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[SANET-MG] marker assisted selection and micro array breeding



I apologize for the long article. It is a talk I gave at the ecological farming conference. MAS is not new to most but a recent development in MAS called micro array breeding is brand new to many. Micro chips containing gene signals for about 30,000 genes from a farm animal or crop plant are used to locate active genes using RNA probes.By comparing active genes from high yielding crops with genes from low yielding crops the genes for yield can be identified and with the aid of computer programs used select high yielding crops at early stages for mass screening. Animals selected by micro array have been bred and are ready for commercial release. Crop plants lag a little but will soon swamp the market.Micro array breeding can improve crops without adding transgenes. Organic agriculture should soon decide whether or not micro array crops are organic.
January 10, 2007
Professor Joe Cummins
Professor Emeritus of Genetic University of Western Ontario
London, Ontario Canada
And Institute for Science in Society
London, UK
Marker Assisted Selection
Quantitative traits are the key to animal and crop plant improvement
Genetically modified (GM) crops are based on inserting synthetic foreign genes, mainly approximating

the genes of bacteria, to impart herbicide tolerance or insect resistance into the genomes of crop

plants. This technology has so far provided little if any increase in yield, stress tolerance or long-

term resistance to microbes or nematodes. Genetic modification using synthetic genes is unlikely to get

very far in enhancing yield or tolerance to stress The Traditional breeding of crops and animals has

been based on the use of genetic markers that are inherited. The main agricultural traits governing

yield (or size), stress resistance or long-term disease protection are quantitative trait loci (QTL,

‘loci’ is another word for genes). One of the founders of the study of population genetics, Ronald A.

Fisher, described QTL as many independent loci that added together to determine traits such as size

[1]. QTL are seldom tightly linked on a chromosome and the loci are dispersed over many chromosomes in

the genome. Selection of QTL traits has been inherently slow and meticulous, but has resulted in major

improvements to crops and livestock. QTL are recognized as being the key to long term animal and field

crop improvement and for that reason farmers should become acquainted with the genes and their

identification.
While Fisher believed that QTL were made up of very many genes each adding small increments to a trait,

recent findings indicate that some QTL may be made up of a relatively small number, say twenty or so,

genetic markers that could be easily selected provided they could be identified. Currently, it appears

that many QTL may have relatively few loci but some important QTL may be closer to the very large

number of genes envisioned by Fisher, in which case, identifying and selecting such traits by the

molecular markers are unlikely to be cost-effective. However, recent developments in the area of micro

arrays suggest that most of the QTLs will be accessible and cost effective in the near future.
Molecular markers are used to aid selective breeding
Molecular markers are obtained by using molecular probes obtained from pieces of a gene. The probes are

used to identify the progeny of crosses bearing the desirable gene. The process of using such markers

is called marker-assisted selection (MAS), which differs from genetic modification because the genes

being selected for crop or animal improvement are not altered in any way. The molecular markers used in

selection are probed using sequences from a gene bank and identified. MAS has proved useful in

selecting specific individual genes bearing a desired trait but they are most effective in dealing with

the important QTLs. The molecular marker for individual QTL, as with individual genes for a single

desirable trait , is the preferred and most effective way to identify desirable traits but in many

instances it is fastest and most economical to employ markers linked to a desirable trait or QTL as a

surrogate for the desired trait.
There is a growing arsenal of molecular markers (polymorphisms) that aid in identifying QTL and

selecting them for crop and animal enhancement. The markers used to probe the progeny of a cross need

not be the QTL genes themselves but they are close to the QTL on the genetic map. Of course the

markers can be used to determine the molecular identity of the QTL, but the molecular marker is used

even when the QTL is identified because the marker is cheaper and quicker to use to identify a large

number of progeny. Recombination may separate the marker from a QTL, but the closer the marker is to

the QTL, the more remote is the chance of separation by recombination. The more polymorphic markers

available for a breeding program the more effective it will be.
There are several types of molecular markers used in MAS; these include restriction fragment length

polymorphism (RFLP), random amplification of polymorphic DNA (RAPD), amplified restriction fragment

length polymorphism (AFLP), single sequence repeats (SSR) and single nucleotide polymorphisms SNPs [2].

RFLP involves the use of restriction enzymes to cut chromosomal DNA at specific short restriction

sites, polymorphisms result from duplications or deletions between the sites or mutations at the

restriction sites. RFLP provided the basis for most early work but requires a relatively large amount

of DNA and is rather expensive in a large screening program [2]. RAPD utilizes low stringency

polymerase chain reaction (PCR) amplification with single primers of arbitrary sequence to generate

strain-specific arrays of anonymous DNA fragments [3]. The method requires tiny DNA samples and

analyses a large number of polymorphic loci [2]. AFLP requires digestion of cellular DNA with a

restriction enzyme, then using PCR and selective nucleotides in the primers to amplify specific

fragments [4]. The method measures up to 100 polymorphic loci and requires a relatively small DNA

sample for each test [4]. SSR analysis is based on DNA micro-satellites (short-repeat) sequences that

are widely dispersed throughout the genome of eukaryotes, which are selectively amplified to detect

variations in simple sequence repeat [5]. SSR analysis requires tiny DNA samples, and has a low cost

per analysis [2]. SNPs are detected using PCR extension assays that efficiently pick up point mutations

