Marker Assisted Selection. Credit, Monsanto via Washington Post.

Marker Assisted Selection. Credit, Monsanto via Washington Post.

Marker aided selection or MAS refers to the use of DNA markers (DNA sequences with a known location) that are tightly-linked to target loci (the specific location of a gene) and can be used as an alternative to screening for phenotypic traits (an observable trait such as leaf colour). As such, plants that possess particular genes can be identified based on their genotype.[1] Species with the desired trait can then be used under conventional plant breeding, for example in hybridisation, using DNA markers to track the gene or genes in question. MAS can also be used to identify and track genes that will be introduced from one variety to another.

The value of MAS is the possibility of identifying the presence of a trait at the seedling or even the seed stage, obviating the need for observing varieties after full maturity to identify if a desired trait is present. As a result, using MAS greatly speeds up the breeding process and makes it cheaper. Further, new varieties can be brought to commercialisation in 4 to 6 generations instead of 10 under conventional breeding processes.[2]

Genetic diversity of cowpea seed. Credit, J. Ehlers, UC Riverside.

Genetic diversity of cowpea seed. Credit, J. Ehlers, UC Riverside.

In developing countries MAS has been used primarily for maize, in part because hybrid variety production allows for protection of improved varieties under intellectual property laws and thus offers the possibility of capturing a return on investments. One notable application of MAS is in breeding for resistance to an insect transmitted virus, known as maize streak, affecting around 60% of planted area and loses of more than 5 million tonnes per year.[3] Prior to the development of MAS technology, it was necessary to grow seeds from breeding programs into plants and then subject them to virus carrying insects to determine whether they were resistant. The costs were prohibitive for national breeding programs. With MAS it can be speedily determined whether the resistance genes are present.

In rice varietal improvement, MAS has been key to the development of submergence-tolerant rice able to withstand submergence in water for a number of weeks.[4] Although rice in Asia is typically grown in standing water, deep flooding for more than a couple of days is detrimental to crop growth and viability. As flooding in Asia is expected to rise as a result of global warming, ‘deepwater’ rice will likely be instrumental in withstanding increased frequencies and severity of flash floods and other extreme weather events.

Contribution to Sustainable Intensification

Conventional plant and animal breeding techniques have clearly contributed a great deal to food security, and will remain a mainstay for breeding for the foreseeable future, but they have practical limitations: it is not an efficient or speedy process – often requiring breeding and observing multiple generations over a decade and sometimes 2 in order to achieve desired results; whilst some desirable characteristics may emerge, others will be lost; yields may increase, but often at the expense of pest or disease resistance. MAS offers the ability to overcome the limitations to conventional breeding by rapidly identifying desired traits and significantly reducing the length of time and randomness of the process. This also reduces the time from varietal development to commercialization, benefiting famers in need of improved cultivars adapted to their specific circumstances, preferences and environments. Immediate benefits are expected to come from breeding for pest and disease resistance. Further, as MAS can be employed to undertake a method of either conventional plant breeding or aid other modern breeding practices, it offers wide applications within the field of plant and animal breeding.

Benefits and limitations
Broad application to conventional and recombinant breeding

Due to the large number of genetic mapping studies for a range of crop species and the identification of a multitude of DNA marker–trait associations, marker-aided selection (MAS) can be used to improve the efficiency and precision of both conventional and recombinant crop breeding.[5] MAS opens new possibilities for deliberately designing new crop and animal breeds speedily with considerable precision. This can lead to profound impacts on the ability of improved varieties to withstand major impediments to food and nutrition security and adapting to climate change.

Disease and pest resistance

In particular MAS is extremely useful for breeding crops with traits controlled by multiple genes such as fruit yield, disease resistance and milk and meat production; traits that would be difficult to measure under conventional breeding.[6] MAS is being widely used to transfer high quality protein traits developed at the International Maize and Wheat Improvement Center (CIMMYT) into African maize hybrids and to transfer leaf streak virus resistance into African maize. The Kirkhouse Trust, a UK charity, also supports the West African Cowpea Consortium (WACC) to develop new cowpea varieties with resistance to the parasite Striga gesnerioides.  The East African Regional Programme and Research for Biotechnology, Biosafety and Biotechnology Policy Department (BIO-EARN), also prioritises MAS technology to locate resistance markers to plant viruses and fungi for crops such as sweet potato, maize, banana and sorghum, and genotype variation in coffee and banana.[7] A pearl millet hybrid with resistance to downy mildew disease was also developed in India.[8]

