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Research reveals origin and genetic diversity of citrus

citrusLÉO RAMOSFor habitual consumers of oranges, mandarins, lemons, and other citrus sold in supermarkets, the Sylvio Moreira Citriculture Center may hold a pleasant surprise. Its greenhouses of saplings and vast orchard of mature plants in the city of Cordeirópolis, state of São Paulo, are home to 1,700 different types of citrus. That includes almost 700 varieties of sweet oranges – those suitable for juicing or in natura consumption – and nearly 300 types of mandarins. Tasting the fruit of different trees at the Citriculture Center will reveal an astonishing wealth of flavors and textures. The research center is affiliated with the Campinas Institute of Agronomy (IAC), an agency within the São Paulo State Department of Agriculture. “All of the material used by the Brazilian citrus industry has passed through here at some point,” summarizes agronomist Marcos Machado, researcher at the Citriculture Center and coordinator of the National Institute of Science and Technology of Genomics for Citrus Improvement (INCT Citros).

Throughout the center’s 85-year history, researchers have crossbred different varieties, mainly in search of disease-resistant plants. Starting with traditional crosses, similar to the ones that resulted in the varieties of citrus sold in markets since their species were domesticated, the center has gradually enriched its arsenal of techniques as genetic information became available. Until now, this knowledge has focused primarily on the use of molecular markers to characterize different crosses, determining which descendants of a cross between two varieties (or species) had received the genetic material targeted by the researchers. But genomics has now come to the Citriculture Center, making way for new possibilities.

Grafting scar: mandarin lime serves as rootstock for orange trees

LÉO RAMOSGrafting scar: mandarin lime serves as rootstock for orange trees LÉO RAMOS

The first major breakthrough, which resulted in a paper published on the Nature Biotechnology website in June 2014, included some unexpected discoveries about the origin of present-day oranges and mandarins. Researchers already knew that citrus fruits are not natural species, but rather hybrids perfected by thousands of years of natural interbreeding. But there is no historical record of this Citrus domestication, which started in Southeast Asia. “We knew there were hybrids, but we had no details,” says biologist Marco Takita, one of the authors of the paper.

The researchers were surprised to discover that the genomes of some types of mandarin, thought to be variants of the ancestral species C. reticulata, actually contain several genetic segments from C. maxima, a different species known as pummelo. Takita explains that the pummelo resembles a giant orange, weighs up to a kilogram (about 2.2 pounds), and is not usually consumed in Brazil. The species is used as a source of genetic diversity in plant breeding programs and, as researchers now know, it took part in the hybridizations that gave rise to the Ponkan mandarin. Because of the fruit’s commercial success in Brazil, the Ponkan genome was sequenced by the Citriculture Center with financial support from INCT Citros, which is funded by FAPESP and the National Council for Scientific and Technological Development (CNPq). “It is important to know that the pummelo served as a genetic source,” says the researcher.

The study also produced another unexpected finding. The Chinese mandarin variety known as Mangshan, thought to have the same origin as other mandarins and tangerines, is actually a separate species, C. mangshanensis, which appears to be distantly related to C. reticulata.

Surprising new knowledge was also brought to light regarding the sweet orange, most commonly sold in Brazilian markets as laranja-pera, -baía, or -lima. These varieties are all hybrids of C. reticulata and C. maxima, and much of their genome is genetically similar to mandarins like the Ponkan. The results revealed a very low level of genetic differentiation between sweet and mandarin oranges after branching off from their common ancestor. “The challenge now is to understand such things as why they are so genetically alike, but taste so different,” says Takita.

Germplasm bank houses a wide variety of citrus

LÉO RAMOSGermplasm bank houses a wide variety of citrusLÉO RAMOS

The sour orange, used for making preserves, is also a hybrid of the species that gave rise to pummelos and mandarins. As if recreating the story from back to front, genomic research has enabled researchers to conclude that citrus fruits appeared in nature in Southeast Asia a few thousand years ago, before spreading across the globe.

A consortium of citrus genome researchers began to be formed in 2005, with active participation from the researchers at the Citriculture Center. After nearly 10 years of work, during which the Center made progress in the sequencing of a Spanish clementine (a type of mandarin), a Chinese research group scored a coup and published the sweet orange genome in the journal Nature. Then, with similar results on hand, the international group decided to expand the study. “We sequenced an additional six genomes and produced a more elaborate discussion that actually questions some aspects of the Chinese study,” says Machado.

