CN113881796A - 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 major QTL for regulating and controlling the vitamin E content of rice, belonging to the technical field of rice breeding and molecular biology, wherein the QTL is positioned on a No. 11 chromosome of rice, the genetic distance is 90.36-97.08cM, and the physical distance is 21077880-22646003 bp; 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 of a major QTL (quantitative trait locus) site for regulating and controlling the content of vitamin E alpha-tocopherol in total tocopherol in rice and application thereof.
Background
Rice (Oryza sativa L.) is one of the important food crops in the world, and people pay more and more attention to the nutritional quality of the rice along with the development of the society and the improvement of the living standard. Rice grains are one of the main sources of daily intake of micronutrients, wherein Vitamin E (VE) plays a crucial role in resisting oxidation in organisms[1-2]Not only is necessary for the metabolism of rice and the resistance to adverse conditions, but also is involved in the nutrition and health of human beings[3-5]。
The biological activities of vitamin E isomers are significantly different, among which alpha-tocopherol, which has the highest activity and is effective in lowering blood cholesterol levels, has been identified as a food additive for fat-soluble vitamins as safe by U.S. Federal regulations[6]. In addition, Tocopherol Binding Proteins (TBPs) in humans have different binding capacities for individual tocopherol isomers, and under the same conditions, have the strongest affinity for alpha-tocopherol and preferentially absorb alpha-tocopherol[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. People have an increasing demand for high-quality rice. The cultivation of rice with both yield and quality has become the main direction of food production in China[8]。
In recent years, with the development of genome mapping technology and molecular marker technology, QTL mapping of plant vitamin E has become a new research hotspot. At present, in plants such as soybean, rape, arabidopsis thaliana and the like, QTL related to vitamin E content or components is located, but for rice, QTL related to vitamin E content control is less researched and still in the starting stage. In the existing research results, the positioning results of the vitamin E main isomer alpha-tocopherol related QTL in rice are as follows:
zhang Xiaona et al[9]A Recombinant Inbred Line (RIL) population constructed by taking Zhenshan 97(ZS97) and Nanyang occupation (NYZ) as parents is taken as an experimental material, the content of vitamin E is determined, and 4 QTLs related to the vitamin E are successfully positioned and are respectively positioned on No.1, No.2 and No. 6 chromosomes. Wherein, the chromosome 1 is positioned with an alpha-tocopherol related qVE-1, the additive effect is 0.8522, and the contribution rate is 8.9%; meanwhile, the gene locus qVE-1(RM5-RM488) and Sookwong and the like[10]The mapped (RM8144-RM3642) have overlapping intervals, so that the two probably originate from the same site. Furthermore, zhang xiana also tested rice plants and its F2 population with nanyang zhang and zhenshan 97 as parent, and detected 6 QTL loci associated with α -tocopherol, qT α 2, qT α 4, qT α 5, qT α 7a, qT α 8, respectively, with contribution rates ranging from 6.4% to 10.3%, located on chromosomes 2, 4,5, 7, 8, respectively, during the period 2010 to 2011.
