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You wake up in the morning, put on a cotton shirt, brew coffee and make
toast. The day has hardly begun and already you have dabbled in some of
the great mysteries of plant evolution. For most crops – including cotton,
coffee and wheat – have long, tortuous biological histories and as a result
carry a confusing mix of genes and chromosomes inherited from different
ancestors. Identifying those ancestors can be a fearsome task. The clues
are certainly there, etched tantalisingly in the DNA of the crop plants.
The problem in the past has been how to read them.

Now, researchers believe they have the answer. The secret, they say,
is to ‘paint’ the plant chromosomes before trying to understand their origins.
The approach owes little to fine art. It involves persuading the DNA in
plant chromosomes to ‘mop up’ fluorescent ‘probes’ – molecules themselves
made of DNA – that can act as a guide to the ancestry and character of the
chromosomes. The palette of colours available to the chromosome painter
includes reds, yellows and blues. The result is a multicoloured display
that can be used as the botanical equivalent of a paternity test. Such
displays are also helping plant breeders to monitor the mixing of DNA that
is crucial to creating cross varieties which combine the beneficial traits
of their parents.

Although chromosome painting is used throughout biology, especially
in medicine, molecular biology and genetics, some of the most dramatic results
are coming from plant science. ‘From a breeding point of view, it’s an
absolute godsend,’ says Michael Wilkinson, who studies potato genetics
at the Scottish Crop Research Institute in Invergowrie. Equally excited
are botanists searching for the wild ancestors of crops. Already, chromosome
painting is helping such researchers to solve the problem of how the tobacco
plant evolved. In future, they will want to use it to trace the ancestors
of potato, coffee, peanut and cassava.

The technique is based on a phenomenon called ‘DNA hybridisation’ (not
to be confused with plant hybridisation). Though technically demanding,
it is conceptually straightforward. First, researchers ‘unzip’ part of a
chromosome’s double helix, producing two single strands of DNA. Then they
supply these DNA strands with alternative partners – short fragments of
DNA tagged to molecules such as dyes which can readily be made to fluoresce.
What happens next depends on the DNA sequences of the probes: if any of
them are a good match for the DNA that has been unzipped, they will cling
to it, advertising their whereabouts by fluorescing. Moreover, the probes
can be persuaded to cling to DNA inside cells as well as in test tubes.
Hence the official name of the technique – fluorescence in situ hybridisation,
or FISH for short.

The form of the final canvas depends crucially on the design of the
probe. Some probes are specialists that will highlight just one stretch
of DNA in the chromosomes, sometimes a single gene. They are particularly
useful for mapping the layout of genes on chromosomes. Other probes are
generalists that are capable of lighting up entire chromosomes. In many
cases, researchers use a potpourri of probes produced by chopping up the
entire genome of a particular plant species.

Cellular apartheid

The idea of painting plant chromosomes took shape independently in at
least two laboratories, one in Britain, the other in Canada. In the mid-1980s,
Mike Bennett and Pat Heslop-Harrison, then at the Institute of Plant Science
Research in Cambridge, a laboratory run by the Agricultural and Food Research
Council, were using conventional microscope techniques to investigate how
plant cells store and organise their various chromosomes. Studying hybrid
plants created by crossing a type of barley with a type of rye, the researchers
discovered something that challenged existing dogma: when the hybrid’s cells
divided, its chromosomes were not jumbled together. Chromosomes of barley
origin were kept separate from those of rye origin – and each collection
of chromosomes occupied its own territory in the dividing cell. It was a
classic case of cellular apartheid.

Before investigating this any further, the researchers needed to know
whether the standoff persisted when the cells were not dividing. But here
the standard microscopical methods let them down. ‘We had a dream, ‘says
Bennett, ‘that we would have some simple system for colouring chromosomes
in a way that would allow us to tell which set came from which parent.’
The dream acquired more substance as the team began to sift through the
medical literature. They were joined by Trude Schwarzacher, a biologist
who had worked on fluorescence hybridisation at the Los Alamos National
Laboratory in the US. She had studied cultured cells carrying a mixture
of chromosomes from humans and Chinese hamsters, and as a result knew all
about techniques for identifying human chromosomes with DNA probes.

