NEW YORK (GenomeWeb) — By analyzing the genomes of a number of citrus crops, researchers from the US Department of Energy Joint Genome Institute, the University of Florida, and elsewhere pieced together the complex and interwoven admixture events leading to the domestication of various fruits.
The international team compared and analyzed the genomes of 10 varieties of citrus, including sweet orange, sour orange, mandarins, and pummelos, as they reported in Nature Biotechnology yesterday. From this, they uncovered that mandarins are hybrids of pummelos and an unknown crop, while sweet oranges are complex hybrids of mandarins and pummelos and sour oranges are a hybrid of two unrelated species.
In the US, citrus is a more than a $3 billion industry, and one that is currently threatened by citrus greening, an emerging infectious disease that is destroying entire groves.
By knowing citrus' past, the researchers said that they might be able to develop new breeding strategies for the crop, which is now mostly grown through vegetative propagation, to protect it from disease and other stressors.
"Now that we understand the genetic structure of sweet orange, for example, we can imagine reproducing the unknown early stages of citrus domestication using modern breeding techniques that could draw from a broader pool of natural variation and resistance," University of Florida's Fred Gmitter said in a statement.
As a framework for their analyses, Gmitter and his colleagues sequenced a haploid derivative of a Clementine mandarin, yielding a 301.4 megabase reference genome. Because of the long scaffolds they developed, the researchers were able to construct pseudochromosomes and identify 10 crossovers from the meiosis event that produced the haploid Clementine.
The researchers also sequenced and assembled a draft genome of the diploid sweet orange variety Ridge Pineapple through a combination of deep 454 and light Sanger sequencing.
They further sequenced four mandarins, two pummelos, and one sour orange, and aligned the whole-genome shotgun reads from each of those cultivars to the sweet orange chloroplast and nuclear genomes.
Through the comparisons of the chloroplast genomes, Gmitter and his colleagues found two distinct chloroplast genome types: type M shared by all mandarins and type P shared by pummelos and oranges.
The nuclear genomes, they noted, presented a more complicated story of the citrus family tree.
The two pummelos that the researchers sequenced contained three haplotypes, as one of the pummelos is a known hybrid of two other pummelos.
Within these two sequenced pummelos, the researchers noted modest levels of heterozygosity — 5.7 heterozygous sites per kilobase — as well as a second low heterozygosity peak — about one heterozygous site per kilobase — that they attributed to an ancient bottleneck within the Citrus maxima line.
By re-analyzing previously reported pummelo genomes, the researchers noted that both Thai and Chinese pummelos are derived from the same wild population. This, they said, indicates that pummelos were domesticated through selection from a wild C. maxima population.
Of the mandarins the researchers sequenced, two were so-called traditional mandarins that are not thought to be admixed and two were thought to be hybrids of mandarin and other citrus.
As the researchers reported, these accessions have high levels of heterozygosity — some 17 heterozygous sites per kilobase — but those blocks are interspersed with regions of low heterozygosity.
In the low-heterozygosity regions, the researchers said, both alleles are often distinct from C. maxima and instead are likely derived from C. reticulata, the species from which mandarins are presumed to have arisen.
The blocks of high heterozygosity, meanwhile, have one allele that matches pummelo and one non-pummelo allele, again presumed to be from C. reticulata. These presumptive C. reticulata alleles, the researchers added, are common to multiple mandarin accessions.
Traditional mandarins like Ponkan and Willowleaf, this analysis indicates, are actually interspecific introgressions of C. maxima into C. reticulata.
Though none of these mandarins were pure C. reticulata, the researchers extrapolated wild mandarin alleles from their sequences, and identified some 1.5 million SNPs that differ between C. reticulata and C. maxima.
These SNPs, Gmitter and his colleagues added, can be used as a sort of diagnostic to determine the species ancestry of different segments of the mandarin, pummelo, and orange genomes.
Using that catalog of C. reticulata and C. maxima alleles, the researchers turned to sweet oranges, finding that they share alleles with Ponkan mandarins, and many of those alleles are also shared with Willowleaf and Huanglingmiao mandarins.
"This leads to the surprising conclusion that these three tradi¬tional mandarins, previously considered to be independent selections, in fact show substantial kinship with each other and with an ancestor of sweet orange, thus suggesting much more limited genetic diversity among the traditional mandarins than has previously been recognized," Gmitter and his colleagues said.
The other parent of sweet orange, the researchers added, is harder to untangle, but based on the distribution of heterozygous segments and its pummelo-type chloroplast genome, they suspects that the other parent is a pummelo, but with substantial introgression from wild mandarin.
Sour oranges, though, seem to be a simple cross between a pummelo and wild mandarin, the researchers reported.
However, the Chinese Mangshan, a presumed wild mandarin, appears to be a distinct species. It has neither the M nor the P type chloroplast genome, limited heterozygosity, and a uniform 2 percent homozygous difference across the genome from C. reticulata and C. maxima. From this, they concluded that the Mangshan mandarin is its own species — C. mangshanensis.
Gmitter and his colleagues noted that this analysis of the relationships between citrus crops underscores the limited germplasm that led to these commercially important plants. It also, though, "highlights the opportunities for the creation of new combinations of the ancestral citrus types with new fruit quality traits or even the re-creation of sweet orange with improved disease resistance via sexual hybridization, beyond the current approaches based on somatic mutations and genetic engineering," they added.