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| An old rubber tree in the Amazon rainforest exhibiting old scars from rubber tapping |
Currently 30 years of genetic improvement work is needed to obtain more productive varieties of rubber. Geneticists at the University of Campinas expect to reduce this deadline to less than 10 years and make Brazil self-sufficient in latex production
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Between 1870 and 1910, Brazil was the world's largest producer of latex, the raw material for rubber. Today, the country occupies the 10th place. The 190,000 tons of latex produced in the country in 2016 were sufficient to supply only 35% of domestic demand. More than half of Brazilian production comes from the State of São Paulo. Such prominence of the producers of São Paulo is due to the work of genetic improvement of rubber trees that have been made since the 1970s at the Instituto Agronômico de Campinas (IAC). But the process of rubber tree breeding is too slow. Today, it takes 30 years of continuous work to obtain varieties with high productivity of latex and better adapted to the climate and soils.
Between 1870 and 1910, Brazil was the world's largest producer of latex, the raw material for rubber. Today, the country occupies the 10th place. The 190,000 tons of latex produced in the country in 2016 were sufficient to supply only 35% of domestic demand. More than half of Brazilian production comes from the State of São Paulo. Such prominence of the producers of São Paulo is due to the work of genetic improvement of rubber trees that have been made since the 1970s at the Instituto Agronômico de Campinas (IAC). But the process of rubber tree breeding is too slow. Today, it takes 30 years of continuous work to obtain varieties with high productivity of latex and better adapted to the climate and soils.
An important genomic research at the University of Campinas (Unicamp) is yielding dividends that promise to greatly accelerate the time of improvement of the rubber tree. "Our research on the genome of rubber trees is expected to reduce the breeding time from 30 years to just 10 years," says plant geneticist Anete Pereira de Souza, leader of the Laboratory of Molecular Genetic Analysis at the Center for Molecular Biology and Genetic Engineering, of the Biology Institute, at Unicamp.
The geneticists Lívia Moura de Souza and Luciano dos Santos are responsible for the search of genes of interest for the improvement of the rubber tree. In two articles, one published in 2015 in PLoS ONE, and another one that has just been published in Frontiers in Plant Science, the team reveals the discovery of regions defined by 576 molecular markers of interest to the genomic improvement of the rubber tree.
These are 576 potential opportunities to obtain more vigorous, more productive and disease-resistant seedlings, thus speeding up all the rubber tree breeding work.
Learn why this achievement is so important. But first, a little history.
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Biopiracy in the 19th century
The discovery of the vulcanization procedure in 1839 by the American Charles Goodyear led to the rubber boom. Between 1870 and 1910, Brazil was the main world producer of latex, the sap extracted from rubber trees that is the raw material of natural rubber. The demand for latex grew year-on-year. At that time, rubber accounted for 24% of Brazilian foreign trade.
In 1890, the first attempts at managed cultivation began, with the creation of farms with thousands of rubber trees, lined up side by side. It was a fateful decision.
The rubber tree (Hevea brasiliensis) is a species native to the Brazilian Amazon. Inside the forest, the wild trees are spread sparsely in the forest, far from each other. This distance hinders the spread from one tree to the other of the fungus Microcyclus ulei, the causative agent of a disease called South American leaf blight. The fungus only attacks the youngest leaves, killing them. Unable to produce a new generation of leaves, the rubber tree dies.
This is what happened to the big rubber plantations planted by the Amazon farmers. The most emblematic case was Fordlandia, a model city in the Brazilian state of Pará, on the banks of the Tapajós River, founded in 1928 by the American millionaire Henry Ford for the production of rubber on an industrial scale for its automobiles. The huge clump of Fordland rubber trees proved to be a feast for the Microcyclus fungus. Latex production in Fordlandia was never able to take off, and the project was finally abandoned in 1945.
Long before that, in 1875, the British botanist Henry Alexander Wickham smuggled 70,000 rubber tree seeds from Brazil to England, which were sown in the Royal Botanic Gardens, at Kew. About four percent of these germinated, and in 1876 about 2,000 seedlings were sent to Ceylon (now Sri Lanka) and to Singapore.
