CN104969855B - Method for cultivating rice breeding material with broad spectrum and lasting spike blast resistance - Google Patents
Method for cultivating rice breeding material with broad spectrum and lasting spike blast resistance Download PDFInfo
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Abstract
The invention discloses a method for cultivating a breeding material with broad spectrum and lasting spike blast resistance, belonging to the technical field of rice molecular breeding. The method is characterized in that with a parent of conventional rice or hybrid rice with large production extension area and excellent comprehensive characters as a recurrent parent, broad-spectrum anti-disease genes, Pigm and Pi54, are polymerized in the breeding material through continuous backcross and convergent cross and with combination of marker-assisted selection; meanwhile, representative strains are selected for performing artificial inoculation identification of spike blast, natural inducedidentification in rice blast serious areas and basic agronomic character evaluation during a total growth period, and an advanced line with excellent spike blast resistance is obtained. The agronomic characters of the advanced line bred through the method are similar to or consistent with those of the recurrent parent, and the broad spectrum and lasting resistance to spike blast is enhanced obviously.
Description
Technical field
The invention belongs to Rice molecular breeding technical field, is related to a kind of cultivation wide spectrum, lasting neck blast resistance rice breeding
The method of material.
Background technology
The rice blast caused by funguses Magnaporthe oryzae is one of Oryza sativa L. most serious disease, seriously threatens water
The Sustainable high yield and stable yields of rice.According to statistics, the whole world is every year because the production loss that rice blast is caused reaches 10%-30%, direct economy
About 5,000,000,000 dollars of loss, the grain of loss be enough to support 60,000,000 people.In China, rice blast has every year different degrees of sending out
It is raw, particularly in recent years, in rice workspaces such as southwest, the middle and lower reach of Yangtze River and northeast big generation is continued, year onset area up to 3,300,000~
5700000 ha, lose several hundred million kilograms of Oryza glutinosa, and to China's rice safety production huge hidden danger is brought.It was verified that using disease-resistant base
It is one of Disease management measure the most cost-effective because cultivating resistant variety.But due to the Differentiation of Pathogenicity of Pyricularia oryzae it is tight
Weight, biological strain is more, and variation is frequent, brings difficulty to the selection-breeding of disease-resistant variety, the disease-resistant variety containing single disease-resistant gene
Often popularizing planting 3-5 just " loses " disease resistance and becomes susceptible variety.Therefore, be polymerized several/multiple resistant genes, always
It is considered as to cultivate wide spectrum, another effective way of durable resistance kind.And for the assembly effect of different rice blast ospc genes,
Result of study is not consistent.Such as Chen Hongqi (2008), Jiang et al (2012) and Fu et al (2012) report respectively poly-
The genes of Pi1/Pi2/Pi33 tri-, the genes of Pi1/Pi2/D12 tri-, the strain of the genes of Pi1/Pi2 two are closed to paddy area in the southern part of China rice blast
Pathogenic bacteria strain has resistance of wide spectrum.And IRRI constructs carrying Pi1, Piz respectively with Co39 as background5With the Monogenic lines of Pita,
And the polymerization system of 2-3 resistant gene is carried respectively, although qualification result shows that multiple gene polymerization system is general with stronger
Resistance, but Piz5With the polymerization of the resistant gene of Pita be resistance on the contrary not as monogenic resistance strain (Hittalmani et al,
2000)；Same kit is in the qualification test of China it has also been found that the polymerization system of Pi1 and Pita is not so good as monogenic resistance product
System (what month autumn etc., 2001).
, there is period and position in the generation of rice blast, can be divided into leaf pestilence, section pestilence, the fringe of Seedling pestilence and Adult plant according to it
Pestilence, grain pestilence etc., wherein most commonly seen with Seedling pestilence and fringe pest etc..Seedling pestilence resistance inoculated identification, due to easy to operate, flow process
The advantages of easily standardization, easy realization are directed to the identification of large-scale genetic analyses colony, and extensively applied.At present,
Positioning or the rice blast resistance gene cloned, the overwhelming majority is differentiated using Seedling pestilence authentication method.Pi1, Pi2 mentioned above
With Pi33, D12, and Pi1, Piz5With the multiple gene polymerization effect of Pita, for being also primarily directed to Seedling pestilence resistance.And fringe
Pestilence, to the extent of injury of Rice Production Seedling pestilence, leaf pestilence etc. will be significantly higher than, but because the inoculated identification of fringe pest has error
Greatly, flow process such as is difficult to standardization, inoculation workload is big at the defect, be difficult always it is extensive carry out application, for different genes/gene
The neck blast resistance effect analysis of polymerization, thus also just lack very much.