[6]. The procedure requires little DNA per sample and costs little per sample once the method is

established [2]. One or two methods are used in a typical MAS breeding program. However, probes that

identify the gene to be selected yield the best results.
The methods described above are rapidly being replaced by a technique called micro array analysis. The

technique is exceptionally powerful and has begun to identify a number of QTL that have been difficult

to find using the earlier techniques. The method is proving powerful in research in most aspects of

molecular biology and genetics.. The system is designed to dissect the genes for individual traits. For

example, as of 2005, 7000 QTL controlling various complex traits have been located on different

chromosomes of rice and such markers can now be selected using the micro array techniques. In this

technology glass slides or chips are prepared with DNA oligonucleotide sequences representing say

25 bases the last 600 bases of several thousand different genes dispersed on the chip. Micro arrays

can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass

slides, photolithography using pre-made masks, photolithography using dynamic micro mirror devices,

ink-jet printing , or electrochemistry on microelectrode arrays. The chips are hybridized using RNA or

DNA copies of the RNA messages from genes of the plant or animals studied. The RNA targets are labeled

with fluorescent dyes which are scanned to measure the activity of each gene. In a simple example RNA

messages from a high yielding crop variety would be compared with a low yielding variety to identify

QTL for yield. Because many genes are involved data analysis is complex and handled using computer

programs.(7,8,9).Chips such as the barley gene chip (10) are commercially available but best left in

the hands of researchers. However, the products gleaned from micro array analysis will soon be

available in the form of varieties enriched in desirable QTL.
Does MAS actually work?
MAS does work according to a recent review but its development may , at first, be rather costly. An

Australian estimate of the costs of developing molecular markers in wheat was about $50,00 per gene

to about $90,000 per gene. However, that cost could return significant benefit. The review suggests

that the farmers who will use the selected strains should be brought into the development of effective

strains (11). A detailed description of the fundamental concepts of MAS was provided for beginners

(12). Micro arrays and their use in MAS is featured in a review which stresses the usefulness of the

technique in breeding programs stressing the need for cost benefit estimates to evaluate usefulness of

the micro array over conventional selection (13). The impact of QTL times environment interactions on

genetic responses to MAS pointed out that it was unwise to utilize QTL information from one environment

and execute breeding studies in another (14). The usefulness of gene information over the use of linked

markers was explored and it was indicated that selection based on the QTL themselves was preferred over

the use of linked markers. Furthermore, in cases where many QTL were expressed selection was most

successful when the QTL with large effects were used in selection while those with relatively less

impact on the trait were ignored (15).A direct comparison of phenotype and MAS selection for QTL in

sweet corn concluded that MAS is most effective when traits are difficult and costly to measure ,

incorporating DNA markers into a breeding program expedites selection progress and is cost effective

(16).
MAS in crop plants
QTL specifying yield
The following discussion will focus on a select examples from current literature rather than an

inclusive review of a mass of reports on applications of MAS. For the most part the reports deal with

identification of important QTL and their mapping rather than in production of high yielding strains.

In these reports the terms additive and epistasis are used to refer to the interaction involved in QTL

. Additive means that the QTL interact by adding increments such as yield or stress tolerance to the

crop while epistasis is like dominant and recessive alleles but the effect is between genes at separate

locations in the genome. Epistasis usually means that the genes are in a metabolic pathway leading to a

particular product. The first thing that comes to mind will MAS effectively identify genes for yield?

Linked markers were used to locate QTL for yield and yield related factors in wheat including

shattering and lodging resistance, heading date and plant height , 10 to 30 QTL were identified and

mapped to the chromosomes. The study is described as a benchmark for future identification of QTL for

yield (17). Using linked markers maize yield and its components were studied under different water

treatments at flowering time. Irrigated plots were compared with water stressed plots. Only a few of

the QTL were the same for both water regimes and this included both additive and epistatic loci most of

the QTL were different between the water regimes. The experiment suggests that gene expression differs

under drought conditions. It was concluded that drought tolerance should be considered from a genetic

point of view and considered a factor in QTL selection(18).MAS was used to improve drought adaptation

in maize. Under water stress conditions strains selected by MAS produced 50% more than control

hybrids, while yielding about the same as controls under normal conditions (19). One powerful rice QTL

linked to a DNA marker on chromosome 8 was identified and promoted as a candidate for an applied MAS

breeding program (20). Analysis of QTL in rice expression of the QTL was governed by additive effects,

epsistatic effects and environmental interactions. Biomass yield was expressed as straw yield and grain

yield. QTL governed the correlation between straw yield and grain yield while three QTL may govern a

negative correlation between straw yield and grain yield. 12 QTL for biomass yield had additive

effects, 27 QTL had additive –epistatic effects and 18 of the QTLwere effected by the environments with

additive-epistatic interactions. The study was aimed at genetic improvement of rice using MAS (21).