But would the technique work in plants? Many of Bennett’s and Heslop-Harrison’s
colleagues were sceptical. ‘They thought that the best we could expect
would be to distinguish small, isolated regions of chromosomes,’ recalls
Heslop-Harrison. ‘It came as quite a surprise that we could actually paint
the whole length of the chromosomes relatively uniformly.’

The experiment on barley-rye hybrids had been arranged so that chromosomes
originating from rye would shine with a bright greenish-yellow hue. The
researchers stained the remaining chromosomes – the ones from barley –
with a second fluorescent dye, this time a red one. The upshot was an image
of breathtaking clarity, from which it was clear that the rye and barley
chromosomes remained separated even when the cells were not dividing. Barley
chromosomes tended to be at the centre of the nucleus, rye chromosomes at
the periphery.

No one yet knows whether this separation serves any bio-logical purpose.
But one guess is that a chromosome’s position in the nucleus of the cell
affects the activity of the genes it carries. In the hybrids Bennett and
Heslop-Harrison were studying, for example, there was evidence that the
genes in the outer region of the nucleus exerted more influence on the
plants’ appearance and behaviour.

In science, good ideas have a habit of popping up independently in more
than one place. So it was that in the mid-1980s, a trio of researchers at
the Plant Research Centre in Ottawa – Hoan Le, Ken Armstrong and Brian
Miki – also set out to develop techniques for painting plant chromosomes.
Like the British team, they used probes produced by chopping up plant genomes
to paint the chromosomes of a hybrid, in this case triticale, a hybrid of
wheat and rye. Since then, Armstrong and his colleagues have gone on to
use the technique to study the genetic makeup of wheat varieties and other
cereals produced by plant breeders.

Going for quality

One of the main botanical applications of chromosome painting is in
plant breeding. Plant breeders routinely try and improve crop plants by
crossing them with relatives that harbour useful genes. Breeders are keen
to bring new genes into bread wheat, for instance, to improve its performance
during bread-making, say, or its tolerance to salt. But there is a dilemma.
‘If you transfer a whole chromosome,’ says Heslop-Harrison, ‘you very often
transfer poor quality genes which you don’t want.’ In physical terms, the
breeder wants to transfer the smallest possible chunk of DNA from the donor
to recipient.

Heslop-Harrison and his colleagues are using chromosome painting to
monitor this process. Once the researchers have obtained a promising batch
of plants carrying DNA from some donor, they paint the chromosomes of those
plants using a probe that reveals the newly installed ‘foreign’ DNA. They
can then select plants with the smallest amount of foreign DNA for the next
stage of the breeding programme.

Researchers at the Institute of Grassland and Environmental Research
in Aberystwyth are also practising the art of chromosome painting. Huw Thomas
and his colleagues are studying rye-grass plants that incorporate pieces
of chromosome from other grasses called fescues. Using probes, they are
able to light up these foreign fragments and see how large they are. Mike
Leggett of the IGER is in charge of a similar study aimed at importing
disease resistance into oats from its wild relatives.

Chromosome painting promises to speed up the breeding programme. Suppose
breeders have two lines of rye-grass, each of which has some desirable trait
brought in from a fescue; one might be drought-tolerant, for instance, whereas
the other might be long-lived. How can those two desirable traits be combined?
The tried and trusted method is to cross the two lines, grow all the seedlings
and select plants which are both drought-tolerant and long-lived. The problem
is that it can take years to see if the cross has been successful. Chromosome
painting will enable researchers to inspect their seedlings at an early
stage and select those whose chromosomes light up in two places, showing
that they have incorporated two pieces of fescue DNA. One example comes
from Wilkinson and his colleagues Keith Harding and Joel Allainguillaume,
who are using chromosome painting as part of their effort to select disease-resistant
genes from wild relatives of the potato, of which there are at least 235.