The results showed why the boundaries between citrus species are getting increasingly fuzzy. “Some say there are 163 species of Citrus, while others identify no more than 16,” says the researcher. “Linnaeus classified six,” he adds, referring to the 18th-century Swedish naturalist known as the father of taxonomy and creator of the binomial nomenclature system, which is used by scientists to this day.

Breeding for improvement
Based on the available information, Machado believes that the genomic approach is a way of “aiming high while staying grounded”. By sequencing the genome of the Rangpur or mandarin lime, the Brazilian group has started studying the genealogy of limes, whose earliest ancestor is C. medica. The species was not selected at random: the mandarin lime can withstand drier environments, and therefore is used as root stock in 85% of all orange and mandarin plantations in the state of São Paulo. “The northern part of the state has the best characteristics for growing juice oranges, but its drier climate would require impracticable amounts of irrigation,” Machado explains. Genomic research may help identify the genetic basis for the mandarin lime’s drought resistance and pinpoint the genes associated with certain characteristics. This information could serve as a guideline for breeding decisions, and it might even help researchers achieve gene transfer in the laboratory. Experts call the gene transfer process cisgenics (which differs from transgenics in that it involves same-group species that can generate natural hybrids).

Germination in the laboratory: controlled genetic variation

LÉO RAMOSGermination in the laboratory: controlled genetic variationLÉO RAMOS

Given the economic importance of citrus, these studies are essential not only to meet the demands of the market and steer the search for new fruit varieties with improved flavor and other qualities, but also to fight disease. Two good examples of such afflictions for oranges are citrus variegated chlorosis (CVC), caused by the bacterium Xylella fastidiosa, and huanglongbing, or greening disease, first detected in Brazil 10 years ago and now a threat to this country’s citrus orchards. Mandarins are resistant to CVC, but susceptible to huanglongbing and Alternaria brown spot, a fungal disease that discolors leaves and fruit and causes leaf loss. The genetic homogeneity highlighted in the study published in Nature Biotechnology clearly shows why citrus are easy prey for crop-infesting microorganisms: if one tree falls victim to a disease, others of the same type will also succumb, because of their genetic similarity. For this reason, the Citriculture Center focuses much of its effort on producing varieties that can resist these diseases. “Some hybrids of mandarin species produce fruit that have no direct value for consumers, but generate important genetic variability,” explains agricultural engineer Mariângela Cristofani-Yaly. The results of the Citriculture Center researchers’ attempts to create new root stock and fruit varieties are described in recent publications, such as their papers published in the Journal of Agricultural Science and in Bragantia respectively in 2013 and 2014, .

“First we introduced resistance, and then we resumed our focus on fruit quality,” says the researcher. To assess potential breeding options for producing new varieties of citrus used to make juice or eaten in natura, everyone at the Citriculture Center – researchers, students, and employees – serves as guinea pigs by taking part in sensory evaluation experiments to judge such characteristics as color, flavor, and ease of peeling, as described in the Journal of Agricultural Science paper published in 2013. The plants are also categorized by productivity, juice yield, and fruiting period, among other characteristics.

Academic achievement is just one side of this research center in inland São Paulo State. Its other mission is contributing to the improvement of citrus, an industry in which Brazil stands out as the world’s leading producer of oranges and third largest supplier of mandarins, tangerines, and other varieties. China is the global leader in overall citrus production, but focuses mainly on mandarins. “With the new techniques, we can combine basic and applied knowledge, create a platform for new things,” says Machado.

But the Brazilian market is also a constraint. Much of the country’s orange harvest is used for making juice concentrate, which means that the industry has control over production. Its main objectives are to get more juice out of each fruit, and to pay less for them; this has triggered a crisis among orange growers. According to Machado’s calculations, about 10,000 citrus growers abandoned the business in the past decade. But he believes that the crisis may ultimately have positive consequences for consumers. If the researchers in Cordeirópolis are right, more citrus growers will agree to try planting the new varieties developed by the research center, and markets may start offering a wider variety of citrus meant for eating in natura. A mouth-watering prospect.

1. Genomic platforms applied to citrus breeding (nº 08/57909-2); Grant mechanism Thematic Project; Principal investigator Marcos Antonio Machado (IAC); Investment R$ 3,533,624.89 (FAPESP).
2. Obtaining and evaluation of new varieties of scion and rootstock for citrus (nº 11/18605-0); Grant mechanism Thematic Project; Principal investigator Mariângela Cristofani-Yaly (IAC); Investment R$ 859,670.11

Scientific article
WU, G. A. et alSequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domesticationNature Biotechnology. On-line, 8 jun. 2014 (FAPESP).