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Prof. Piotr Ziółkowski, Institute of Molecular Biology and Biotechnology – helps developing intelligent breeding strategies for crops

High-yield barley plant that is drought and pathogen resistant at the same time – sounds like science-fiction? Not at all – we are in need of those kinds of crops now. Prof. Piotr Ziółkowski, the PI in the Institute of Molecular Biology and Biotechnology (Faculty of Biology, AMU), is conducting studies, which could help developing intelligent breeding strategies for crops.

Prof. Piotr Ziółkowski, together with his team, is conducting fascinating projects, which have a good chance of opening new doors for modern agricultural studies. Scientists are working to identify factors controlling meiotic recombination. Experiments are heading towards the creation of intelligent breeding strategies and the obtained results might have practical utility.

Biologists perfectly know what „meiotic recombination”, or simply crossing-over, means. However, since this article will be read by non-specialists as well, let us explain what recombination is. Without meiosis, as we remember from biology lessons, formation of gametes (germ cells) wouldn’t be possible. All eukaryotic cells, including those in humans, contain two sets of chromosomes – one from a mother, one from a father – which fuse together during fertilization. Without meiosis, with each new generation the chromosome number would double, which would lead to chaos. Because of meiosis, the number of chromosomes in gametes decreases by half, so in successive generations chromosome set does not change.

So, what is the purpose of recombination? It leads to reshuffling and proper exchange of genes in chromosomes, which we got from mother and father. Because of that, the chromosomes which will finally end up in a gamete are the mosaic of chromosome fragments obtained from both parents.

Recombination is strictly controlled – in most eukaryotes there are no more than 2-3 crossing-over events per each chromosome pair.

Selection versus variation

Prof. Piotr Ziółkowski got interested in genetic variation when he was on his postdoctoral fellowship at the University of Cambridge. He conducted research together with Prof. Ian Henderson, trying to provide answers to: are the same species individuals subject to differences in meiotic recombination rates?  How to increase the frequency of recombination and target it into specific places on a chromosome? Those issues are important for plant breeders, who care about generating species exhibiting specific features, for instance high productivity and drought-resistance at the same time. This is not a simple task, as very small genetic variation of crops stands in the way, which results from thousands of years of artificial selection by men.

Wheat spikes of the same variety, but growing on different fields, are identical. Farmers were crossing cereal plants guided primarily by the yield they could obtain, and not realizing that artificial selection towards one feature automatically eliminates variability within other features, e.g. drought resistance. If the climate in a given historical period was wetter, such changes in the population structure were not noticed by breeders. However, with the arrival of dry years, there was a lack of adequate crop variation and there was no way to supplement it. In natural wild populations, very high variation means that regardless of the conditions, some individuals in those populations survive, thus ensuring the survival of the species. – Prof. Piotr Ziólkowski explains.

Inteligent breeding

Therefore, breeders are trying to enrich the diversity by introducing new gene versions (gene alleles) from unrelated lineages or from wild populations. The problem is that by doing so, they can also lose some yield and at the same time transfer some primitive undesirable features, e.g. the way the chaff is combined with the grain, which hinders the processing. Boosting the recombination frequency seems like a good solution, because it increases the chance that only the desired chromosome fragment will be transferred. The second strategy is to direct the recombination to a specific place on the chromosome, where the gene interesting for the breeder is located. Both strategies would be groundbreaking, especially in the cultivation of crops with very large genomes. In the case of cereals, it turns out that 2-3 recombination events per one chromosome pair are definitely not enough to facilitate effective breeding.

As a result, the breeder creates a new variety for several years, using huge populations, in which he is looking for the right combination of alleles! Often this process ends in a failure, because not only is the recombination random, but in many cases it occurs at the very end of the chromosome. Actually, about 80 percent of the chromosomes in cereals do not recombine at all, so there is no physical possibility of transferring some genes between different lines.

Progress in the field may be influenced by a new project of Poznań scientists, which aims to examine what factors influence recombination rate and localization. The point is that the recombination is not random – scientists want to direct it to a specific place on the chromosome.

