Tools to solve a thorny problem

The global blackberry (Rubus subgenus Rubus) industry has experienced rapid growth during the past 15 years, driven by increased consumer demand, advanced production methods, year-round product availability, and new cultivars. Despite the growing economic importance of blackberries and their excellent nutritional properties, few genomic resources exist to facilitate molecular breeding.

Blackberries!

The application of molecular breeding in blackberry is a ‘thorny’ problem due to polyploidy, multisomic inheritance, and heterozygosity. While the red and black raspberries in subgenus Idaeobatus are diploids (2n = 2x = 14), cultivated blackberries in the subgenus Rubus are bred at the tetraploid (2n = 4x = 28) and higher order polyploid levels (Clark et al., 2007).

Today, we share the chromosome-length genome assembly for the diploid blackberry ‘Burbank Thornless’ (R. ulmifolius inermis, PI 554060), generated using plants donated by the USDA-ARS National Clonal Germplasm Repository in Corvallis, OR. The assembly was created using PacBio and Hi-C sequencing data. The PacBio data was assembled using FALCON software. The Falcon assembly was phased into haplotypes using FALCON-Unzip (see Chin, Peluso et al., 2016), with error correction on the phased assembly performed using Arrow. The Hi-C scaffolding was performed using the standard DNA Zoo workflow, based on in situ Hi-C (Rao, Huntley et al., 2014) prepared from fresh leaf samples. The tools used for Hi-C data processing included: Juicer (Durand, Shamim et al., 2016), 3D-DNA (Dudchenko et al., 2017) and Juicebox Assembly Tools (Dudchenko et al., 2018).

‘Burbank Thornless’ was chosen because it is believed to be closely related to ‘John Innes’, the source of the recessive gene for thornlessness in ‘Merton Thornless’, which has been used widely in fresh-market blackberry breeding programs (Coyner et al., 2005; Scott et al., 1957). Botanically speaking, it would be more accurate to describe this accession as prickle-free than thornless. Blackberries have prickles, outgrowths from epidermal tissue, instead of thorns or spines, which are connected to the vascular systems of the plant (more details on this here). Regardless of what they are called, anyone who has spent time picking berries in wild bramble patches can appreciate that picking from thornless cultivars is a much smoother experience!

Thorny vs thornless blackberry canes.

This work is part of a collaborative effort between the University of Arkansas, USDA-ARS, North Carolina State University, DNA Zoo, NIAB-EMR, Pairwise Plants, and the Wellcome Sanger Institute. Thanks to all involved: Erez Aiden, Rishi Aryal, Hamid Ashrafi, Nahla Bassil, Mario Caccamo, Brian Crawford, Michael Dossett, Olga Dudchenko, Felicidad Fernandez-Fernandez, Gina Fernandez, Jodi Humann, Sook Jung, Dorrie Main, Dan Mead, Cherie Ochsenfeld, Gina Pham, Melanie Pham, Tom Poorten, Dan Sargent, Aabid Shariff, Margaret Worthington, Xiaoyu Zhang. Find out more on the Genome Database for Rosaceae (GDR) webpage dedicated to the 'Burbank Thornless' blackberry genome assembly, here!

See below the whole-genome alignment plots that compare the genomes of the Burbank Thornless (R. ulmifolius) to the black raspberry (R. occidentalis V. 3, VanBuren et al., 2018) and woodland strawberry (Fragaria vesca V. 4, Edger et al., 2017). All three genomes are highly collinear. Inversions on chromosomes 1 and 7 were found between woodland strawberry and both Rubus species. Interestingly, the inversions between R. occidentalis and F. vesca previously documented on chromosomes 4 and 6 were not seen in the new assembly of Burbank thornless. These inversions likely represent errors in the chromosome-scale assembly of R. occidentalis.