Rice is used as staple food for more than half of the world population, and the nutritional 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 benefits and huge economic benefits, but the research on the QTL positioning of the main isomer alpha-tocopherol existing in the vitamin E in the rice is limited at present. The rice vitamin E content phenotype identification cost is high, the vitamin E content in rice is low, and the accurate measurement difficulty is high. Therefore, QTL sites and related molecular markers for controlling the vitamin E content in rice grains need to be further and 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-;
[2] almeida J., Quadran L., As is R., Setta N, Godoy F.D., Berm dez L, Otaiza S.N., Corr E E a Silva J.V., Fernie A.R., Carrari F.and Rossi M.2011, Genetic disection of vitamin E biosynthes in tomato J.journal of Experimental Botany,62(11) 3781-3798 (Verliena Almada. Almadder, Lindello Quadra, Remeng Alsi. tala. Sauta, Fabena. Goudo, Fraena. Grada. Loudo, Fraena. Lambda, Losslekura, Sadamia, Saidera. Thailand, Hitacea Hitachi, Hitachi, Higashi, plant Genetic analysis [ 3781, Higashi, plant Higashi, plant Higashi, Biotashi, Biolabs. Sjoassay [ 3781, plant Higashi, Biolabs. Sjoassay [ 3781, Biolabs. Sjogashi [ 3781, Biolabs. Sjohnson, Higashi;
[3] jing Stand, Shuan Wu, Munhua, Rou Yun Xixia, Yang lianxin, general deterioration of rice quality caused by atmospheric environmental change [ J ] Chinese agricultural science, 2018,51(13): 2462-; string L Q, Hu S W, Mu H R, Wang Y X, Yang L X. Change of exothermic environmental leads to determination of edge quality [ J ]. Scientia Agricutural scienica, 2018,51(13): 2462-;
[4] ross M W, Robin D G.Breeding for microorganisms induced crop from a human nutrition [ J ]. Journal of Experimental botanic, 2004,55(396):353-364 (Ross Welch, Robin Graham. cultivation of essential food crops from the point of human nutrition [ J ]. Journal of Experimental botanic, 2004,55(396): 353-364);
[5] uauy C, Distelfeld A, Fahima T, Blech A, Dubcovsky J.A NAC gene regulating sensory improvents grams pathway, zinc, and iron content in wheat [ J ] Science,2006,314: 1298-;
[6] tigu F, Zhang J L, Liu G X, Cai Z, Li Y.A high hly active panthetate synthesis from Corynebacterium glutamicum enzymes the production of D-panthetic acid with high production [ J ]. Applied Microbiology and Biotechnology,2018,102: 6039-Strobinzyme 6046 (Fisher. Zygon. Richardson, Charcot, Yucai, Lixiyin.) A highly active pantothenate synthetase extracted from Corynebacterium glutamicum can produce D-pantothenic acid [ J ]. Applied Microbiology and Biotechnology 2018,102: 6039-Strobistic 6046 with high yield;
[7] pan Weidong, Li Xiaofeng, Chenshuangyan, Liugong, 2006, cloning of plant vitamin E synthesis-related enzyme genes and research progress of functions thereof in vivo, botanical bulletin, 23(1): 68-77;
[8] influence of different temperatures from the ear to the milk stage on the hormone seed hormone content, starch accumulation and the synthesis key enzyme activity of the rice Nanjing 9108 seed kernel [ J ] Chinese Rice science, 2019,33(1): 57-67;
[9] zhang Xiaona, location and analysis of vitamin E content and composition related QTL in brown rice [ D ] agricultural university in Huazhong, 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): 4620-4-4625 (puzong, cuntian and zheng, Zhongchuan greenling, chai-tian three, mu cun jun, shankou tortoise field, Xiaodao yangyiang, Gongguang Xiong, 2009, crossfertilization promoting synthesis of oryza sativa trienol and F thereof2QTL analysis of progeny [ J]Journal of agricultural and food chemistry 57(11) 4620-.
Disclosure of Invention
The invention aims to solve the technical problem of providing a main effect QTL for regulating 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 content and can improve the screening efficiency.
In order to solve the technical problems, the invention provides a main effect QTL for regulating the ratio of vitamin E alpha tocopherol to total tocopherol of rice, wherein the main effect QTL is positioned on a No. 11 chromosome of rice and is named as qVE alpha-toco/total-toco, the genetic distance of a positioning interval is 90.36-97.08cM, and the physical distance is 21077880-22646003 bp.
The invention also provides a molecular marker of the main effect QTL for regulating and controlling the content of the vitamin E in the rice (the molecular marker of the main effect QTL which is closely linked):
the main effect QTL is positioned 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', respectively;
a downstream primer: 5'-CCCATGCTCCTAGCTGAGTC-3', respectively;
the primer pair of the molecular marker Indel Tococ-2 is as follows:
an upstream primer: 5'-TCGTTGTCCTTAAACTGGTCTG-3', respectively;
a downstream primer: 5'-TCAGGCATGGATGGCTTAG-3' are provided.