Rubber trees acclimated so well to Southeast Asia that, when the South American leaf blight spread through Brazilian rubber plantations, Southeast Asia eventually became the world's largest producer of natural rubber.
Although technically there was no Brazilian law prohibiting the export of rubber tree seeds in 1876, there was, however, a requirement for the issuance of an export license, which Wickham obtained under false pretenses, claiming that the seed load he would send to London would be botanical material intended for a herbarium. By his feats, Wickham came to be decorated with the title of knight of the British empire.
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Production of latex in Brazil, today
World rubber production in 2016 was 12.4 million tonnes. Thailand and Indonesia, the two largest producers, account for 60% of production. The seven biggest producers, all Asian, concentrate 90% of production. The 8th largest producer is Guatemala and the 9th is Ivory Coast. Brazil occupies the 10th place, accounting for only 1.5% of world production.
Brazilian production comes from states in the Southeast, Midwest and Northeast regions. Production in the Amazon is minimal due to the presence of leaf blight, which has remained restricted to the region for more than a century - and has never infected rubber plantations in other countries.
The 190 thousand tons of latex produced in Brazil in 2016 were sufficient to supply only 35% of domestic demand. More than half of the production comes from the State of São Paulo. In its natural habitat, the Amazon, the rubber tree is adapted to grow under a regime of high temperature and humidity. The same does not occur in southeastern Brazil. On the other hand, in the southeastern region the climate is dry and cold enough to prevent the proliferation of the South American leaf blight. The fungus does not survive in the cold.
The success of the rubber crop in São Paulo is mainly due to the breeding of rubber trees that have been made since the 1970s at the Instituto Agronômico de Campinas (IAC), by researchers such as veteran Paulo de Souza Gonçalves, who works with rubber trees since 1974.
Gonçalves explains that the work in the IAC aims to select the seedlings with greater productive potential, that is, those capable of producing more liters of latex during the tapping of the tree. Rubber tapping is the method used to extract the sap (latex) from the trunks. The latex is harvested by slicing a groove into the bark of the tree at a depth of a quarter inch with a hooked knife and peeling back the bark.
"Another objective of the improvement is to obtain trees that produce well in regions of drier and colder climates, which are acclimatized more and more to the conditions of the Brazilian southeast, in order to expand the production of latex per hectare planted with rubber trees."
If, on the one hand, the improvement work of the rubber tree in the IAC has yielded good results, on the other hand the whole process until obtaining varieties interesting to the planting is too time consuming. The methodology of classical genetics breeding, the one promoted by Gonçalves in IAC, involves a life of dedication. "30 years is the time of improvement needed to obtain new production varieties. You have to have a lot of patience," says Gonçalves.
"In the first place, we must select the most productive rubber trees," explains Gonçalves, who in the 1970s and 1980s participated in several expeditions to collect rubber tree seedlings in the Amazon. "We asked the rubber tappers to take us to the trees where they got more latex. There are in the Amazon, lost in the middle of the forest, wild trees from which we can extract in a single tapping 4, 5, 6, even 8 or 9 liters of latex!"
Once a productive specimen is found, the work of improvement involves three steps. In the first, the productive plant is crossed with another, which may not be as productive, but is more tolerant of cold or drier climates. From this crossing germinate thousands of plants that will grow and develop over two and a half to three years.
"Then comes the second step, when we select for multiplication by cloning those plants that are the most vigorous," explains Gonçalves. From each of them 100 to 200 specimens are cloned, which in turn are replanted in the experimental stations and allowed to grow until the age of seven years.
"When trees reach age seven, we start bleeding to do latex production tests. After a year, we select those trees that are the most productive, usually 5% of the total."
This is when the third stage of improvement begins. The most productive specimens are again multiplied by cloning and distributed among the rubber farms of the State of São Paulo, in order to test their performance in different soils and diverse micro-climates. "Then we have to wait another ten or eleven years in order to be able to select those specimens who are most productive for a given type of soil or microclimate." These specimens are then cloned and finally recommended for large-scale planting," says Gonçalves.
