Origin and adaptation to high altitude of Tibetan semi-wild wheat

Understanding plant evolution and domestication accelerates crop breeding by identifying new germplasm for improving world food security. Hexaploid wheat (*Triticum aestivum*), a staple crop, originated in the Near East. Its adaptation to the Tibetan Plateau, characterized by high UV-B irradiation, low temperature, and hypoxia, is poorly understood. A unique Tibetan semi-wild wheat (*Triticum aestivum* ssp. *tibetanum*) exists on the plateau, phenotypically resembling local landraces but with brittle rachis. This suggests a de-domestication process, yet lacking genetic evidence. This study aims to investigate the genomic changes associated with high-altitude adaptation in Tibetan wheat and confirm the de-domestication hypothesis using genomic sequencing and population analysis.

Previous research has explored the agronomic traits of Tibetan wheat accessions, noting the phenotypic similarity between semi-wild and landrace varieties, particularly concerning rachis brittleness. Studies on rachis brittleness have identified genes on chromosome group 3, especially chr3D, and alleles of the *Q* locus on 5A as being involved in this trait. However, a comprehensive understanding of genomic variations influencing this phenotype in Tibetan wheat at a population scale is still lacking. Other studies have examined genomic adaptations in high-altitude plants like *Eutrema* species, *Crucihimalaya himalaica*, and *Maca*, finding enrichment in stress tolerance and DNA repair genes. Research on wheat domestication has focused on identifying domestication genes and pathways but hasn’t extensively studied the de-domestication process in high-altitude environments.

This study involved *de novo* genome assembly of Tibetan semi-wild wheat accession Zang1817 using Illumina paired-end, mate-pair, and 10X Genomics data. The assembly was validated and scaffolded using DeNovoMAGIC3 software and the IWGSCv1 reference genome. Gene prediction utilized homology-based, ab initio, and RNA-Seq data. Repetitive element annotation was performed with RepeatMasker. A comparative genomics analysis was conducted between Zang1817 and the CS reference genome (IWGSCv1) to identify present/absent variations (PAVs) and SNPs. Resequencing data from 245 additional wheat accessions (including Tibetan semi-wild, Tibetan landraces, and global cultivars) were used. Population structure analysis employed neighbor-joining trees, PCA, and ADMIXTURE. Genome-wide association studies (GWAS) were used to identify loci linked to rachis brittleness, employing a mixed linear model to account for population structure and kinship. Demographic modeling assessed the origin of Tibetan semi-wild wheat. Finally, analysis of haplotype diversity of candidate genes associated with high-altitude adaptation was conducted across a larger dataset of wheat accessions, including those from high-altitude regions in Nepal.

The *de novo* assembly of the Zang1817 genome yielded 384,307 scaffolds with an N50 of 37.62 Mb and a total size of 14.71 Gb. 118,078 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed significant PAVs between Zang1817 and the CS reference genome, primarily in non-coding regions. The Zang1817 genome shows extensive expansion of α-gliadin genes. Resequencing of 245 wheat accessions revealed that high-altitude adaptation in Tibetan wheat involves extensive genome reshaping. A *TaHY5*-like mediated signaling pathway is implicated in the high-altitude adaptation, showing a high frequency of the AT haplotype in Tibetan wheat compared to lower altitude accessions. Several genes involved in stress response, DNA repair, and photosynthesis also showed haplotype divergence. Population structure analysis strongly supports the hypothesis that Tibetan semi-wild wheat originated from Tibetan landraces through de-domestication, showing a close genetic relationship in phylogenetic trees and PCA plots. A bottleneck effect following de-domestication is also suggested. GWAS analysis identified two loci significantly associated with rachis brittleness: a 0.8-Mb deletion region on chromosome 3D containing *Brt1/2* homologs, and a region containing the *TaQ-5A* gene on chromosome 5A. A 161-bp transposable element insertion in *TaQ-5A* is also implicated.

This study provides strong evidence for the de-domestication of Tibetan semi-wild wheat from local landraces. The adaptation to high-altitude environments involved both extensive genome-wide reshaping and specific selection at loci influencing key traits. The *TaHY5*-like mediated pathway plays a crucial role in the response to high-altitude stress. The findings highlight the importance of considering de-domestication events when studying crop evolution and adaptation. The identified loci associated with rachis brittleness provide valuable targets for future research into the genetic mechanisms of this trait. Further functional validation is needed to confirm the role of the identified genes in high-altitude adaptation.

This research provides comprehensive genomic evidence supporting the de-domestication of Tibetan semi-wild wheat from local landraces. The study identifies key genetic adaptations to high-altitude environments, focusing on the *TaHY5*-like pathway and loci associated with rachis brittleness. The findings offer valuable insights into crop adaptation and provide targets for future breeding efforts aimed at developing wheat varieties suitable for high-altitude environments. Further research could focus on functional validation of candidate genes and a more detailed analysis of gene interactions involved in adaptation.

The study relied on association mapping, which cannot definitively establish causality. Further functional validation experiments are necessary to confirm the roles of the identified candidate genes. The sample size for some analyses, while substantial, may not fully capture all the genetic diversity within Tibetan wheat populations. The study primarily focuses on Tibetan wheat; extending the research to other high-altitude wheat populations could provide further insights into generalizable adaptation mechanisms.