The invention also provides the application of the major QTL for regulating the content of the vitamin E of the rice in the breeding of rice varieties:
the molecular marker is utilized to detect the QTL related to the vitamin E content in the rice variety or strain, so that the rice with the high ratio of the alpha tocopherol to the total tocopherol is bred through the molecular marker.
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 banding (analyzing the ratio of vitamin E alpha-tocopherol to total tocopherol of rice grains by banding);
aiming at hybrid rice obtained by using Huazhan as a parent;
when the strip tends to the parent Huazhan, the ratio of vitamin E alpha tocopherol in the total tocopherol of the rice seed of the strain is high; otherwise, the vitamin E alpha tocopherol accounts for low total tocopherol ratio.
Description of the drawings: the other parent is a rice variety with low vitamin E alpha tocopherol in total tocopherol, such as Hokka 2, Nipponbare, etc.
The improvement of the breeding method of the rice with high vitamin E content of the invention is as follows:
the reaction system for PCR amplification (11. mu.L) was: 1 uL of upstream primer, 1 uL of downstream primer, 2 uL of DNA template, 6 uL of mix enzyme, ddH2O 1μL；
The Mix enzyme was purchased from Onghamia bio (2 × Taq Master Mix);
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.
The invention also provides a high vitamin E content rice breeding kit: primer pairs comprising molecular markers as described above. Namely, the primer pair of the molecular marker can be used for preparing a rice breeding kit with the high alpha-tocopherol to total tocopherol ratio. The kit for rapidly, simply and conveniently screening the ratio of the vitamin E alpha tocopherol in the total tocopherol to the grains can utilize molecular markers to assist breeding and improve the rice quality.
The invention can be used for screening rice fine varieties and identifying rice germplasm resource molecules.
The method comprises the steps of taking japonica rice variety Hokka 2 as a female parent and indica rice variety Huazhan as a male parent for hybridization, taking a recombinant inbred line population obtained after continuous inbreeding of a hybrid F1 generation as a material, adopting a high performance liquid chromatograph to measure vitamin E alpha-tocopherol and total tocopherol, simultaneously utilizing an encrypted genetic map constructed by the population to carry out QTL mapping analysis on data, detecting a QTL with an LOD value up to 4.92, and naming qVE alpha-total/total-total, wherein the locus is positioned on a rice 11 chromosome, the genetic distance is 90.36-97.08cM, and the physical distance is 21077880-46003 bp.
The main effect QTL for regulating the ratio of the vitamin E alpha-tocopherol to the total tocopherol of the rice can be applied to the breeding of rice varieties, and the breeding process of the rice varieties with the ratio of the vitamin E alpha-tocopherol to the total tocopherol can be accelerated by developing molecular markers closely linked with the main effect QTL and detecting the related QTL for controlling the ratio of the vitamin E alpha-tocopherol to the total tocopherol in the rice varieties or strains.
The molecular marker Indel Tococ-1 and the molecular marker Indel Tococ-2 are molecular markers closely linked with a major QTL of the ratio of the rice vitamin E alpha-tocopherol to the total tocopherol, and the content of the vitamin E in rice grains can be predicted by detecting the molecular markers, so that the breeding progress of rice varieties with the ratio of the high vitamin E alpha-tocopherol to the total tocopherol is accelerated.
In conclusion, the major QTL qVE alpha-toco/total-toco for regulating the ratio of the vitamin E alpha-tocopherol to the total tocopherol in the rice grains is positioned, 2 pairs of molecular markers which are closely linked with the QTL locus are obtained by applying the QTL locus, the ratio of the vitamin E alpha-tocopherol to the total tocopherol in the rice variety can be predicted by utilizing the molecular markers, and the breeding of the ideal plant type of the rice is accelerated.
Drawings
The following describes 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 a main effect QTL positioning process for regulating and controlling the ratio of vitamin E alpha-tocopherol to total tocopherol in rice grains;
FIG. 2 is a RIL histogram;
wherein Nekken represents the rice variety Hongshan No.2, and HZ represents the rice variety Huazhan;
FIG. 3 shows the position of major QTL qVE alpha-toco/total-toco on chromosome 11 for regulating the ratio of vitamin E alpha-tocopherol to total tocopherol in rice grain;
FIG. 4 shows the electrophoretogram generated by the amplification of the primer pair labeled Indel Tococ-1 in the parent and its F1 generation and RIL population;
wherein, 1 is Huazhan, 2 is Hot Shang No.2, 3 is the hot Shang No. 2/Huazhan filial generation F1, and 4-12 is the rice strain material with higher content in RIL group (F12) of the hot Shang No. 2/Huazhan hybridization combination.