Unrealized potential

Despite the considerable resources that have been invested in this field, with few exceptions, marker-aided selection (MAS) has not yet delivered its expected benefits in commercial breeding programmes for crops, livestock, forest trees or farmed fish in developed or developing countries.[9] Although the potential benefits of using markers linked to genes of interest in breeding programmes have been obvious for many decades, the realization of this potential has been limited. This is due in part because 1) not all markers are applicable across all populations within a particular crop; 2) not all markers can be transferred; 3) false selection may occur that is only apparent once the markers and genes of interest are combined; and 4) although costs have declined, they still remain high. In developing countries, where investments in molecular markers have been far smaller, delivery of benefits has lagged even further behind. However, it is expected that as the technology is further developed and improved, the drawbacks will be overcome.[10]

Trade-offs between MAS and conventional selection

Some results have been published recently from studies at the International Maize and Wheat Improvement Center (CIMMYT) in Mexico on the relative cost-effectiveness of conventional selection and marker aided selection (MAS) for different maize breeding applications. Often conventional breeding is less expensive, but MAS is quicker. For situations when the choice between conventional breeding and MAS involves a trade-off between time and money, the cost-effectiveness of using MAS depends on four parameters: marker screening; the time saved by MAS; the size and temporal distribution of benefits associated with accelerated release of improved germplasm; and finally, the availability of operating capital to the breeding programme. Since “all four of these parameters can vary significantly between breeding projects, suggesting that detailed economic analysis may be needed to predict in advance which selection technology will be optimal for a given breeding project.”[11]

Application of MAS in developing countries

Molecular markers are widely identified in developing country plant breeding despite the uptake and realized potential being slow. The spectrum of crops for which markers have been identified is wide, covering many plants relevant to food security in developing countries, but many important species are still neglected. Initially, there was a relatively fast uptake of MAS in maize, but less so for wheat and barley and other important cereal crops.[12] Today, molecular markers are effectively applied to a broad range of crop species, among them crops important to food security such as barley, beans, cassava, chickpea, cowpea, groundnut, maize, potato, rice, sorghum, and wheat.[13]

Although MAS is already routinely employed by private seed companies, its wider use in the public sector, particularly in developing countries is still constrained.[14] The successful application of MAS in plant and animal breeding necessitates a high level of expenditure in terms of establishment and maintenance costs and requires skilled human resources, as well as substantial investment in equipment, laboratories and supportive infrastructure.[15] The low rate of adoption of MAS in developing countries can be attributed mainly to a shortage of well-trained scientists and personnel, inadequate equipment such as imaging hardware and data analysis software, and generally resource-constrained breeding programmes.  The scarcity of genomic resources for less-studied crops such as tropical legumes and minor cereals such as millet also present a challenge.

Generally, the cost of MAS will continue to be a major obstacle for its application.[16] Fortunately, the emergence of affordable large-scale marker technologies such as Diversity Arrays Technology (DArt), the sharp decline of sequencing costs, and concerted efforts of country and international breeding programmes, such as in the CGIAR, most crops with significant economic importance to developing countries have been sufficiently mapped for the purposes of applying MAS technologies.[17] Unfortunately, there are few examples where these technologies have benefitted smallholder farmers, but 2 notable examples include the development of water-submergent tolerant rice and the incorporation of 4 bacterial blight resistance genes into hybrid rice varieties in India.[18]

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Case Studies

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Case Study 1: Marker aided selection (MAS) for drought tolerance and disease resistance in pearl millet in India
Pearl millet with genetic potential. Credit ICRISAT

Pearl millet with genetic potential. Credit ICRISAT

Pearl millet (Pennisetum glaucum) is extremely important as a major grain staple and as animal feed in some of the driest areas of Asia and Africa. Grown primarily by smallholder farmers in marginal areas under rain-fed conditions, improving yields and resistance to drought and disease through ‎modern breeding is important to millions of livelihoods.[1] New hybrids have been developed that offer higher yields, but also greater vulnerability to certain diseases. In India, pearl millet is grown on 9 million hectares of which 70% are planted with hybrid cultivars.[2]  Since pearl millet hybrids first reached farmers’ fields in India in the late 1960s, each of the hybrid varieties grown have been attacked by a downy mildew plant disease that can result in up to 80% crop loss.[3]

