New research reveals major difference between American and Chinese chestnut genomes

The American and Chinese chestnut chromosomes are ultimately not that similar, at least in a key region of the genome: the nucleolus organizing region (NOR).

The discovery, published in Scientific reportshas major implications for anyone wishing to confer downy mildew resistance to American chestnut trees through hybridization with Chinese chestnut.

“This is an unprecedented finding in the field of plant cytology,” says Nurul Faridi, a Forest Service geneticist and lead author of the study.

Traditional backcrossing, involving hybridization between two species, aims to combine an ideal blend of traits from two species without genetic engineering. Backcrossing can only be successful when the chromosomes of the two species are compatible. Since Chinese-American chestnut hybrids are viable, people assume that the two species are highly compatible. But the new study reveals significant differences in the NOR of the two species.

NOR is part of every plant and animal cell. It contains the genetic instructions needed to make ribosomes, the molecular machines that produce proteins essential to life.

The NOR is located near the end of the short arm of a particular chromosome. It is present in both species, but in the Chinese chestnut it contains a type of DNA called heterochromatin and makes up about 25% of the chromosome.

The structure and composition of this DNA surprised researchers: it is very condensed, devoid of genetic content and transcriptionally inactive. In contrast, the American Chestnut satellite is very small and appears euchromatic. Euchromatic regions of DNA are transcriptionally active.

Faridi first noticed a small pair of Chinese chestnut chromosomes exhibiting very bright fluorescence with a specialized microscope, a UV filter and a dye that binds to DNA.

Faridi used fluorescent in situ hybridization (FISH) to further analyze the discovery.

“Our high-quality FISH images provide unequivocal evidence of this unique DNA arrangement,” says Faridi. “These images are not just images; they demonstrate the dynamic nature of genetic material.”

Faridi has worked with FISH since 1991 and has extensive experience in preparing plant chromosomes for analyses. Well-separated chromosomes from enzyme-digested root tips that are mostly free of cell walls, nuclear membranes, and cytoplasmic debris are best for FISH.

Most FISH images are obtained from animal cells, because plant cells, and especially trees, are more difficult to use. Faridi discovered that chestnut trees are much more difficult to work with than pines and poplars.

Researchers will use a technique called oligonucleotide FISH to further their research. Oligo-FISH uses short, specific DNA probes acquired during DNA sequencing. Since the entire genomes of American and Chinese chestnuts have been sequenced, oligo-FISH will allow researchers to conduct detailed genetic studies to discern subtle genomic differences. The technique is particularly useful for studying hybrids because it can indicate which parent a gene comes from.

Progress in developing American chestnut hybrids exhibiting the height of the American chestnut and the blight resistance of the Chinese chestnut has been significant. However, the most advanced hybrids currently do not have enough resistance to downy mildew to be restored, previous Forest Service research has shown.