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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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