– We could transfer specific gene variants from wild lines to crops thanks to “recombination targeting”. First of all, it would be effective even without having to use gigantic populations, which generates huge costs. The project is heading towards intelligent breeding, i.e. we deal with breeding processes in a way that was impossible until now. We know which genes we want to transfer and we do it.

Evolution is not in a rush

As we have already mentioned, nature itself ensures the greatest variability in the population. This is done by the appropriate mechanisms that direct recombination to the most variable chromosomal regions. This means that if one chromosome fragment is almost identical in both parents while the other region is significantly different between them, then the crossover will be directed to more variable regions. This is one of the main discoveries made
by Prof. Piotr Ziółkowski while he was a postdoc in Prof. Henderson lab. Moreover, he has also identified a gene encoding an enzyme, which plays a crucial role in the recombination as its increased dosage in the cell contributes to a proportional increase in the recombination frequency in meiosis.

– Evolution is not in a rush, as it does not matter how fast gene mixing occurs. What is more, this process in nature cannot take place too quickly as it breaks down combinations of evolutionarily beneficial alleles. It is different in the case of cultivation, in which new breeding varieties are desirable as soon as possible. Therefore, increasing the recombination frequency can be critical in supporting farmers. Currently, the identified pro-recombination factor is tested to be introduced in barley and wheat cultivation.

Recently, a lot of progress has been made to understand the phenomenon of recombination. The next step is to control and target it to the exact regions. Biologists from AMU have created tools that allow measuring recombination frequency in very short intervals on the chromosome. This is a so-called reporter system.

– How can you statistically measure where crossover occurs if the recombination frequency is very low? To determine this, it would be necessary to test millions of individuals, map crossover sites and draw the resulting system – Prof. Ziółkowski explains. – Instead, we have created a system based on reporter genes separated by a short distance on the chromosome. They encode fluorescent proteins, green (GFP) and red (dsRed), which are produced in seeds. By targeting factors affecting recombination to the site between those two reporter genes, we can influence the crossover rate at that site. Later, based on the segregation of the fluorescent reporters we are able to measure the differences in recombination frequency. Thus, it is possible to quickly determine which factor, and to what extent, stimulates recombination. By testing various proteins and different strategies of delivering them to the chromosome, we will be able to find the most promising and worth using strategies in modern breeding.

Recombination and GMO

While buying firm and juicy tomatoes in the store, we rarely think why they look like that. The progress we have been making for thousands of years has brought about big changes. Traditional breeding is responsible for this, which at the genetic level does not differ significantly from GMOs, although it does not cause as much controversy. Also in this case, we shuffle genes from different varieties, arranging them in the way we choose. Imagine that in the ancestor of corn, one cob contained up to 7 grains, or wild tomatoes, which are the size of berries. Another example is rice, that in nature immediately spills the seeds as soon as they develop, preventing them from harvesting. Maize as we know it today is a hybrid maize, obtained by crossing two distant lines each time to increase the yield. Not only the introduction of new genes from another species is treated as GMO, but also the increase in expression of naturally occurring factors in a given organism, even though a similar phenomenon occur in natural selection. Despite this, GMOs in Europe have been fortified with many regulations that hinder its use.

Increasing the recombination frequency in chromosomal regions that differ between parents is a natural process that does not require genetic modification. Researchers from AMU are testing whether this phenomenon occurs in crops other than the model organism, which is Arabidopsis. It can become a promising breeding strategy, using the selection of appropriate parents for crosses. However, the strategy of directing recombination to specific places on a chromosome requires the use of the CRISPR technique, which will probably be identified by the European Union as GMO.

– We mainly carry out basic science, as in our research we are aiming to understand the mechanisms of shaping the recombination process by various factors. The application aspect is very interesting for us, but in this situation our hands are tied. Perhaps in the future this problem will be solved – Prof. Piotr Ziółkowski sums up.