Final Report Summary - LOWASRICE (Breeding for low grain arsenic rice)
Project objectives
Objective 1: Map QTLs for low-grain arsenic in crosses with locally preferred cultivars to identify markers suitable for marker-assisted breeding.
Objective 2: Progress to near completion the production of locally adapted low-arsenic rice by using the advanced backcross breeding approach.
Objective 3: Test the hypothesis that arsenic uptake in rice is linked to silicon transport.
Objective 4: Develop screening infrastructures and expertise in West Bengal and international networks suitable for sustained breeding of low-arsenic rice for both local needs and for international collaboration. The three-year project will be split into two parts: the first two years in Aberdeen, the final year in Calcutta. Crosses conducted in Calcutta before the start of the project involved genotypes not ideally suited to project activities since Pusa Basmati and Kallinga III were revealed to be only moderately high in grain arsenic when more data became available from other projects. Local land races Noyan Moni and Bon Ahu were therefore identified as more suitable parents for crosses. This meant that some project activities were delayed in relation to the original workplan. In Calcutta, during the first year low-grain arsenic tropical japonica cultivars Azucena and Lemont were crossed with local, high-grain arsenic cultivars Noyan Moni and Bon Ahu; these F1s were advanced to eight backcross 1 populations by recrossing with the local cultivar to give [Azucena x Noyan Moni] x Noyan Moni, [Noyan Moni x Azucena] x Noyan Moni , [Lemont x Noyan Moni] x Noyan Moni, [Noyan Moni x Lemont] x Noyan Moni , [Azucena x Bon Ahu] x Bon Ahu, [Bon Ahu x Azucena] x Bon Ahu , [Lemont x Bon Ahu] x Bon Ahu, [Bon Ahu x Lemont] x Bon Ahu.
Project outcomes
BC1s were sown at the end of May 2010, 12 plants per cross. The BC1 plants were 'selfed' and the resulting plants cultivated. In February 2011, dried leaves of one cross [Lemont x Noyan Moni] x Noyan Moni] were sent to Aberdeen for DNA extraction. For each of the 12 BC1s crossed, leaves of 20 BC1F2s were sent off to give 240 leaf samples. The seeds of each of these 240 genotypes were collected as bulked BC1F3 seeds and used to set out a replicated experiment in a high-arsenic field to test grain arsenic. This was sown in December 2011 to be harvested in April 2012. The grain samples will be analysed for arsenic content in grain during July 2012.
In Aberdeen, DNA was extracted from Azucena, Lemont, Noyan Moni and Bon Ahu and a SSR (microsatellite) polymorphism screen was initiated. A total of 530 SSR markers were tested for polymorphism with approximately 18 % proving useful when assessed with high-resolution agarose gels. Using funding from another project, Aberdeen are creating a 384 SNP marker chip using the Illumina BeadXpress system. The DNA of 200+ of the BC1F2s will be assessed on this chip proving between a 100 and 300 markers by July 2012. Marker work is also continuing in Calcutta on genotyping 240 mapping population with more than 100 SSR markers in QIAxcel Advanced System nucleic acid analyser (QIAgen). The aim of this marker work is to have 150+ markers for 200 genotypes by the start of summer 2012. A molecular map will then be completed and, using data from grain arsenic, mapping of quantitative trait loci (QTLs) for will be achieved. The F2 of a second population of Lemont (As tolerant) x Kalinga II (As susceptible) (not one of the original crosses) is available.
Once results from QTL mapping are available, individual plants of the mapping population can be used to enter a breeding programme for low-grain arsenic (objective 2). Addressing objective 3, an undergraduate project in Aberdeen tested the hypothesis that silicon reduced the toxicity of arsenic to rice plants. The results of field experiments in Bangladesh coordinated in Aberdeen (another project) revealed a correlation between shoot arsenic concentration and silicon concentration, suggesting a common pathway of uptake into the shoots. However, silicon in the shoot or grain was not related to grain arsenic, suggesting silicon is not important in determining grain arsenic levels.
During the second year, Professor Dasgupta took an opportunity to gain experience in gene knock-out technology through the REFUGE programme see http://www.refuge-platform.org/ for details. After designing sequences for RNAi-based gene silencing of three candidate aquaporin genes, he spent four weeks in CIRAD, Montpellier, France working with their molecular biologists to make constructs to transform rice cultivars Bala and Azucena. The products of that transgenic work were grown on by CIRAD and seeds of 117 plants have been sent to Aberdeen for molecular and physiological characterisation.
In relation to objective 4, Prof. Dasgupta is a collaborator on a large UK-funded grant on alternative wetting and drying in rice led by Dr Price (the scientist in charge of LOWASRICE).
Although some of the main objectives of this project are behind the workplan schedule, it is anticipated that most objectives will be met. It is anticipated that the mapping of low-grain arsenic will be achieved and breeding low-grain arsenic in local cultivars will be advanced by the end of the project. In addition, activities (specifically the RNAi knockout work in Montpellier) which were not originally part of the workplan but which represented a significant training and networking opportunity, have been conducted.
Project impacts
The project still has nearly one year to run and it is not expected that major dissemination activities or impacts will accrue until the end when grain arsenic is measured allowing QTLs to be identified and low-grain arsenic cultivars can be entered into a breeding programme. Experience gained on molecular marker technology in the LOWASRICE project has enabled Prof. Dasgupta to get funding from the government of West Bengal to strengthen the molecular biology laboratory in Calcutta University.
Contact details: Prof. Tapash Dasgupta, Department of Genetics and Plant Breeding, University of Calcutta, 35 B.C. Road, Kolkata 700 019 West Bengal, India; email tapashdg@rediffmail.com for more details; scientist in charge: Dr Adam Price, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 3UU, UK; email a.price@abdn.ac.uk for more details.