In recent years, with the development of molecular biology, Sequencing of Rice Genome is completed, increasing rice blast resistance
Gene is positioned, and has identified more than 80 rice blast resistance gene, wherein have more than more than 20 disease-resistant gene being cloned.
We are using the rice blast resistance gene feature and compact linkage molecule labelling cloned to the breeding in the current production of China
Main body parent carries out genotype detection, preliminary by multivariate regression models and correlation analysiss with reference to Seedling pestilence Resistance Identification result
Specify that and there is good Seedling pestilence resistance positioned at the Pigm and the Pi54 for being located at the 11st chromosome long arm of Oryza sativa L. Short arm of chromosome 6
It is complementary.
Pigm is located at Oryza sativa L. Short arm of chromosome 6, and the wide spectrum rice blast resistance gene equipotential such as Pi2, Pi9 or closely connects
Lock, its paddy area in the southern part of China different strains are had good Seedling pestilence resistance (Deng et al, 2006).Pi54 is positioned at the 11st dye
Colour solid is long-armed, is one of NBS-LRR member in Pik gene clusters, and the different rice blast bacterial strains to India have good Seedling
Pestilence resistance (Sharma et al, 2005；Ramkumar et al, 2011)., but not yet studies have reported that the two genes at present
Whether neck blast resistance effect is possessed after polymerization.
The content of the invention
It is an object of the invention to provide a kind of breeding method of anti-fringe pest rice breed.By backcrossing, molecule mark
The methods such as note assisted Selection, agronomic performance evaluation and Resistance Identification formulate the rice breed of anti-fringe pest.In order to realize this
Bright technical purpose, the technical solution used in the present invention is as follows：
A kind of cultivation wide spectrum, the method for lasting neck blast resistance breeding material, its feature includes step in detail below：
(1) recurrent parent respectively with the hybridization of resistant gene donor paddy prunus mume (sieb.) sieb.et zucc. No. 4 and K3, obtain hybridization F1Generation, respectively with F1-
Pigm、F1- Pi54 is represented；The resistant gene of wherein No. 4 carryings of paddy prunus mume (sieb.) sieb.et zucc. is Pigm, and the resistant gene that K3 is carried is Pi54；
(2) F is planted1- Pigm and F1- Pi54 seeds, florescence respectively with recurrent parent backcrossing obtain be returned 1 generation, respectively with
BC1F1- Pigm and BC1F1- Pi54 is represented；
(3) BC is planted1F1-Pigm、BC1F1- Pi54 seeds, in seedling stage each BC is extracted1F1Individual plant DNA in segregating population, and
Enter performing PCR detection using with the molecular marker of target gene close linkage, select containing Pigm and Pi54 genes and economical character
The heterozygous individual plant similar with recurrent parent continues to obtain BC with recurrent parent hybridization in florescence2F1Segregating population, and with
BC2F1- Pigm and BC2F1- Pi54 is represented；
(4) repeat step (3), until in 6 generations of backcrossing, obtain BC6F1- Pigm and BC6F1- Pi54 is simultaneously planted, and seedling stage carries
Take each BC6F1Individual plant DNA in segregating population, and enter performing PCR detection using with the molecular marker of target gene close linkage, respectively
Select the BC containing Pigm and Pi54 genes6F1Heterozygous individual plant carries out convergent cross in florescence, obtains high-quality hybrid F1Kind
Son, and with BC6F1-Pigm/Pi54Represent；
(5) BC is planted6F1-Pigm/Pi54Colony, seedling stage extracts each individual plant DNA in segregating population, and using tight with target gene
Close chain molecular marker enters performing PCR detection, selects the heterozygous individual plant containing Pigm and Pi54 genes simultaneously to harvest seed, obtains
Obtain dual-gene BC6F2For colony, with BC6F2-Pigm/Pi54Represent；
(6) by BC6F2-Pigm/Pi54More than 1000 plants of population size is planted into, the individual plant in seedling stage extracts segregating population
DNA, and proceed PCR detections using with the molecular marker of target gene close linkage, select Pigm and Pi54 dual-gene pure
Close and economical character and recurrent parent individual plant the most similar；
(7) the target single-strain seed for harvesting the dual-gene homozygosis of Pigm and Pi54 plants into BC6F3Strain, carries out neck blast resistance
Artificial vaccination and sick nursery spontaneous induction identification, while carry out agronomic performance evaluation；Select artificial vaccination and sick nursery spontaneous induction
The strain of disease-resistant and basic economical character and recurrent parent no significant difference is showed in identification, neck blast resistance is obtained and is changed
Good breeding material.
