grain has low alpha-tocopherol to total tocopherol ratio.
The detection of the alpha-tocopherol and the total content of the total tocopherol in the rice grains of the plant line is carried out by adopting a high performance liquid chromatograph (GB 5009.82-2016), and the obtained results are as follows:
therefore, the vitamin E content of the rice grains of the tested strain is compared with the predicted result through the band type analysis, and the predicted result is matched with the actual detection result.
Example 3 application of rice vitamin E content related QTL in rice breeding
Hybridizing a rice variety male parent Nippon with low vitamin E alpha-toco/total-toco content with a female parent Huazhan to obtain corresponding F1, and backcrossing with Nippon as a recurrent parent to BC 3 F 1 And (3) replacing. Extraction of BC 3 F 1 The generation of partial single-strain DNA was then amplified by PCR using the primers Indel Tococ-1 and Indel Tococ-2, and electrophoretically detected.
And analyzing the band type of the electrophoresis detection band, wherein the band tends to the parent Huazhan, so that the rice grain of the strain has high vitamin E content. By using the method for screening and directional selection, the rice with high vitamin E content and retaining the fine properties of Japanese sun can be obtained, and the breeding efficiency is greatly improved.
In practice, the bands tended to be planted on 9 individual plants of the parent Huazhan, the ratio of the finally obtained alpha-tocopherol to the total tocopherol content was 0.8755,0.8754,0.8758,0.8759,0.8755,0.8754,0.8760,0.8751,0.8756, and the ratio of the parent Huazhan to the total tocopherol content was 0.8758, so that the results obtained by the present invention were confirmed to be identical to the actual results.
In conclusion, the main effect QTL for regulating and controlling the rice grain with high ratio of vitamin E alpha-tocopherol to total tocopherol can effectively accelerate the progress of optimizing rice varieties. In the rice molecular assisted breeding process, rice with rice grains and high ratio of vitamin E alpha-tocopherol to total tocopherol can be cultivated, and the quality and yield of the rice can be 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.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Sequence listing
<110> Zhejiang university of teachers and students
<120> Rice qVE alpha-toco/total-toco molecular marker and application
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Claims (4)
1. The molecular marker of the major QTL related to the regulation of the vitamin E content of rice is characterized in that:
the main effect QTL is located between a molecular marker Indel Tococ-1 and a molecular marker Indel Tococ-2;
the primer pair of the molecular marker Indel Tococ-1 is as follows:
an upstream primer: 5'-TGGAAATCGCATGACCAGTA-3';
a downstream primer: 5'-CCCATGCTCCTAGCTGAGTC-3';
the primer pair of the molecular marker Indel Tococ-2 is as follows:
an upstream primer: 5'-TCGTTGTCCTTAAACTGGTCTG-3';
a downstream primer: 5'-TCAGGCATGGATGGCTTAG-3';
the major QTL is located on chromosome 11 of rice and is named asqVEα-toco/total-tocoThe genetic distance between the positioning intervals is 90.36-97.08cM, and the physical distance is 21077880-22646003bp;
and detecting QTL related to vitamin E content in rice varieties or lines by using molecular markers, so that rice with high ratio of alpha tocopherol to total tocopherol is bred through the molecular markers.
2. A breeding method of rice with high vitamin E content is characterized in that: extracting rice DNA, carrying out PCR amplification on the DNA by using the primer pair of the molecular marker as claimed in claim 1, carrying out electrophoresis detection on an amplified product, and analyzing the vitamin E content of the rice by a band type;
aiming at hybrid rice obtained by using Huazhan and hot-grinding No.2 as parents or using Huazhan and Nippon as parents;
when the strip tends to be occupied by parent, the rice grain of the strain has a high ratio of vitamin E alpha tocopherol to total tocopherol; conversely, it is indicated that the ratio of vitamin E alpha tocopherol to total tocopherol is low.
3. The method for breeding rice with high vitamin E content according to claim 2, wherein:
the PCR amplification reaction system is as follows: 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 2. Mu.L of DNA template, 6. Mu.L of mix enzyme, ddH 2 O 1μL；
The reaction procedure for PCR amplification was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 30s, amplification for 38 cycles; finally, the extension is carried out for 10min at 72 ℃.
4. A rice breeding kit with high vitamin E content is characterized in that: a primer pair comprising the molecular marker of claim 1.
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