Up to this point, 20 years have passed since the beginning of the breeding process. It will take another ten years before the improved clones and distributed to the farms are in full production.
How can molecular biology studies developed in Unicamp laboratories shorten this enormous 30 year period?
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| Nursery with rubber tree seedlings |
100 thousand genetic variations
All the material collected among the wild rubber trees in the 1970s and 1980s by researchers such as Paulo Gonçalves are deposited in germplasm banks at IAC and the Brazilian Agricultural Research Corporation (Embrapa). There are thousands of seeds collected in rubber trees in the interior of the forest in the Brazilian States Acre, Amazonas, Mato Grosso, Pará and Rondônia.
Geneticist Lívia Moura de Souza visited germplasm banks in the cities of Belém, Brasília and Ilha Solteira, and also in French Guiana, in order to obtain material for molecular research. She worked with the leaves of a total of 1,117 trees. Many regions where collections of seeds and leaves have been made 40 years ago today are totally deforested, as is the case of the State of Rondônia. "This is where the most productive material used by rubber planters comes from. The place where the collection was made today no longer exists. It was all deforested. This means that the genetic material that may have been in the region's rubber trees today is only available in germplasm banks. In nature, it no longer exists," says Livia. "The only way to gain access to this material is in the collections, herbs, and germplasm banks I have visited."
The rubber tree (Hevea brasiliensis) belongs to the genus Hevea, which comprises 11 species. Lívia worked with leaves of four species: H. brasiliensis, H. guaniensis, H. benthamiana and H. pauciflora. Although commercial latex is only extracted from H. brasiliensis, the other species are very important in the study, since they may be sources of genes of interest with possible use in the improvement of the rubber tree.
"In the first of the two works published, we wanted to understand the diversity of material at our disposal," says Livia. The material is divided into two distinct groups. One group is totally wild, from the trees that grew alone in the Amazon rainforest. The other group involves leaves collected between trees of the improved material planted in the experimental breeding stations of plants.
"We tried to collect the leaves with the use of a slingshot. It did not work, because the trees were very tall," recalls Anete Pereira de Souza. "We had to use a crane to get some leaves from each tree so we could analyze its molecular markers."
As regards the group of the improved material, Livia observes: "once one selects molecular traits (alleles, in the genetic jargon) that improve the plant, other are eliminated. Our work aims at the search for these rare alleles, those that are not found anymore in the improved material."
Using a technique called microsatellite molecular markers, Livia found 408 alleles in both groups. Of these, 89 were rare alleles (25% of the total), because they were present only in genetic material from wild rubber trees. Among the cultivated rubber trees, those rare alleles were lost during the breeding process.
"This first job was to verify that the genetic diversity of the wild rubber group is far superior to that of the improved rubber trees," says Lívia.
The next step of the research, which resulted in the second article, involved the further genetic study of all the material. Of the 1,117 specimens whose leaves were collected in germplasm banks, Livia excluded those that were most related, selecting only those 368 specimens with the most divergent genome. The material of 254 improved specimens was added to them, totaling a set of 626 specimens analyzed.
Here the chosen research method was genotyping with SNPs. Genotyping is the process to identify the genetic makeup (genotype) of each of the 626 specimens. SNPs ("snips"), is an acronym for "single nucleotide polymorphisms". SNPs are one of the most common types of genetic variation markers.
Genotyping with SNPs resulted in the discovery of approximately 100,000 SNPs. Of these, 77,600 SNPs were found in material from wild rubber trees, while 21,300 SNPs came from improved rubber material. "As you can see, there are far more SNPs in germplasm banks than in the improved rubber population," says Livia. "This gives a good idea of the diversity of the genetic basis of wild rubber trees."
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| Leaf collecting for genomic research, with the help of crane (divulgation) |
The Genomic Promise
It is precisely in this great diversity that Livia de Souza, Luciano dos Santos and Anete Pereira de Souza, and collaborators of different research institutions in Brazil and France, expect to detect the genes of interest for the improvement of the rubber tree, providing more vigor, better adaptation to cold and low humidity, greater resistance to diseases and pests.