Wherein, with the preferred ZJ58.7 of molecular marker of target gene Pigm close linkages, closely connect with target gene Pi54
The preferred M11.54 of molecular marker of lock.
Shown in the preferred SEQ ID NO.1 of molecular marker ZJ58.7 forward primers, the preferred SEQ ID NO.2 institutes of reverse primer
Show.
Shown in the preferred SEQ ID NO.3 of molecular marker M11.54 forward primers, the preferred SEQ ID NO.4 institutes of reverse primer
Show.
Described recurrent parent is that excellent conventional variety or two-line hybrid rice parent raise rice No. 6.
The beneficial effects of the present invention is：
1) present invention is selected target gene using molecular marker assisted selection in each backcross generations, without the need for field
Inoculated identification, simplifies the procedure of breeding.
2) present invention is constantly adopted while segregating generation adopts molecular marker assisted selection for target resistant gene
The individuality that the method choice economical character of range estimation is the most similar to recurrent parent is hybridized and selfing, obtains the strain of just one-step forming
Carry out agronomic performance evaluation after system again, reduce chain and genetic background reply degree the detecting step of burden, improve breeding choosing
Select efficiency.
3) two target resistant genes that the present invention is utilized, the two genes are also equipped with fringe while possessing Seedling pestilence resistance
Pestilence resistance, the anti-spectrum degree of overlapping of its fringe pest is little and with strong complementarity, by continuous backcross, and binding molecule marker assisted selection, realize
Polymerization in breeding material, the polymerization of structure has the dual-gene breeding materials of Pigm/Pi54, can slow down the prominent of Pyricularia oryzae
Variable Rate, it is ensured that the wide spectrum of target material neck blast resistance, persistency.
Description of the drawings
Fig. 1 is homozygotic selection schematic diagram dual-gene for target resistant gene Pigm and Pi54.Swimming lane 1 is donor parent
This paddy prunus mume (sieb.) sieb.et zucc. No. 4 or K3；2 raise rice No. 6 for recurrent parent；3-20 is BC6F2The part individual plant of inbreeding population.Red arrow indicates mesh
Mark dual-gene homozygote.
Fig. 2 is showed for the neck blast resistance of target Improved lines YD6-Pigm/Pi54.Fig. 2A is detached using South Rice Region of China
The neck blast resistance artificial infection idenfication result of 120 single-ascospore strains；Fig. 2 B are enshi, Jiangxi Suichuan and Anhui Jinzhai County 3
Sick nursery neck blast resistance spontaneous induction qualification result；Fig. 2 C are detached using Hubei, Hainan and the province of Jiangsu 3 between 2007-2014
The neck blast resistance artificial infection idenfication result of single-ascospore strain.
Specific embodiment
Embodiment 1
(1) carrying (Yiwen Deng, the et al.Genetic of donor parents paddy prunus mume (sieb.) sieb.et zucc. No. 4 of resistant gene Pigm
characterization and fine mapping of the blast resistance locus Pigm(t)
tightly linked to Pi2and Pi9in a broad-spectrum resistant Chinese
variety.Theoretical and Applied Genetics,2006,113(4):705-713) with carrying resistant gene
Donor parents K3 (Yamada M, the et al.Proposal of a new method for differentiating of Pi54
races of Pyricularia oryzae Cavara in Japan.Ann Phytopathol Soc Jpn,1976,42
(2):216-219) raise rice No. 6 with recurrent parent respectively to hybridize, obtain F1, respectively with F1-Pigm、F1- Pi54 is represented.
(2) F is planted1- Pigm and F1- Pi54 seeds, respectively recurrent parent raised the backcrossing of rice No. 6 and obtained being returned for 1 generation florescence,
Respectively with BC1F1-Pigm、BC1F1- Pi54 is represented.