From 1999-2002, the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) with the John Innes Centre and the Plant Sciences Research Programme of the Department for International Development (DFID) developed biotechnological solutions to help reduce the incidence of this disease in hybrid cultivars. After mapping the genomic regions of pearl millet that control downy mildew resistance, straw yield potential, and grain and straw yield under drought stress conditions, breeders used conventional breeding and marker-aided selection (MAS) to transfer several genomic regions with improved downy mildew resistance to 2 parental lines of popular hybrid millet. MAS was then used to derive 2 new varieties – ICMR 01004 and ICMR 01007 – with 2 different gene blocks for downy mildew resistance. In trials, these varieties have had grain and straw yields equal to or better than their parent lines whilst showing a vast improvement in their resistance to downy mildew. [4]

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Case study 2: Deepwater rice, International Rice Research Institute (IRRI)
A deepwater rice field near Dhaka, Bangladesh. Credit Md Johir Uddin

A deepwater rice field near Dhaka, Bangladesh. Credit Md Johir Uddin

The use of marker aided selection (MAS) was key to the development of submergence-tolerant rice, a potentially revolutionary rice variety able to withstand submergence in water for a number of weeks.[1] Rice in Asia is typically grown in standing water, but deep flooding for more than a couple of days is detrimental to crop growth and viability. Deep flooding affects more than 25% of global rice-producing land, a proportion expected to rise as a result of global warming. Flash flooding can submerge rice plants, often at the seedling stage, for several weeks.

Deepwater rice is known for its ability to elongate its internodes. These have hollow structures and function as snorkels to allow gas exchange with the atmosphere to prevent drowning. In 2009, a pair of genes responsible was identified by a team at the Nagoya University in Japan.[2] They were named SNORKEL1 and SNORKEL2. Under deep-water conditions, ethylene – a plant hormone – accumulates in the plant and induces expression of the two genes. Their products then trigger remarkable internode elongation through growth hormones, causing the rice plant to grow by up to eight metres in the presence of rising water levels.

Another gene Sub1A with a similar function has also been discovered by the International Rice Research Institute (IRRI) in the Philippines.[3] The resulting rice was named Scuba rice. It responds to ethylene by limiting the elongation of the internodes. This conserves carbohydrates so permitting regrowth when the flood recedes. The rice becomes dormant during the flooding then continues growing once floodwaters recede.[4]

There is potential to utilise both sets of genes so that high-yielding rice varieties can withstand both flooding that is deep and quick, where submergence genes are appropriate, and floodwaters that climb in a progressive and prolonged fashion, for which snorkel genes are better suited.[5]

MAS-based breeding is already underway. Markers for the Sub1 locus have now been used to integrate Sub1A from IRRI deep-water rice into a widely grown Indian variety, Swarna. The resulting crosses when grown in the field in the Philippines exhibited submergence tolerance, but the yields, plant height, harvest index and grain quality remained the same. New submergence-tolerant varieties are now being produced in this way in Laos, Bangladesh and India, and in Thailand where a submergence-tolerant jasmine rice is being bred.[6] In one farmer’s fields during IRRIs Indian field trials, 95% to 98% of the scuba rice plants recovered while only 10% to 12% of the traditional varieties survived. Within 1 year of its release, scuba rice was adopted by more than 100,000 Indian farmers.[7] As of 2012, 3 million farmers were using the new variety, Swarna-Sub1.

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Case study 3: Bringing Marker-aided selection to West Africa for improved cowpea
MAS technology for West African cowpea breeders. Credit Kirkhouse Trust

MAS technology for West African cowpea breeders. Credit Kirkhouse Trust

Cowpea is one of the most drought tolerant of all grain legume crops and it serves as a major source of protein for millions in sub-Saharan Africa. Similar to other ‘minor’ food crops little investment has been directed towards the improvement of the cowpea. Yet, the cowpea is vulnerable to the parasitic weed Striga gesneroides. With no chemical options for disease control, one feasible solution is improved breeding.

In order to identify genes for cowpea resistance, ‎marker aided selection (MAS) can infinitely speed up the process compared to conventional breeding and varietal selection. The Kirkhouse Trust, a UK-based charity, is helping to bring MAS technology to West African cowpea breeders. With support from the Trust, a consortium of cowpea breeders has been formed across the region, all provided with equipment, chemical reagents and training. Progress has been uneven, attributed in part to a lack of trained personnel, but Burkina Faso has succeeding in building a functional laboratory.

The major focus of the Trust has been to support in situ programmes, but it has also invested in a cowpea genome sequencing programme, the foundation for designing genetic markers.  Junior and senior breeding staff have been trained through a series of 3 to 6 month courses, supported by visits from researchers from the University of Virginia, regional workshops, and working visits for technicians to the Ouagadougou laboratory.[1]

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