By the late 1980s, it was clear that chromosome painting would have
much to offer plant breeders. But the idea that it would also help to solve
long-standing problems in plant evolution and taxonomy emerged more slowly.
That development came after Bennett’s move in 1987 to the Royal Botanic
Gardens in Kew, where he is now Keeper of the Jodrell Laboratory.

One of the great themes in plant evolution is the way new species can
be created from hybridisation (the sort that happens between plants, not
strands of DNA). One famous example is common cord grass (Spartina anglica),
a vigorous resident of mud flats, which arose when pollen of one species
strayed from its proper course and fertilised a receptive plant from a second
species. Here, the identities of the parents are known, but this is not
the case with other plants.

Could chromosome painting help? The answer came when Bennett was supervising
Simon Bennett (no relation), then a research student at the University of
Oxford. Simon Bennett was studying two closely similar grasses, Milium vernale
and Milium montianum. He suspected that M. montianum descended from a hybrid
of vernale and an unknown species. But after two years of conventional tests,
the answer was still elusive.

‘Then one day the penny dropped,’ says Mike Bennett. The researchers
realised that the painting technique could be used to unravel M. montianum’s
parentage. Working with Ann Kenton at Kew, they took DNA from the putative
parent (M. vernale) chopped it up and used it as a probe to paint the chromosomes
of its supposed descendant (M. montianum). The team reasoned that if any
of M. montianum’s chromosomes had come originally from M. vernale they would
cling tightly to the probe and fluoresce yellow. Obligingly, eight of its
22 chromosomes lit up, implying that they had indeed come originally from
M. vernale, or something very like it. The donor of the other 14 chromosomes
is still a mystery – it may be extinct.

The speed of the technique compared with traditional methods was remarkable.
‘It took a week to get to the answer,’ says Bennett. ‘One Friday you have
the idea. By the next Friday, you’re ready to publish. It’s real eureka
³¦´Ç³Ü²Ô³Ù°ù²â.’

Wild weed

Success with Milium prompted the Kew team to take on other challenges,
one of which was the origins of cultivated tobacco Nicotiana tabacum. Tobacco
originated in the Bolivian Andes of South America when two species hybridised.
Judging from conventional microscopy and biochemical techniques, one of
these species must have been an ancestor of N. sylvestris, a wild tobacco.
But the identity of the other parent has been the subject of debate, with
two candidates locked in contention, N. tomentosiformis (the favourite)
and N. otophora.

Bennett, Kenton and their two colleagues Alex Parokonny and Yuri Gleba
set to work to see if chromosome painting could resolve the argument. They
rapidly confirmed the role of N. sylvestris. When they probed tobacco chromosomes
with DNA from N. sylvestris, half of them fluoresced yellow. And the other
parent? Here the Kew team uncovered a more complex story: both contenders
contributed to tobacco’s parentage.

Chromosome painting lights up half of tobacco’s chromosomes in yellow
against a background of red – firm evidence of the plant’s split personality.
But a closer look reveals a wealth of extra detail. Since the two brands
of chromosome came together, they have occasionally swapped pieces, and
so some of the red chromosomes have yellow ends and vice versa. This process
is well known to geneticists as ‘translocation’. But chromosome painting
picks it out more swiftly than any other techniques.

‘This is not interesting just in a stamp-collecting way,’ says Bennett.
‘There are many practical applications.’ For example, since closely related
species will tend to share the same chromosome rearrange-ments, the fine
structure of the fluorescent regions could in principle be used to trace
evolutionary relationships between plants.

Researchers now want to apply chromosome painting to many other plants
– coffee, potato, peanut and cassava. Chromosome painting may help to decode
those histories, just as it did with tobacco, although there are likely
to be extra complications in some cases. For example, the ancestors of a
crop plant may be extinct, or may have changed character since they donated
chromosomes to the crop.