(3) BC is planted1F1-Pigm、BC1F1- Pi54 seeds, in seedling stage each BC is extracted1F1The DNA of individual plant in segregating population,
And enter performing PCR inspection using with molecular marker ZJ58.7 and M11.54 (table 1) of target resistant gene Pigm, Pi54 close linkage
Survey, select containing target resistant gene and economical character and recurrent parent raise the similar heterozygous individual plant of rice No. 6 florescence after
It is continuous to raise No. 6 backcrossing acquisition BC of rice with recurrent parent2F1Segregating population, respectively with BC2F1-Pigm、BC2F1- Pi54 is represented.
The sequence information of labelling used by the assisted Selection of table 1
(4) above step (3) is repeated, until in 6 generations of backcrossing, obtain BC6F1- Pigm and BC6F1- Pi54 is simultaneously planted, Seedling
Phase extracts each BC6F1The DNA of individual plant in segregating population, and using with the molecular marker ZJ58.7 of target gene close linkage and
M11.54 proceeds PCR detections, and the BC containing Pigm and Pi54 genes is selected respectively6F1Heterozygous individual plant is carried out in florescence
Convergent cross, obtains high-quality hybrid F1More than 50, seed, and with BC6F1-Pigm/Pi54Represent.
(5) BC is planted6F1-Pigm/Pi54Colony, seedling stage extracts the DNA of each individual plant in segregating population, and utilizes and target gene
The molecular marker ZJ58.7 and M11.54 of close linkage enters performing PCR detection, selects simultaneously containing the heterozygosis of Pigm and Pi54 genes
Type individual plant harvests seed, obtains dual-gene BC6F2For colony, with BC6F2-Pigm/Pi54Represent.
(6) by BC6F2-Pigm/Pi54More than 1000 plants of population size is planted into, the individual plant in seedling stage extracts segregating population
DNA, and proceed PCR detections using with the molecular marker ZJ58.7 and M11.54 of target gene close linkage, select double-basis
Because homozygosis (Fig. 1) and economical character and recurrent parent raise the most similar individual plant more than 10 of rice No. 6.
(7) harvest and raise the target single-strain seed of dual-gene homozygosis under No. 6 backgrounds of rice and plant into BC6F3Strain, using from difference
Collect and separate and belong to 7 representative bacterium of A, B, C, D, E, F, G group (for Chinese rice blast differential variety) in ecotope
Strain carries out combined inoculation in boot stage by injection, and its inoculation method and Evaluation standard of resistance are referring to Luo Chuping (2009) (sieve
Chu's equality. rice blast inoculation technique and Jiangsu Province's novel wheat cultivars Resistance Identification in 2009. Jiangsu's agriculture science, 2009,6:
178–179).10 individual plant optimum periods that center row is continuously planted are selected to carry out basic economical character tune for each strain simultaneously
Look into, including plant height, heading stage and yield component traits (single-strain tassel number, number of grain per ear, setting percentage, mass of 1000 kernel and single plant yield).
Select neck blast resistance to raise rice No. 6 compared with recurrent parent to obtain significantly improveing (reaching anti-level), basic economical character and raise rice No. 6
The strain of similar (zero difference significance), obtains target material resistance improvement strain YD6-Pigm/Pi54。
(8) wide spectrum, the checking of lasting neck blast resistance of strain are improved.Using Sichuan, Chongqing, Hubei, Jiangxi, Anhui, Hunan,
The South Rice Region of China such as Guangdong, Hainan, Jiangsu are collected, detached 120 single-ascospore strains, in boot stage by injection to improveing strain
YD6-Pigm/Pi54Raising rice No. 6 with recurrent parent carries out single fungus strain inoculated identification, as a result shows that improvement strain neck blast resistance frequency is high
Up to 96.67 ± 2.34%, and recurrent parent is only 18.33 ± 6.17% (Fig. 2A)；In enshi, Jiangxi Suichuan and Anhui gold
The rice blast such as stockaded village retransmit area for improvement strain YD6-Pigm/Pi54Rice No. 6 being raised with recurrent parent and carrying out spontaneous induction identification, it resists
Property evaluation criterion adopt International Rice institute (2002) (IRRI.Standard Evaluation System for Rice (SES)
.2002,(4ed.,pp.15–16).Los Banos.Philippines:International Rice Research
Institute (IRRI)) formulate 0-9 levels grade scale perform, as a result shows improve strain tassel blast level it is equal in 3 sick nurseries
Less than 3 grades, anti-level is reached, and recurrent parent is above 7.5 grades in 3 sick nurseries, shows as sense or high sense (Fig. 2 B).This
Outward, during 2007-2014, Hubei, Hainan, totally 30, the province of Jiangsu 3 are selected from the Pyricularia oryzae storehouse collected and preserve every year
Unit cell bacterial strain (often saves 10), in boot stage by injection to improveing strain YD6-Pigm/Pi54Rice No. 6 is raised with recurrent parent to enter
Row list fungus strain inoculated identification, as a result shows improvement strain neck blast resistance frequency stable more than 90%, and recurrent parent is below
30% (Fig. 2 C).Result above shows that target improvement strain has wide spectrum, lasting neck blast resistance, fully confirms the present invention's
Effectiveness.