Once 100,000 SNPs were found in the analyzed material, the next objective was to try to know in which chromosomes they are located. The genome of the rubber tree has 18 chromosomes and has been partially sequenced - but, according to Livia, this genome has not yet been well assembled. The whole analysis of the genomic data of the research was carried out by geneticist Luciano dos Santos, assisted by colleagues from the Luiz de Queiroz College of Agriculture, University of São Paulo.
"Luciano was able to locate the chromosomes where there are 576 SNPs. The next step of the work will be to perform the genomic selection of the mapped SNPs. We need to find out which characters each of those SNPs confers to the plants, ie, what phenotypic characteristics they refer," says Livia.
"In the medium term, when we can figure out the function of each of these SNPs, this will shorten and greatly improve the work of rubber tree improvement," says Souza. "Instead of continuing to cross productive plants with resistant plants to obtain many years later the improved material, we will be able to select the plants carrying the SNPs and, consequently, the specific regions and genes that give those characteristics of interest to the breeders."
In other words, the rare genes of interest and now absent in the improved rubber trees will be incorporated by crossing the genome of rubber trees to produce a new generation of seedlings. The plants that will generate upper trees will be identified by genomic prediction, based on analysis of the SNPs (molecular markers) that they contain, which identify the superior characteristics associated with the regions that contain these SNPs. These trees already selected early by genomic selection, will grow and develop to become, at the age of seven, adult trees with the selected characteristics. Then, when they are ten years old, they will be already in the production phase, to produce more latex per planted hectare, in poorer soils, in colder climates and drier environments.
When all this happens, the genomic improvement will save around 20 years of work of the rubber tree breeders. Who knows, then, the resulting increase in latex production could make Brazil once again self-sufficient in the product? Perhaps the country can even achieve some lost positions in the world rubber market? Who knows?
Since 2007 the research of the rubber tree in the laboratory of Anete Pereira de Souza has been financed by research agencies such as Fapesp, CNPq and Capes. At the moment, Souza seeks funding for the application of genomic selection to obtain rubber clones with greater efficiency and in a shorter time. The investment required will result in more productive rubber trees adapted to cold and dry regions. As a consequence, there will be expansion of the area of rubber plantations in regions where there is no danger of the leaf blight. "With greater planting area and higher productivity, Brazil could become self-sufficient in rubber production and perhaps even an exporter," says the researcher.
According to Souza, "Brazil's self-sufficiency in the production of rubber is of interest to everyone in Brazil, from the tire and rubber industries to the farmers and rubber mills. Farmers in regions previously not adapted to the planting of rubber trees can diversify their crops, planting rubber trees in idle areas on the property."
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CONTACT WITH THE AUTHORS:
Profa. Dra. Anete Pereira de Souza
Laboratório de Análise Genética Molecular
Instituto de Biologia (IB)
Universidade Estadual de Campinas (UNICAMP)
Campinas-SP
Telefone: (5519) 3521-1132
Celular: (5519) 99111-6547
e-mail: anete@unicamp.br
Lívia Moura de Souza
Laboratório de Análise Genética Molecular
Instituto de Biologia (IB)
Universidade Estadual de Campinas (UNICAMP)
(5519) 3521-1156
(5511) 97602-0561
e-mail: liviamoura31@gmail.com
SCIENTIFIC REFERENCES:
de Souza LM, Le Guen V, Cerqueira-Silva CBM, Silva CC, Mantello CC, Conson ARO, et al. (2015) Genetic Diversity Strategy for the Management and Use of Rubber Genetic Resources: More than 1,000 Wild and Cultivated Accessions in a 100-Genotype Core Collection. PLoS ONE 10(7): e0134607. doi:10.1371/journal.pone.0134607
de Souza LM et al (2018) Linkage Disequilibrium and Population Structure in Wild and Cultivated Populations of Rubber Tree (Hevea brasiliensis). Front. Plant Sci. 9:815.
doi: 10.3389/fpls.2018.00815
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0)
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