Overcoming the Challenges of Complex Plant Genomes

Plant genomes exhibit a remarkable range in size, stretching from incredibly compact genomes¹ to some of the largest known eukaryotic genomes². Significant progress has been made in understanding plant genomics since the first plant genome (Arabidopsis thaliana) was sequenced over 20 years ago³. Since that date, advances in sequencing technologies and analytical tools have expanded our knowledge, facilitating crop development, conservation efforts, and our understanding of evolution and biodiversity.

Confronting the Complexities of Plant Genomes
While animal genomes are known to be challenging, plant genomes bring their own unique complexities that have presented difficulties to researchers over the years. According to Nicholas Gladman, Ph.D., Research Plant Molecular Geneticist at the US Department of Agriculture (USDA), Agricultural Research Service (ARS), isolating high-quality plant DNA for long-read sequencing has historically been one of the biggest challenges when working with plant genomes. As outlined in a recent review that he authored⁴, extracting high molecular weight DNA was a consistent problem and a limiting step for many plant researchers.

“Thankfully, we have great collaborators that have iterated over this roadblock and have gotten sufficient single molecules for the PacBio or Nanopore platforms,” stated Gladman, “But it does seem like every plant species requires significant methods of development, even for different tissues within the same species.”

In addition to isolation difficulties, Gladman pointed out that one of the biggest headaches dealing with plant genomes has always been that a large percentage of the genome for many species is made up of a broad class of repetitive elements that are hard to confidently assemble into a suitable reference assembly. “Sometimes you have 60%, 70%, or even more than 80% of a plant genome that is comprised of various repetitive or recalcitrant elements that have historically made assembly a headache.” These repetitive elements are important contributions to the genome’s structural complexity but the similarity between the sequences makes genome assembly and accurate interpretation of genomic data increasingly difficult.

The fact that numerous plant and crop species are polyploid presents another significant challenge beyond what is typically encountered in their animal counterparts. “Crops like wheat, oat, coffee, strawberry, potato, and lots of species from the cabbage and mustard seed family are tetraploid or hexaploid (or even higher multiple polyploids),” explained Gladman.

“This creates obvious problems when trying to create a good reference genome when you always have multiple large, highly identical paralogous sequences within the genome; until the last 5–6 years, it made polyploid assembly nigh impossible if you were using short-read sequencing methods alone.” He also specified that polyploids, along with the repetitive content of plant genomes, offer immense potential for breeding and trait discovery. However, they pose considerable challenges for genomicists and molecular biologists to manage and decipher.

Progress, innovation, and the future
Despite these known issues, the scientific community has made significant strides in the field of plant genomics. “The continued quality improvement of long-read sequencing has been the biggest boon for me and for the research community,” explained Gladman. “Along with the ever-decreasing costs for both long-read and short-read sequencing, it has made it much easier to go out and make a high-quality reference genome from any cultivar or organism you want to investigate.” Some of these recent achievements with the use of long-read technologies include complete telomere-to-telomere assemblies of important plants such as Arabidopsis thaliana⁵, Musa acuminata⁶, and Zea mays⁷.

Additionally, Gladman noted that other technologies, like single-cell RNA-seq along with continued advancements in chromatin and epigenetic profiling, also provide a lot of valuable insights for basic and applied research programs.

Guy Naamati, Ensembl Plants Project Leader at EMBL-EBI, similarly acknowledged the difficulties researchers have recently overcome despite the great variation in plant genomes. “The first wheat genome was sequenced in 2017 using the propriety algorithm DeNovoMAGIC,” said Naamati. “This was a big breakthrough, but since then different types of technology and algorithms have allowed us to sequence large genomes with public code, such as Illumina PE, MP, 10X Genomics Chromium, and Hi-C. This has also allowed more and more pan-genomics in the last few years.”

Pan-genomes, which effectively capture a broader scope of genetic diversity, have proven especially advantageous for plant researchers, given that a single assembly falls short in representing all relevant genes of a species. The use of pan-genomes has assisted the plant community with understanding functional genomics⁸, metabolic diversification⁹, and heritability¹⁰. Furthermore, resources such as SorghumBase and Gramene, which host some of this diverse genomic data, have become invaluable tools that allow for comprehensive studies of genetic variations and phenotypic diversity.

Considering the recent advancements in plant genomics, it's exciting to imagine the possibilities of the future landscape. “Currently, more and more pan genomes are being sequenced, but the plant community is still trying to understand ways to best visualize and utilize this vast source of knowledge,” stated Naamati. “I envision this improving significantly in the next few years and this can have a big impact on plant science and breeding. Also, with projects such as the Darwin Tree of Life and the African Biogenome, more and more plant species are being sequenced, which is a great benefit to the community.” These large collaborative ventures underscore the scientific community's commitment to understanding these diverse organisms.

For those new to plant genomics research, Gladman advised seeking input from a variety of experts throughout their work, as each can offer their unique knowledge. “There are a lot of challenges ahead, but we are in a truly great time to be researchers with all the wonderful genomic resources at our disposal.”

References:

1. Leushkin EV, Sutormin R, Nabieva E, Penin AA, Kondrashov AS, Logacheva MD. The miniature genome of a carnivorous plant Genlisea aurea contains a low number of genes and short non-coding sequences. BMC Genomics . 2013;14(1). doi:https://doi.org/10.1186/1471-2164-14-476
2. Stevens KA, Wegrzyn JL, Zimin A, et al. Sequence of the sugar pine megagenome. Genetics. 2016;204(4):1613-1626. doi:https://doi.org/10.1534/genetics.116.193227
3. The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant arabidopsis thaliana. Nature. 2000;408(6814):796-815. doi:https://doi.org/10.1038/35048692
4. Gladman N, Goodwin S, Chougule K, Richard McCombie W, Ware D. Era of gapless plant genomes: innovations in sequencing and mapping technologies revolutionize genomics and breeding. Current Opinion in Biotechnology. 2023;79:102886. doi:https://doi.org/10.1016/j.copbio.2022.102886
5. Wang B, Yang X, Jia Y, et al. High-quality Arabidopsis thaliana genome assembly with nanopore and HiFi long reads. Genomics, Proteomics & Bioinformatics. 2022;20(1):4-13. doi:https://doi.org/10.1016/j.gpb.2021.08.003
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7. Chen J, Wang Z, Tan K, et al. A complete telomere-to-telomere assembly of the maize genome. Nature Genetics . 2023;55(7). doi:https://doi.org/10.1038/s41588-023-01419-6
8. Zhang F, Xue H, Dong X, et al. Long-read sequencing of 111 rice genomes reveals significantly larger pan-genomes. Genome Research. 2022;32(5). doi:https://doi.org/10.1101/gr.276015.121
9. Zhou X, Liu Z. Unlocking plant metabolic diversity: A (pan)-genomic view. Plant Communications. 2022;3(2):100300. doi:https://doi.org/10.1016/j.xplc.2022.100300
10. Zhou Y, Zhang Z, Bao Z, et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature. 2022;606(7914):527-534. doi:https://doi.org/10.1038/s41586-022-04808-9