Claims (5)
1. a kind of to cultivate wide spectrum, the method for lasting neck blast resistance breeding material, its feature includes step in detail below：
(1) recurrent parent respectively with the hybridization of resistant gene donor paddy prunus mume (sieb.) sieb.et zucc. No. 4 and K3, obtain hybridization F1Generation, respectively with F1-Pigm、
F1- Pi54 is represented；The resistant gene of wherein No. 4 carryings of paddy prunus mume (sieb.) sieb.et zucc. is Pigm, and the resistant gene that K3 is carried is Pi54；
(2) F is planted1- Pigm and F1- Pi54 seeds, florescence respectively with recurrent parent backcrossing obtain be returned 1 generation, respectively with
BC1F1- Pigm and BC1F1- Pi54 is represented；
(3) BC is planted1F1-Pigm、BC1F1- Pi54 seeds, in seedling stage each BC is extracted1F1Individual plant DNA in segregating population, and utilize
Enter performing PCR detection with the molecular marker of target gene close linkage, select containing Pigm and Pi54 and economical character and samsara parent
This similar heterozygous individual plant continues to obtain BC with recurrent parent hybridization in florescence2F1Segregating population, and with BC2F1- Pigm and
BC2F1- Pi54 is represented；
(4) repeat step (3), until in 6 generations of backcrossing, obtain BC6F1- Pigm and BC6F1- Pi54 is simultaneously planted, and seedling stage extracts each
BC6F1Individual plant DNA in segregating population, and enter performing PCR detection using with the molecular marker of target gene close linkage, select respectively
BC containing Pigm and Pi54 genes6F1Heterozygous individual plant carries out convergent cross in florescence, obtains high-quality hybrid F1Seed,
And with BC6F1-Pigm/Pi54Represent；
(5) BC is planted6F1-Pigm/Pi54Colony, seedling stage extracts each individual plant DNA in segregating population, and utilization closely connects with target gene
The molecular marker of lock enters performing PCR detection, selects the heterozygous individual plant containing Pigm and Pi54 genes simultaneously to harvest seed, obtains double
The BC of gene6F2For colony, with BC6F2-Pigm/Pi54Represent；
(6) by BC6F2-Pigm/Pi54More than 1000 plants of population size is planted into, the DNA of individual plant in seedling stage extracts segregating population,
And proceed PCR detections using with the molecular marker of target gene close linkage, select the dual-gene homozygosis of Pigm and Pi54 and
Economical character and recurrent parent individual plant the most similar；
(7) the target single-strain seed for harvesting the dual-gene homozygosis of Pigm and Pi54 plants into BC6F3Strain, the people for carrying out neck blast resistance
Work is inoculated with and the identification of sick nursery spontaneous induction, while carrying out agronomic performance evaluation；Select artificial vaccination and the identification of sick nursery spontaneous induction
In show the strain of disease-resistant and basic economical character and recurrent parent no significant difference, obtain neck blast resistance and obtain what is improved
Breeding material.
2. it is according to claim 1 it is a kind of cultivation wide spectrum, the method for lasting neck blast resistance breeding material, it is characterised in that：
Molecular marker with target gene Pigm close linkages is ZJ58.7, is with the molecular marker of target gene Pi54 close linkages
M11.54。
3. it is according to claim 2 it is a kind of cultivation wide spectrum, the method for lasting neck blast resistance breeding material, it is characterised in that：
As shown in SEQ ID NO.1, reverse primer is as shown in SEQ ID NO.2 for molecular marker ZJ58.7 forward primers.
