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In a really neat piece of work based around a remarkably simple bit of engineering and some textbook genetics, a team of scientists has found a way to regenerate a plant’s parents through breeding — a technique they call “reverse breeding”. This clever bit of research, which is described in a paper appearing in Nature Genetics, should be applicable to a wide range of crop species, opening up the possibility of significant advances in crop improvement and breeding programmes.

Most crops today are grown from hybrid (or F1) seed, which is  produced by using two highly inbred lines as parents.  The resulting plants have very little genetic variation and quite uniform growth, which is important for modern, mechanized farming.  The hybrids also tend to perform better and have higher yields than non-hybrids.  The source of this “hybrid vigor” is poorly understood, though one possibility is that the genetic input from each parental line serves to mask the shortcomings of the other, resulting in hybrid plants that are superior to either parent.  Unfortunately, hybrid vigor and uniformity is lost in subsequent generations because of the mixture of genetic material during sex.  Breeders therefore have to continuously re-create the hybrids by crossing the inbred parental lines.  This means that any improvements have to be bred into the parental lines; it also restricts the breeders’ ability to take advantage of outbreeding in their breeding programme.

The loss of hybrid vigor is because of genetic variation introduced during meiosis, the production of eggs and sperm.  Most of this variation isn’t from mutation but is the result of two processes called assortment and crossing over.  Our DNA is packaged into chromosomes which are arranged in pairs, each of which consists of one maternal and one paternal chromosome.  Assortment simply means that each pair is randomly divided during meiosis, resulting in each sperm or egg cell having a random mixture of maternal and paternal chromosomes.  In addition, before this division happens the chromosomes of each pair “cross over”; they swap bits of DNA between themselves and so are no longer intact maternal or paternal chromosomes, but rather a mixture of DNA from both.

Using playing cards as an analogy, everyone starts with a hand made up of pairs of red and black cards, representing maternal and paternal chromosomes.  During meiosis, only half of the hand goes into each sperm or egg cell; assortment is simply shuffling before passing along cards during meiosis — there’s no guarantee that the cards chosen will be the same colour, but the individual cards are all still intact.  By contrast, crossing over would mean cutting out parts of one card from a pair and gluing them into the other before shuffling.  The resulting cards would be a mish-mash of red and black, making it impossible to recreate the original hand.  If this step could be avoided, then recovering the original (parental) hand would just be a question of reshuffling enough times.

That’s precisely what the researchers did.  Since crossing over scrambles chromosomes into a mix of maternal and paternal DNA, it becomes impossible to regenerate the original parents that were bred to create the hybrid.  By blocking crossing over, the researchers created plants which passed along their maternal and paternal chromosomes intact, making meiosis nothing more than a reshuffling of chromosomes.  Each sperm or egg cell therefore contained a random combination of intact maternal and paternal chromosomes; reverse breeding to regenerate the original parental lines is then simply a matter of juggling chromosomes.  This task was made much easier by the fact that it’s possible to recreate an entire plant from a single sperm or egg; there are various techniques for doing this, but the basic idea is to double the DNA, making each chromosome duplicate itself to form a pair.  By generated plants from sperm and eggs which hadn’t undergone crossing over, the researchers were able to start with a hybrid plant where every pair of chromosomes was composed of one chromosome from each parent and breed plants in which each pair of chromosomes was made of two identical copies from the same parent. Recovering the original parents was then simply a matter of crossing these plants in the right combination.  In fact, this technique doesn’t just recreate the original parents; it also recreates several sets of possible parents — that is, pairs of plants which, if bred together, would produce the hybrid.  For example, the second and third reverse bred plants in the figure aren’t the original parents, but a cross between them would also generate the hybrid.

In order to block crossing over, the researchers reduced the expression of a gene called DMC1 which produces an enzyme that is important for this process. Since DMC1 is widely conserved between different plant species, it should be straightforward to apply this technique in different crops even though this study used Arabidopsis thaliana, a small relative of mustard which serves as the model organism for plant genetics. By making sure that the DMC1 reduction was dominant, the researchers were able to be sure that the regenerated plants weren’t harboring an unseen copy; this is quite important since plants with decreased DMC1 also had lower fertility, which would be unacceptable in crops.

It’s clear that this work will have important practical applications for plant breeders. It also makes it quite straightforward to create chromosome-substitution lines — that is, plants which have one chromosome from variety A and the rest from variety B; this will be a useful tool for breeders and for plant biologists in general. My favourite thing about this study, though, isn’t the practical benefits it will generate but rather its striking simplicity; like a lot of the best scientific work, it seems so obvious and straightforward in retrospect that I wish I’d been clever enough to think of it.

Ref:
Wijnker, E., van Dun, K., de Snoo, C., Lelivelt, C., Keurentjes, J., Naharudin, N., Ravi, M., Chan, S., de Jong, H., & Dirks, R. (2012). Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant Nature Genetics, 44 (4), 467-470 DOI: 10.1038/ng.2203

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