FIG. 5 shows the electrophoretogram generated by the amplification of the primer pair labeled Indel Tococ-2 in the parent and its F1 generation and RIL population;
wherein, 1 is Huazhan, 2 is Hot Shang No.2, 3 is the hot Shang No. 2/Huazhan filial generation F1, and 4-12 is the rice strain material with higher content in RIL group of the hot Shang No. 2/Huazhan hybridization combination.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 major QTL location for modulating the ratio of vitamin E alpha-tocopherol to total tocopherol in rice grain
1. Acquisition of test materials
The method comprises the steps of hybridizing by taking the Hongyao No.2 as a receptor parent and taking the rice variety Huazhan as a donor parent, and finally obtaining 120 stably inherited lines (F12, all lines have stable phenotypes) by using a single-event transmission method (namely, bagging F1 to allow single plants to be subjected to seed treatment until the phenotypes of progeny lines are not separated), wherein a recombinant inbred line RIL group is formed as shown in figure 1.
60 seeds of each parent and each strain (F12) are selected, the seeds are soaked for 2 days after the surfaces of the seeds are disinfected, the seeds are wrapped by a wet towel, the seeds are placed in a constant temperature box at 37 ℃ for accelerating germination for 48 hours, and the seeds with consistent exposure are selected and sowed. 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. Determination of vitamin E content data of rice grains
Measuring the content of vitamin E alpha-tocopherol and tocopherol in rice grains by using High Performance Liquid Chromatography (HPLC);
the results are shown in fig. 2, the ratio data of vitamin E alpha-tocopherol to total tocopherol shows continuous normal distribution and wide range, more super-parent individuals exist, and the genetic characteristics of quantitative traits are shown.
The abscissa in fig. 2 represents the alpha-tocopherol to total tocopherol ratio and the ordinate represents the number of RIL populations.
3. QTL localization analysis
The Quantitative Trait Locus (QTL) interval mapping is carried out on the content of the vitamin E of the rice by utilizing a genetic map constructed by a large number of SNP and Indel marks developed in the early stage of a laboratory, the relation between the marks of the whole chromosome set and the quantitative trait phenotype values is analyzed by 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 molecular marker with LOD >3 is detected, 1 QTL exists between 2 markers corresponding to the highest LOD value.
Finally, a major QTL between the Indel Tococ-1 marker and the Indel Tococ-2 marker on the 11 th chromosome is found in the entire genome of oryza sativa, the LOD value of the vitamin E content of the oryza sativa seeds is as high as 4.92, the genetic distance is 90.36-97.08cM, the physical distance is 21077880-22646003bp, and the result is named as qVE alpha-toco/total-toco (figure 3).
Example 2 molecular marker assisted selection
Setting a molecular marker Indel Tococ-1 and a molecular marker Indel Tococ-2 respectively at the upstream and downstream of a QTL site qVE alpha-toco/total-toco, and designing primers;
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 the rice leaves of the parent strain of the rice plant of the hot grinding No.2, Huazhan and the F1 generation thereof and the RIL group (obtained in the step 1 of the embodiment 1), 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 (> 100 ng/. mu.L), 6 μ L of Mix enzyme (Onck. Biopsis, 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.
Detecting the PCR amplification product in 4% agarose gel electrophoresis,
the results of electrophoresis detection using a primer pair labeled with Indel Tococ-1 are shown in FIG. 4;
the results of electrophoresis were shown in FIG. 5 using primer pairs labeled with Indel Tococ-2.
And analyzing the band type of the electrophoresis detection band, wherein if the band tends to the parent Huazhan, the ratio of vitamin E alpha-tocopherol to the total tocopherol of the rice grain of the line is high, and if the band tends to the heat research No.2, the ratio of the alpha-tocopherol to the total tocopherol of the rice grain is low.
