NEW YORK (GenomeWeb) – By sequencing the almond genome, an international team of researchers has homed in on the genomic alteration that makes domesticated almonds sweet, unlike their bitter wild counterparts.
Almonds, Prunus dulcis, are thought to have been domesticated in the Fertile Crescent in the early Holocene and have more recently been introduced in North America and the Southern Hemisphere. But wild almond species are bitter and toxic, as their cotyledons accumulate the cyanogenic diglucoside amygdalin.
By sequencing the P. dulcis genome, researchers led by the University of Copenhagen's Lindberg Møller uncovered a cluster of genes encoding transcription factors linked to kernel taste. As they reported yesterday in Science, further functional analysis found that a point mutation in one of these prevents the transcription of two cytochrome P450 genes to yield a sweet almond.
"Here, we report the assembly of the draft genome of almond," Møller and his colleagues wrote in their paper. "The sequence information was used to unravel the genetic differences between bitter and sweet kernel genotypes."
He and his colleagues sequenced the Lauranne almond cultivar using a combination of Pacific Biosciences long-read sequencing and Illumina mate-pair and paired-end sequencing, using the long reads for genome assembly and the short reads for gap filling and scaffolding. In all, the final assembly included 4,078 scaffolds, slightly more than half of which were organized into eight pseudomolecules.
They estimated based on almond RNA sequencing and peach, P. persica, transcript data that the P. dulcis genome encompasses 27,817 genes, slightly less than half of which could be assigned a Gene Ontology term.
With the almond genome in hand, the researchers then sought to find the sweet kernel locus that confers domesticated almonds' taste. They used markers linked to the locus and generated a large mapping population of 475 F1 individuals segregating at that site. By aligning those markers to the new almond genome, they identified a 46-kilobase stretch of the almond genome associated with being sweet. This region harbored 11 genes, including five genes thought to encode basic helix-loop-helix transcription factors.
An RT-PCR time course analysis of tegument tissue from the sweet Lauranne cultivar and a bitter almond strain uncovered no differences in the expression of bHLH1, bHLH2, and bHLH4, and no expression of bHLH3 and bHLH5. But when they compared the sequences of the expressed bHLH1, bHLH2, and genes, they found that the sweet sample harbored bHLH1 with an indel that led to a truncated protein, bHLH2 with a substitution and insertion, and bHLH4 with no polymorphism.
Through a series of functional analyses, the researchers found that the alterations in bHLH2 affect its activity. In particular, they reported that while the Leu-to-Phe change at position 346 doesn't affect the protein's ability to dimerize, it does lead to a nonfunctional dimer. It then cannot activate downstream transcription of two cytochrome P450 genes, PdCYP79D16 and PdCYP71AN24, which are part of the amygdalin biosynthetic pathway.
In addition to informing almond breeding practices, the researchers suggested that their work could also be applied to uncover when and where almonds were first domesticated by analyzing alterations in these bHLH transcription factor genes.