The First T2T Genome of Grape Provides New Insights into Its Genetic Breeding and Evolutionary Studies

Since the first human genome was published in 2000, many reference genomes have been assembled successively in various species. However, the highly repetitive sequences (telomeric, mitotic, ribosomal DNA) clustered in the genome have resulted in many missing fragments in the assembled genomes. Fortunately, thanks to improvements in sequencing technologies and computational algorithms, a new era of genome assembly has been ushered in: the telomere-telomere genome (T2T genome). Now, more than 30 T2T genomes have been sequenced and completed, but grapes still do not have a corresponding T2T genome, which hinders the functional study of their mitoses and telomeres.

Recently, a research paper, entitled “The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding”, was published online by Horticulture Research.

The availability of a fully annotated T2T reference genome for grapevine will be particularly useful for the study of traits related to grapevine breeding, such as disease resistance, fruit quality, and yield. With this new resource, researchers will be able to identify candidate genes responsible for these traits and develop more effective breeding strategies. Furthermore, the T2T genome will also be useful for comparative genomics studies, allowing researchers to study the evolution of grapevine and its relationships to other plant species.

PacBio HiFi sequencing is a technique that produces highly accurate and complete genome sequences. By analyzing the structure and function of telomeres and mitogenic regions in the grapevine genome, the researchers were able to gain new insights into the genetic mechanisms that control grapevine growth and development. They also explored the correlation between repetitive sequences and the characteristics of mitogenic regions, which could help researchers understand how these regions evolve over time. Furthermore, by identifying the gene functions of gene clusters and heterozygous regions in the grapevine T2T genome, the researchers were able to provide new insights for grapevine genetic breeding and disease resistance research. This knowledge could be used to develop new grapevine cultivars with improved traits, such as disease resistance or higher yield.

Assembly and annotation of the grape T2T (PN T2T) genome

The researchers were able to obtain a highly pure autogenous grape genome with only one gap using HiFi sequencing and chromosome construction via Mummer. After filling the gap with HiFi reads, they obtained the grape T2T genome, which has a size of 494.78 Mb and a contig N50 of 26.89 Mb. They also evaluated the genome integrity using BUSCO, and it was 98.5%.

Furthermore, the researchers compared the PNT2T with the 12x.v2 grapevine genome and found that the T2T grapevine genome had the best continuity and also improved some of the assembly errors. They annotated a total of 37534 genes and 41064 transcripts and found that the genome contained 62.47% of repetitive sequences and 63.9% of TEs, mainly composed of long terminal repeat LTRs. They also detected 276 rDNA sequences, which account for 0.019% of the genome.

Overall, the researchers obtained a highly continuous and accurate grape T2T genome, which can serve as an important reference for future studies in grape genetics and breeding.

Identification of Telomeres and Centromeres

Telomeres are specialized structures found at the ends of chromosomes that help protect the DNA from damage or degradation. They consist of repetitive sequences of DNA, with the most common sequence being TTTAGGG in vertebrates. In the case of grapes, the researchers identified 150 kb sequences at both ends of each chromosome that contained telomeric repeat units ranging from 5-12 bp in length. The most abundant telomeric repeat units, TTTAGGG/CCCCTAAA, were found in all chromosomes, and a total of 36 telomeres were identified, with the longest being 31 Kb on chromosome 8 and the shortest being a 180 bp repeat on chromosome 7.

The researchers also identified a 107 bp repeat sequence as a characteristic fragment of the centromeres, which was present on all 19 chromosomes, ranging in total length from 1.4 Kb to 3.8 Mb. This sequence was highly conserved between chromosomes, indicating its importance in the overall chromatin structure.

The distribution and scattering of transposons, along with the proximity of tethers, suggest that specific sequence-defined repeat superfamilies are correlated or anti-correlated with tether proximity at different levels, forming a density gradient. This feature may reflect the overall chromatin structure on a chromosome-scale level.

Finally, the researchers identified a total of 343 genes in the grapevine thylakoid region, which were mainly enriched in biological pathways related to protein binding, plastid component composition, RNA modification, protein autophosphorylation, DNA integration, DNA recombination, and photomorphology. These findings provide important insights into the structure and function of grapes’ genetic material and may have implications for the development of new grape varieties with improved traits.

Identification of Gene Clusters in the Grapevine T2T Genome

The identification of gene clusters in the grapevine T2T genome is an important step towards understanding the genetic basis of plant defense mechanisms. Through protein comparisons, the researchers found 377 gene clusters in the genome, which often involve local rearrangements and can span tens or hundreds of genes.

On chromosome 16, there are 599 gene-enriched structural domains, which mainly include WAKs (wall-associated receptor kinase galacturonan binding), PPR repeats, leucine-rich, ABC transporter proteins, mesenchymal structural domains, peptidase families, protein kinases, and reverse transcriptase. On chromosome 18 (25-36 Mb), there are 1237 gene-enriched structural domains, mainly including mesenchymal domains.

These gene clusters and structural domains provide an important resource for further research into the genetic basis of plant defense mechanisms. For example, the WAKs and PPR repeats on chromosome 16 are known to be involved in plant-pathogen interactions and RNA editing, respectively. Similarly, the mesenchymal domains found on both chromosomes 16 and 18 are thought to play a role in plant defense against biotic and abiotic stresses.

This result provides a foundation for further investigation into the genetic basis of plant defense mechanisms, with potential implications for improving crop yield and resilience in the face of environmental challenges.

Genetic heterozygosity in grapes after self-fertilization

It is well established that self-fertilization in plants can lead to reduced genetic heterozygosity, which can have negative consequences on plant fitness and adaptation to changing environments. In grapes, self-fertilization can occur naturally or be induced by human intervention during the breeding process.

This study used resequencing data to identify heterozygous SNPs on grapevine chromosomes and analyzed the genes in the top 5% of these heterozygous regions. The researchers found that these genes were significantly enriched in pathways related to key physiological activities of the plant, such as response to water deficiency, protein phosphorylation, cell division, response to oxidative stress, and response to salt stress.

This suggests that genetic heterozygosity plays an important role in grapevine adaptation to environmental stress, and that self-fertilization may have negative consequences on the plant’s ability to cope with these stressors. However, it is worth noting that this study only focused on a small number of genes and pathways, and further research is needed to fully understand the effects of self-fertilization on grapevine genetic diversity and plant fitness.

Reference

1. Shi X, Cao S, Wang X, et al. The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding[J]. Horticulture Research, 2023: uhad061.