The high performance liquid chromatography (GB 5009.82-2016) is adopted to detect the total content of alpha-tocopherol and total tocopherol in the strain rice grains, and the obtained results are as follows:
therefore, the vitamin E content of the rice grains of the tested strain is compared with the result predicted by the banding pattern analysis, and the predicted result is displayed to be matched with the actual detection result.
Example 3 application of rice vitamin E content-related QTL in rice breeding
Hybridizing a rice variety with lower vitamin E alpha-toco/total-toco content on a male parent Nipponbare with a female parent Huazhan to obtain corresponding F1, and backcrossing to BC by taking the Nipponbare as recurrent parents3F1And (4) generation. Extraction of BC3F1The DNA of a part of the individual strain is generated, and then PCR amplification is performed using primers for Indel Tococ-1 and Indel Tococ-2, and electrophoresis detection is performed.
And analyzing the band type of the electrophoresis detection band, wherein the band tends to the parent Huazhan, and the content of the vitamin E in the rice seeds of the strain is high. The method is used for screening and directional selection, so that the rice with high vitamin E content and retained excellent Nipponbare characters can be obtained, and the breeding efficiency is greatly improved.
Actually, the bands are planted towards 9 individuals of parent Huazhan, the ratio of the alpha-tocopherol to the total content of the tocopherol obtained by the final planting is 0.8755,0.8754,0.8758,0.8759,0.8755,0.8754,0.8760,0.8751 and 0.8756, and the ratio of the alpha-tocopherol to the total content of the tocopherol obtained by the parent Huazhan is 0.8758, so that the obtained result is proved to be matched with the actual result.
In conclusion, the major QTL for regulating the high ratio of the vitamin E alpha-tocopherol to the total tocopherol in the rice grains can effectively accelerate the process of optimizing rice varieties. In the process of rice molecular assisted breeding, rice with high ratio of rice grain vitamin E alpha-tocopherol to total tocopherol can be cultivated, and simultaneously, the quality and the yield of the rice are optimized. 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.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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Claims (6)
1. The major QTL for regulating and controlling the ratio of the vitamin E alpha tocopherol in the total tocopherol is characterized in that: the main effect QTL is positioned on the rice chromosome 11 and named as qVE alpha-toco/total-toco, the genetic distance of the positioning interval is 90.36-97.08cM, and the physical distance is 21077880 and 22646003 bp.
2. The molecular marker of the major QTL for regulating the vitamin E content of rice as claimed in claim 1, wherein the molecular marker comprises:
the main effect QTL is positioned 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', respectively;
a downstream primer: 5'-CCCATGCTCCTAGCTGAGTC-3', respectively;
the primer pair of the molecular marker Indel Tococ-2 is as follows:
an upstream primer: 5'-TCGTTGTCCTTAAACTGGTCTG-3', respectively;
a downstream primer: 5'-TCAGGCATGGATGGCTTAG-3' are provided.
3. The application of the major QTL for regulating the vitamin E content of rice as claimed in claim 1 or 2 in the breeding of rice varieties is characterized in that:
the molecular marker is utilized to detect the QTL related to the vitamin E content in the rice variety or strain, so that the rice with the high ratio of the alpha tocopherol to the total tocopherol is bred through the molecular marker.
4. 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 a primer pair of the molecular marker as claimed in claim 2, carrying out electrophoresis detection on an amplification product, and analyzing the vitamin E content of the rice by banding;
aiming at hybrid rice obtained by using Huazhan as a parent;
when the strip tends to the parent Huazhan, the ratio of vitamin E alpha tocopherol in the total tocopherol of the rice seed of the strain is high; otherwise, the vitamin E alpha tocopherol accounts for low total tocopherol ratio.
5. The method for breeding rice with high vitamin E content according to claim 4, wherein:
the reaction system of PCR amplification is as follows: 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.
6. A high vitamin E content rice breeding kit is characterized in that: a primer pair comprising the molecular marker of claim 4.
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