Whole-genome alignment plots between the new genome assembly for Burbank thornless blackberry (Burbank_HiC aka Burbank_genome_v1_purged_primary_contigs_HiC) and the genome assemblies of woodland strawberry (Fragaria vesca v.4, from Edger et al., 2017) and black raspberry (R. occidentalis v.3, from VanBuren et al., 2018). The new chromosome scale Burbank thornless assembly, as well woodland strawberry and black raspberry genomes, are publicly available at the Genome Database for Rosaceae (https://www.rosaceae.org/).

Citations:

Clark, J.R., Stafne, E.T., Hall, H.K., Region, N., and Finn, C.E. (2007). Blackberry breeding and genetics. Plant Breed. Rev. 29, 19–144.

Coyner, M.A., Skirvin, R.M., Norton, M.A., and Otterbacher, A.G. (2005). Thornlessness in blackberries: a review. Small Fruits Rev. 4, 83–106.

Edger, P.P., VanBuren, R., Colle, M., Poorten, T.J., Wai, C.M., Niederhuth, C.E., Alger, E.I., Ou, S., Acharya, C.B., Wang, J., et al. (2017). Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity. Gigascience 7, 1–7.

Scott, D.H., Darrrow G.M., and Ink D.P. (1957). ‘Merton Thornless’ as a parent in breeding thornless blackberries. Proc. Amer. Soc. Hort. Sci. 69,268-277.

VanBuren, R., Wai, C.M., Colle, M., Wang, J., Sullivan, S., Bushakra, J.M., Liachko, I., Vining, K.J., Dossett, M., Finn, C.E., et al. (2018). A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome. Gigascience 7, 1–9.

No probllama

The llama (Lama glama) is a domesticated South American camelid, widely used as a meat and pack animal by Andean cultures since the Pre-Columbian era.

Today, we release the chromosome-length assembly for the llama named Fiesta, living at the Houston Zoo. Fiesta is pretty famous! Check out this viral video of her enjoying the leaf blower. This is a $1K short-read assembly.

This is the third camelid in our collection, the other two being the dromedary and the alpaca, both upgrades from previously available genomes. Check the whole-genome alignments below: looks like the camel karyotype has been very stable over the last 45MY or so!

Whole-genome alignment plots between the new genome assembly for the llama (Lama_glama_HiC) and the dromedary (PRJNA234474_Ca_dromedarius_V1.0_HiC) and alpaca (Vicugna_pacos-2.0.1_HiC) genome assemblies. All assemblies have the same diploid chromosome number of 74.

An elephantine undertaking

The Asian elephant (Elephas maximus) is the largest land mammal in Asia and the most endangered species of elephant (1). Once ranging from Iran to Southeast Asia, the Asian elephant is now extinct in West Asia, Java, and most of China (1). Remaining populations are highly fragmented with ongoing declines from habitat loss and poaching (2).

The only surviving members of the Proboscidea order, the elephantids first appeared in Africa 5-10 million years ago (3, 4). Asian elephants are most closely related to the extinct mammoths and are one of three remaining extant elephant species, along with the two species of African elephants (Loxodonta africana and Loxodonta cyclotis) (5).

Asian elephants have complex social interactions and live in small herds of related females. They communicate over long distances through low-pitched sound and olfactory cues, and use their trunks for tactile communication. As megaherbivores, Asian elephants range over large areas to graze on grass and browse, which can bring them into conflict with humans.

EEHV

Further hampering global elephant conservation efforts are infections from elephant endotheliotropic herpesviruses (EEHV). This widespread and highly fatal hemorrhagic disease is responsible for 80% of all Asian elephant calf fatalities, and results in the deaths of 1 in 5 elephants born in captivity (6, 7). Improved genomic resources for the Asian elephant may improve our understanding of the genetic factors that contribute to decreased susceptibility to EEHV infections, and may ultimately result in improved treatments.