Identification of QTL for yield is the first step in selecting crops with genetically improved yield

capacity. The studies indicate that environmental factors are important in ensuring consistent high

yield.
QTL for food
A QTL present in wheat and in rice contributes to a high protein content in grain. Grain protein

affects the quality of bread and pasta and contributes to human health. The gene was found to

accelerate grain fill by earlier flag leaf senescence (22,23). A number of linked markers were used

to detect QTL associated with bread milling yield, dough rheology and baking quality of wheat. The goal

of the study was to provide basic information for MAS to improve bread quality (24). Rosaceae fruit

crops such as apple almond, peach, cherry and plums have had a number of QTL and other useful markers

mapped. The useful traits included pest resistance, fruit quality, self incompatibility and other

economic traits. MAS provides early selection several years before the characters can be evaluated in

the field (25). QTL associated with parthenocarpy (seedless) in cucumbers were identified using

linked markers. Four QTL were selected for MAS (26).
All of the MAS projects above seem to lead to results of considerable importance. It is presently

difficult to locate publications on MAS projects that have led to commercial releases but such reports

should soon be forthcoming. Since MAS depends on improvement of existing crops by selection there

should be no need for the regulatory approval employed with transgenic crops or crops with genes

modified within a laboratory from within a crop. As a word of caution, the term MAS has been employed

to enhance selection of transgenic modifications. As for example in the selection of a rice gene that

confers submergence tolerance (27). The term MAS has not been limited to improvements without genetic

modification so we must be careful about what we approve
MAS in animal breeding
Animal breeders have been quicker to apply micro arrays to identification of QTLs than have plant

breeders, at least as far as scientific publications are concerned. In animal breeding micro arrays

have been applied beyond QTL identification to practical selection experiments. Micro arrays have been prepared to identify genes involved in milk production. The chips used in

analysis identified a number genes for enzymes, growth factors and milk proteins. Many significant

associations between combined genotypes and milk production were observed. It was proposed that the

chips may be used for dairy cattle paternity anlysis (28). Earlier marker assisted selection of QTL

based on linked markers in dairy cattle showed an increased genetic gain in use of MAS(29). QTL

for carcass traits in Japanese Black cattle were identified using linked markers. The QTL governed

carcass mass , rib thickness and marbling (30). A bovine QTL viewer has been prepared providing a web

accessible database of bovine QTL including data from both milk and beef cattle (31). In pigs micro arrays have proved useful in identifying QTL related to back fat tissue (32). Human micro

array chips were found to be available and suitable for gene expression analysis in pigs (33). A pig

stress related marker was identified and by selecting against its active allele both meat quality

And drip loss was improved (34).
Micro arrays have been applied to gene activity in the shell glands of chickens. High and low egg

producing chickens were compared and a number of more active genes were identified in the glands of

the high egg producing chickens. The more active genes will be used in MAS (35). Fat chickens are

considered undesirable , micro array analysis was conducted on livers of fat and lean chickens. The

more active genes in lean chickens may be used in MAS (36). MAS based on a multi-trait economic index

for chicken genes was devised proving useful so long a relatively large (around 159 animal) progeny was

used in the prediction equation (37).
MAS is beginning to be the focus of attention in both plant and animal breeding. Two years ago I wrote

an article about MAS that pointed out the valuable aspects of the technique but questioned whether or

not the MAS would be effective when applied to QTL with relatively small contributions from numerous

genes.(38). In the intervening years micro arrays appeared and became available for animal and crop

improvement. The use of micro arrays seems to have opened the door to the immediate use of MAS in QTL

breeding for important characteristics.
MAS and QTL should enter the vocabulary of farmers. Corporations including Monsanto and Syngenta have

invested heavily in the program. A number of gene chips have begun to appear on the market for crops

such as maize, rice, beans, etc. In food animals chips for cattle, pigs, sheep , chickens and even

fish. As indicated earlier pig breeders have used human chips for selections. Farmers in developing countries and even some farmers in the developed world face the growing control

of seed production by a few multinational corporations. One solution has been to help the farmer breed

varieties tuned to the local environment and free of the greedy demands of seed corporations. It is

highly unlikely that indigenous farmers will take to MAS and molecular genomics. However, those

scientists working with indigenous farmers would recognize markers linked to valuable agronomic traits

and pass on that knowledge to the indigenous plant breeders to assist them in making selections that

are beneficial.
In the long run it seems likely that MAS will play an important role in plant breeding. MAS should not

affect organic certification because transgenes are not introduced into the crop. Molecular genetics

is used only in analyzing the crosses. Nevertheless, MAS has far more to offer in crop and animal

improvement than genetic modification. It has ,as well, grown clear that transgenic crops may be

labeled as being produced using MAS and that is something to guard against. There are newer

developments in which the methods used to produce transgenic crops are used to produce crops or genes

are taken out of a crop and then the genetic code is reprogrammed followed by the reintroduction of the

reprogrammed genes into the crop. Organic farmers should decide about organic certification of such

crops and not leave the matter to government bureaucrats and corporation lawyers.
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