De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes

Abstract

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

Keywords: Adaptation; Asian germplasm; Bread wheat; Genome assembly; Norin 61; Polyploidy.

Fig. 1

Norin 61 assembly validation. (A) KAT plot comparing the sequence content in the raw reads (curve profile) and in the assembly (colored areas). The peak at X ≈ 21× contains the single-copy regions that are assembled in one copy (red area). The shoulder at 40–60× contains duplicated genomic sequences that are mostly assembled (as expected) in two copies. (B) Presence and copy number of universal SCOs in the Norin 61 assembly and gene annotation.

Fig. 2

Nondenaturing fluorescence in situ hybridization of a metaphase spread of Norin 61. A pseudocolored image of the stacked pictures taken with red (Oligo-pSc119.2-1; shown in blue), green (Oligo-pTa535; shown in red), near-infrared (Oligo-pTa713; shown in green) and blue (counterstaining with DAPI; shown in gray) filters.

Fig. 3

Analysis of chromosomal introgression in Norin 61. (A) Dot plot comparison of the terminal 100 Mb of chromosome 3B from Norin 61 (horizontal) and CS. Norin 61 contains an introgression at approximately position 25–46 Mb (indicated with a blue bar). Discontinuity in the diagonal denotes lack of sequence conservation in that region, and Norin 61 contains a unique ≈3.9 Mb segment (shift to the right in the dashed blue rectangle). (B) Dot plot comparison of the terminal 100 Mb of chromosome 1A from Norin 61 (horizontal) and CS. The introgression was identified through the analysis of repetitive sequences and ranges between 0 and 47.7 Mb (indicated with a blue bar). Sequences are particularly diverse in the 5′ terminal 25 Mb. (C) Self dot plot alignment of the 3.9 Mb 3B segment that is unique to Norin 61. The alignment of the segment with itself identifies a large array of five tandem repeats (series of parallel lines). (D) Phylogenetic analysis of proteins encoded by gene family 3B_25-46-1 whose members are identified in the repeat array shown in (C). Note that the individual genes cluster in two clades that cannot be further resolved because of the high similarity of the protein sequences.

Fig. 4

(A) Maximum-likelihood tree of 108 FT homologs based on amino acid sequences. The bootstrap values are shown on the branches. N61 and CS stand for Norin 61 and CS, respectively. (B) Multiple sequence alignment of FT1 genes on CS and N61. The red stars highlight the positions with the 1-bp deletion in FT-D1 of Norin 61 and the 1-bp insertion in FT-B1-3 of CS. The black boxes indicate the conserved sequences. The white regions show the SNPs or indels between sequences.

Fig. 5

Sequence features of Ppd-1 genes with the 5-kb upstream region extracted from the CS and Norin 61 pseudomolecules. (A) Both CS and Norin 61 have the photoperiod-sensitive Ppd-A1b allele. Three insertions/deletions were detected. (B) The red region spans the 2,089-bp deletion of the Ppd-D1a allele of Norin 61 compared with the Ppd-D1b allele of CS. The boxes indicate CDSs. The red lines show the sequence polymorphisms between CS and Norin 61 assemblies.

Fig. 6

Characterization of the Fhb1 locus in CS and in FHB-resistant varieties. (A) Sequence dot plot of the region surrounding Fhb1 in CS (X-axis chromosome 3B: 7.99–8.88 Mb) and Norin 61 (Y-axis, chromosome 3B: 11.41–12.37 Mb). The gap in the bottom part of the diagonal denotes the region with different sequence composition (spanning about 340 kb). (B) Dot plot comparing CM-82036 derived from Sumai 3 (X-axis, from Schweiger et al. 2016; BAC assembly, GenBank accession: MK450312) and Norin 61 (Y-axis, chromosome 3B: 11.41–12.37 Mb) showing the almost complete identity of the sequence. (C) Annotation of the ≈390 kb Norin 61 region containing Fhb1 (chromosome 3B: 11.89–12.31 Mb). The newly annotated genes (red boxes) and the span of the Norin 61 region differing from CS are shown below the published annotation of Norin 61 (Walkowiak et al. 2020, first three tracks). The TaHRC gene is highlighted in purple. (D) Schematic structure of the deletion in the TaHRC gene. The boxes indicate the third exon. The red dashed line shows the sequence polymorphisms between CS and Norin 61 assemblies.

Fig. 7

Schematic features of Tamyb10 sequences of CS and Norin 61. (A) Tamyb10-A1. CS has the recessive allele (Tamyb10-A1a), characterized by a large deletion, including the start codon. Norin 61 has the (wild type) dominant allele (Tamyb10-A1b). (B) Tamyb10-B1. The CS has the recessive allele (Tamyb10-B1a) has a 19-bp deletion and Norin 61 has the dominant Tamyb10-B1b allele. (C) Tamyb10-D1. Both CS and Norin 61 have the identical dominant allele (Tamyb10-D1b). The boxes indicate CDSs. The red dashed lines show the sequence polymorphisms between CS and Norin 61 assemblies.