Reviving the Woolly Mammoth

Woolly mammoths (Mammuthus primigenius) were cold-tolerant members of the elephant family that ranged across the Northern Hemisphere during the last ice age and went extinct between 4,000-10,000 years ago. Woolly mammoths had a number of adaptations to cold including dense, long hair, increased adipose tissue, shortened ears and tails, and hemoglobin polymorphisms that allowed them to thrive in the frigid mammoth steppe ecosystem (8, 9). Well preserved frozen remains found in the permafrost of Siberia and Alaska provide the rare opportunity to apply functional genomics to examine adaptive evolution in this extinct species. Since woolly mammoths were most closely related to the Asian elephant, this genome will allow us to better characterize the genetic changes that allowed this iconic species to thrive in the cold, and may one day allow us to de-extinct the species (10).

Assembly

Today, we share the chromosome-length assembly for the Asian elephant generated using samples donated by the Houston Zoo Asian elephant herd: Methai, Tupelo, Shanti, Tess, Thai, Tucker and Duncan. Check this live camera feed at the Zoo to meet the elephants! The assembly was generated using the $1K workflow, see (Dudchenko et al., 2018) for details.

One of the elephant's closest living relative is the rock hyrax, a small, furry herbivore native to Africa and the Middle East. Manatees and dugongs are also closely related to the elephant. The manatee, the rock hyrax and the elephant share a common ancestor, Tethytheria, which died out more than 50 million years ago. Despite initial appearances, hyraxes still have a few physical traits in common with elephants. These include tusks that grow from their incisor teeth (versus most mammals, which develop tusks from their canine teeth), flattened nails on the tips of their digits, and several similarities among their reproductive organs. See below how the new assembly relates to the chromosome-length rock hyrax genome assembly in the DNA Zoo collection (an upgrade from the draft assembly from Lindblad-Toh et al., 2011)!

Whole-genome alignment plot beetween the new Asian elephant genome assembly (Elephas_maximus_HiC) and the chromosome-length upgrade for the rock hyrax genome assembly (Pcap_2.0_HiC).

REFERENCES

1. A. Choudhury, Lahiri Choudhury, D.K., Desai, A., Duckworth, J.W., Easa, P.S., Johnsingh, A.J.T., Fernando, P., Hedges, S., Gunawardena, M., Kurt, F., Karanth, U., Lister, A., Menon, V., Riddle, H., Rübel, A. & Wikramanayake, E., Elephas maximus . The IUCN Red List of Threatened Species 2008: e.T7140A12828813. http://dx.doi.org/10.2305/IUCN.UK.2008.RLTS.T7140A12828813.en (2008).

2. S. H. Blake, S., Sinking the Flagship: the Case of Forest Elephants in Asia and Africa. Conservation Biology 18, 1191-1202 (2004).

3. J. Shoshani, Understanding proboscidean evolution: a formidable task. Trends Ecol Evol 13, 480-487 (1998).

4. V. J. Maglio, Origin and evolution of the Elephantidae, Transactions of the American Philosophical Society, (American Philosophical Society, Philadelphia,, 1973), pp. 149 p.

5. E. Palkopoulou et al., A comprehensive genomic history of extinct and living elephants. Proc Natl Acad Sci U S A 115, E2566-E2574 (2018).

6. L. K. Richman et al., Elephant endotheliotropic herpesviruses EEHV1A, EEHV1B, and EEHV2 from cases of hemorrhagic disease are highly diverged from other mammalian herpesviruses and may form a new subfamily. J Virol 88, 13523-13546 (2014).

7. S. Srivorakul et al., Possible roles of monocytes/macrophages in response to elephant endotheliotropic herpesvirus (EEHV) infections in Asian elephants (Elephas maximus). PLoS One 14, e0222158 (2019).

8. K. L. Campbell et al., Substitutions in woolly mammoth hemoglobin confer biochemical properties adaptive for cold tolerance. Nat Genet 42, 536-540 (2010).

9. V. J. Lynch et al., Elephantid Genomes Reveal the Molecular Bases of Woolly Mammoth Adaptations to the Arctic. Cell Rep 12, 217-228 (2015).

10. G. M. Church, E. Regis, Regenesis : how synthetic biology will reinvent nature and ourselves (Basic Books, New York, 2012), pp. ix, 284 p.

Image: https://www.britannica.com/animal/elephant-mammal#/media/1/184366/53263