Objective 1: Map QTLs for low-grain arsenic in crosses with locally preferred cultivars to identify markers suitable for marker-assisted breeding.
Objective 2: Progress to near completion the production of locally adapted low-arsenic rice by using the advanced backcross breeding approach.
Objective 3: Test the hypothesis that arsenic uptake in rice is linked to silicon transport.
Objective 4: Develop screening infrastructures and expertise in West Bengal and international networks suitable for sustained breeding of low-arsenic rice for both local needs and for international collaboration. The three-year project will be split into two parts: the first two years in Aberdeen, the final year in Calcutta. Crosses conducted in Calcutta before the start of the project involved genotypes not ideally suited to project activities since Pusa Basmati and Kallinga III were revealed to be only moderately high in grain arsenic when more data became available from other projects. Local land races Noyan Moni and Bon Ahu were therefore identified as more suitable parents for crosses. This meant that some project activities were delayed in relation to the original workplan. In Calcutta, during the first year low-grain arsenic tropical japonica cultivars Azucena and Lemont were crossed with local, high-grain arsenic cultivars Noyan Moni and Bon Ahu; these F1s were advanced to eight backcross 1 populations by recrossing with the local cultivar to give [Azucena x Noyan Moni] x Noyan Moni, [Noyan Moni x Azucena] x Noyan Moni , [Lemont x Noyan Moni] x Noyan Moni, [Noyan Moni x Lemont] x Noyan Moni , [Azucena x Bon Ahu] x Bon Ahu, [Bon Ahu x Azucena] x Bon Ahu , [Lemont x Bon Ahu] x Bon Ahu, [Bon Ahu x Lemont] x Bon Ahu.
Project outcomes
BC1s were sown at the end of May 2010, 12 plants per cross. The BC1 plants were 'selfed' and the resulting plants cultivated. In February 2011, dried leaves of one cross [Lemont x Noyan Moni] x Noyan Moni] were sent to Aberdeen for DNA extraction. For each of the 12 BC1s crossed, leaves of 20 BC1F2s were sent off to give 240 leaf samples. The seeds of each of these 240 genotypes were collected as bulked BC1F3 seeds and used to set out a replicated experiment in a high-arsenic field to test grain arsenic. This was sown in December 2011 to be harvested in April 2012. The grain samples will be analysed for arsenic content in grain during July 2012.
In Aberdeen, DNA was extracted from Azucena, Lemont, Noyan Moni and Bon Ahu and a SSR (microsatellite) polymorphism screen was initiated. A total of 530 SSR markers were tested for polymorphism with approximately 18 % proving useful when assessed with high-resolution agarose gels. Using funding from another project, Aberdeen are creating a 384 SNP marker chip using the Illumina BeadXpress system. The DNA of 200+ of the BC1F2s will be assessed on this chip proving between a 100 and 300 markers by July 2012. Marker work is also continuing in Calcutta on genotyping 240 mapping population with more than 100 SSR markers in QIAxcel Advanced System nucleic acid analyser (QIAgen). The aim of this marker work is to have 150+ markers for 200 genotypes by the start of summer 2012. A molecular map will then be completed and, using data from grain arsenic, mapping of quantitative trait loci (QTLs) for will be achieved. The F2 of a second population of Lemont (As tolerant) x Kalinga II (As susceptible) (not one of the original crosses) is available.
Once results from QTL mapping are available, individual plants of the mapping population can be used to enter a breeding programme for low-grain arsenic (objective 2). Addressing objective 3, an undergraduate project in Aberdeen tested the hypothesis that silicon reduced the toxicity of arsenic to rice plants. The results of field experiments in Bangladesh coordinated in Aberdeen (another project) revealed a correlation between shoot arsenic concentration and silicon concentration, suggesting a common pathway of uptake into the shoots. However, silicon in the shoot or grain was not related to grain arsenic, suggesting silicon is not important in determining grain arsenic levels.
During the second year, Professor Dasgupta took an opportunity to gain experience in gene knock-out technology through the REFUGE programme see http://www.refuge-platform.org/ for details. After designing sequences for RNAi-based gene silencing of three candidate aquaporin genes, he spent four weeks in CIRAD, Montpellier, France working with their molecular biologists to make constructs to transform rice cultivars Bala and Azucena. The products of that transgenic work were grown on by CIRAD and seeds of 117 plants have been sent to Aberdeen for molecular and physiological characterisation.
In relation to objective 4, Prof. Dasgupta is a collaborator on a large UK-funded grant on alternative wetting and drying in rice led by Dr Price (the scientist in charge of LOWASRICE).
Although some of the main objectives of this project are behind the workplan schedule, it is anticipated that most objectives will be met. It is anticipated that the mapping of low-grain arsenic will be achieved and breeding low-grain arsenic in local cultivars will be advanced by the end of the project. In addition, activities (specifically the RNAi knockout work in Montpellier) which were not originally part of the workplan but which represented a significant training and networking opportunity, have been conducted.
Project impacts
The project still has nearly one year to run and it is not expected that major dissemination activities or impacts will accrue until the end when grain arsenic is measured allowing QTLs to be identified and low-grain arsenic cultivars can be entered into a breeding programme. Experience gained on molecular marker technology in the LOWASRICE project has enabled Prof. Dasgupta to get funding from the government of West Bengal to strengthen the molecular biology laboratory in Calcutta University.
Contact details: Prof. Tapash Dasgupta, Department of Genetics and Plant Breeding, University of Calcutta, 35 B.C. Road, Kolkata 700 019 West Bengal, India; email tapashdg@rediffmail.com for more details; scientist in charge: Dr Adam Price, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 3UU, UK; email a.price@abdn.ac.uk for more details.