Bennett’s group, working with Michael Wilkinson at the Scottish Crop
Research Institute, has already begun work on potato. One idea is that the
cultivated potato Solanum tuberosum arose as a hybrid between an ancient
domesticated form, S. Stenotomum, and a weedy relative S. Sparsipilum. But
there are lots of competing theories and the researchers are not giving
anything away before they publish their results.

Teams elsewhere are interested in an assortment of other species. Heslop-Harrison
and his colleagues, for instance, are using chromosome painting to study
the evolution of plants such as brassicas and garden crocuses. Chromosome
painting is also likely to add to botanists’ understanding of more subtle
aspects of plant evolution.

The sex lives of plants are more exotic than those of animals, and the
genetic consequences can be extraordinary. Take the case of Festuca rubra
and Vulpia fasciculata, a pair of grasses that frequent sandy, seaside places.
Festuca puffs out liberal amounts of pollen and, pollen being what it is,
the two tend to hybridise. The hybrid plants thrive but are almost completely
sterile. Only occasionally do they manage to breed with nearby Festuca.

Nevertheless, such sexual shenanigans can profoundly affect the genetic
make-up of plants. In this case, genes (or longer segments of chromosomes)
could move from Vulpia to Festuca, with the hybrid acting as a genetic halfway
house. Clive Stace and John Bailey of the University of Leicester are trying
to find out more about this genetic traffic. The idea is to take Festuca
plants and use chromosome painting to search for any trace of Vulpia DNA
among their chromosomes. If the plants contain any Vulpia DNA, parts of
their chromosomes should light up – and the team will have firm evidence
of genetic traffic between the species. ‘It isn’t as easy as it sounds.
In practice there are 101 variables that you have to get right,’ says Stace.

But if they succeed, it will be further evidence that chromosome painting
is becoming an indispensable aid to researchers trying to unravel the mysteries
of plant evolution. ‘Chromosome painting for me was a dream come true,’
says Mike Bennett. ‘This method is now available as another major weapon
in the armoury to address questions of origins of species.

Stephen Young is a freelance writer based in Wales.

* * *

How to become a chromosome painter

Suppose the plant under test is a hybrid plant produced by crossing
barley and rye. If the aim is to paint the chromosomes that came originally
from rye, then the probe would be DNA from rye cells, broken into pieces.
This probe would latch on to rye chromosomes in the hybrid, making them
fluoresce in whatever colour the painter chooses. Yellow is a common choice.
The painter would then add another dye that attaches to the remaining chromosomes
and makes them fluoresce in a contrasting colour, such as red. So the end
result in this case – the finished canvas, so to speak – has the rye chromosomes
in yellow and the barley ones in red. It is the ultimate still life.

Sometimes the painter has to adopt a slightly more subtle approach,
especially if the chromosomes in the hybrid come from two very closely related
sources. That closeness means that the probe might bind to chromosomes
of both kinds unless steps were taken to prevent it. In a case like this,
the painter adds nonfluorescent ‘blocking DNA.’ In the case of a barley-rye
hybrid, this would take the form of chunks of barley DNA. The blocking DNA
would be most likely to bind to the barley chromosomes, leaving the rye
ones free to link up with the probe.

It seems a minor miracle that one can spread chromosomes on a slide,
subject them to such a huge number of chemical indignities and emerge with
a coloured portrait that is both elegant and informative. It has to be said
that the technique, with its 27 different steps, demands considerable deftness,
not to say motivation. Adapting it so that it can be applied to any particular
species often requires much skill. And it is still expensive. Yet researchers
are turning increasingly to chromosome painting in all manner of areas.
‘If you want to answer a question – when you’ve done this, you know the
answer,’ says Mike Bennett from the Royal Botanic Gardens in Kew. ‘You don’t
just narrow down the probabilities, you get very close to certainty.