4. it is according to claim 2 it is a kind of cultivation wide spectrum, the method for lasting neck blast resistance breeding material, it is characterised in that：
As shown in SEQ ID NO.3, reverse primer is as shown in SEQ ID NO.4 for molecular marker M11.54 forward primers.
5. a kind of cultivation wide spectrum according to any one of Claims 1 to 4, the method for lasting neck blast resistance breeding material,
It is characterized in that：Described recurrent parent is that excellent conventional variety or two-line hybrid rice parent raise rice No. 6.
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| CN105830912B (en) * | 2016-05-10 | 2018-09-21 | 江苏丘陵地区镇江农业科学研究所 | A kind of breeding method of high yield, lasting neck blast resistance rice |
| CN105961185B (en) * | 2016-05-10 | 2019-01-11 | 江苏丘陵地区镇江农业科学研究所 | A method of cultivating the rice varieties of blast resisting |
| CN108782227A (en) * | 2018-07-03 | 2018-11-13 | 江苏里下河地区农业科学研究所 | A kind of breeding method of high grain weight blast resisting two-line hybrid rice restorer |
| CN108935078A (en) * | 2018-08-10 | 2018-12-07 | 湖北省农业科学院粮食作物研究所 | A kind of breeding method for the transgenic paddy rice polymerizeing multiple target character |
| CN109042304B (en) * | 2018-09-04 | 2022-05-20 | 江苏里下河地区农业科学研究所 | Breeding method of rice blast resistant high-quality high-yield two-line hybrid indica rice combination |
| CN109924121B (en) * | 2018-12-11 | 2021-05-28 | 华智生物技术有限公司 | Method for improving rice blast resistance of rice |
| CN111312335B (en) * | 2020-02-24 | 2023-07-21 | 吉林省农业科学院 | Soybean parent selection method and device, storage medium and electronic equipment |
| CN112889664B (en) * | 2021-01-19 | 2022-06-03 | 江苏里下河地区农业科学研究所 | Method for cultivating broad-spectrum and durable resistant rice breeding material by polymerizing complementary rice blast resistant genes |
| CN115104528B (en) * | 2022-08-15 | 2023-09-22 | 济宁市农业科学研究院 | Method for improving rice blast resistance |
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| CN102577925A (en) * | 2012-02-17 | 2012-07-18 | 江苏省农业科学院 | Breeding method for breeding rice variety resistant to rice panicle blast in Jiangsu province |
| CN103497996B (en) * | 2013-09-22 | 2015-11-25 | 江苏里下河地区农业科学研究所 | A kind of for detecting the molecule marker InDel 587 of paddy plum No. 4 blast resistant genes Pigm (t) |
| CN104542250A (en) * | 2014-12-30 | 2015-04-29 | 天津天隆农业科技有限公司 | Multi-gene high rice blast resistance material breeding method adopting marker assisted selection |
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Address after: 225325 8 Industrial Park, Baima Town, Gao Gang, Taizhou, Jiangsu Patentee after: Jiangsu Lixiahe Prefecture Institute of Agricultural Science Address before: 225007 568 North Road, Yangzi River, Yangzhou, Jiangsu Patentee before: Jiangsu Lixiahe Prefecture Institute of Agricultural Science |
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Application publication date: 20151014 Assignee: Jiangsu gold land Seed Industry Co., Ltd Assignor: JIANGSU LIXIAHE REGION AGRICULTURAL Research Institute Contract record no.: X2020980000803 Denomination of invention: Method for cultivating rice breeding material with broad spectrum and lasting spike blast resistance Granted publication date: 20170517 License type: Exclusive License Record date: 20200318 |
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Address after: No.112-6, building 13, Tongyun Trade City, Guangling District, Yangzhou City, Jiangsu Province, 225002 Patentee after: JIANGSU LIXIAHE REGION AGRICULTURAL Research Institute Address before: 225325 8 Industrial Park, Baima Town, Gao Gang, Taizhou, Jiangsu Patentee before: JIANGSU LIXIAHE REGION AGRICULTURAL Research Institute |
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