Pan-genome and transcriptome analyses provide insights into genomic variation and differential gene expression profiles related to disease resistance and fatty acid biosynthesis in eastern black walnut (Juglans nigra)

Abstract

Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America. Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi-C technologies. The genome size was 540.8 Mb, with a scaffold N50 size of 35.1 Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600 kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9 Mb in J. regia and 18.3 Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

Figure 1

Morphology, genome map, population structure, and demographic history of Juglans nigra. a morphology of J. nigra female flowers (1), male flowers (catkins) (2), leaf (3), mature fruit (4), and nut (5). b Circos map of the J. nigra genome assembly: A, number of chromosomes; B, gene density; C, LTR density; D, Gypsy; E, Copia; F, miRNA; G, rRNA; H, snRNA; I, tRNA; J, syntenic relationships among chromosomes. c Model-based population structure analysis of 74 section Rhysocaryon accessions (K from 2 to 4). d PSMC estimates of the effective population sizes of J. nigra (yellow), Juglans microcarpa (blue), and Juglans hindsii (green); grey shading indicates Riss-Würm interglacial period (13–11 Mya) and the glacial maximum 3–1 Mya.

Figure 2

Analysis of transposable elements (TEs) in Juglans nigra. a The size and percentage of the entire genome occupied by TEs in J. nigra and in 16 other plant species. b Genomic constituents in J. nigra in comparison with those in Juglans microcarpa, Juglans regia, J. sigillata, and Juglans mandshurica. All five constituents, especially Gypsy, were abundant in walnut genomes. c Insertion breaks of Gypsy and Copia elements in J. nigra. d Temporal patterns and insertion time of LTR-RT in J. nigra as compared to those in J. microcarpa, J. regia, J. sigillata, and J. mandshurica.

Figure 3

Juglans nigra genome evolution. a Expanded, contracted, and rapidly evolving gene families in 18 species; pie charts on each branch of the phylogenetic tree indicate the proportion of genes showing expansion (blue), loss (red), and rapid evolution (orange), the numbers near the nodes shows number of expanded or contracted gene families. The font color indicates the number of genes gained (blue), lost (red), and rapidly evolving (orange). b The proportion of multicopy genes, unique paralogues, single-copy orthologues, other paralogues, and unclustered genes among 18 species; rows correspond to species as listed in panel a. c The number of PR, PPR, GATA, CYP450, and NBS-LRR genes among the species shown in panel a; colors are intended as a visual guide to gene number. d Venn diagram of the gene space of five woody perennial species in Fagales (Jn = J. nigra, Jr = Juglans regia, Jm = Juglans microcarpa, Cmo = Castanea mollissima, and Cca = Carya cathayensis). Regions of overlap indicate shared orthologs. e KEGG analysis of gene families that were expanded, contracted, and rapidly evolving in the J. nigra genome. e KEGG analysis of 159 unique paralogs of J. nigra.

Figure 4

Comparative analysis of the genomes of Juglans nigra, Juglans regia, and Juglans microcarpa and lipid biosynthesis in J. nigra. a Circos map of InDels and repeats for each J. nigra chromosome versus the J. regia and J. microcarpa assemblies: (1) number of chromosomes; the (2) deletions, (3) insertions, (4) repeat expansion, and (5) repeat contraction detected between J. nigra genome and J. regia genome; the (6) deletions, (7) insertions, (8) repeat expansion, and (9) repeat contraction detected between J. nigra, J. regia, and J. microcarypa. b KEGG analysis of InDels between the first development stage (ET1) and third development stage (ET3) of J. nigra embryo development. c Venn diagram of the DEGs among three development stages of black walnut embryo (80 DAF, 111 DAF, and 140 DAF). d KEGG analysis of DEGs (281 genes) between the first development stage (ET1) and third development stage (ET3) of J. nigra embryo development. e Transcriptional profiles of lipid biosynthetic genes in developing J. nigra embryos. The expression levels of lipid biosynthesis genes in the developing J. nigra embryo based on transcriptome data. The nine colored panes in each horizontal row represent three stages with three biological replications (80 DAF, 111 DAF, and 140 DAF). Blue to red colored squares indicate log₁₀FPKM values.

Figure 5

Collinearity analysis of resistance-related genes and comparative analysis of the NBS-LRR genes in Juglans nigra and Juglans regia. a Genome collinearity between J. nigra, J. regia, and Juglans microcarpa. Regions of collinearity smaller than 100 kb are filtered out. The green and purple lines indicate syntenic blocks more than 600 kb in length. b The KEGG enrichment analysis of InDels of J. nigra and J. regia. c Gene structure and collinearity of JnNBS-LRR126 and homologous NBS-LRR genes in J. regia, J. microcarpa, Juglans mandshurica, Vitis vinifera, and Arabidopsis thaliana. d The phylogenetic tree and protein sequences of JnNBS-LRR126 and other NBS-LRR genes.

Figure 6

NBS-LRR gene cluster collinearity and expression profiles in Juglans nigra and Juglans regia. a Genome-wide synteny analysis of NBS-LRR genes for J. nigra and J. regia. Orthologous and paralogous NBS-LRR genes were mapped onto the chromosomes and then linked. Red lines and blue lines indicate orthologous and paralogous gene pairs in chromosomes 1, 3, 4, 6, 7, 8, and 11 in J. nigra and J. regia. Grey lines indicate orthologous and paralogous gene pairs in other remaining chromosomes. bNBS-LRR gene chromosomal distribution and gene collinearity of chromosome 3 in J. nigra and J. regia. The large-scale deletion (0.02 Mb) on chromosome 3 of the J. nigra genome contained one disease resistance gene JnNBS-LRR80 (red), while the J. regia genome contains eleven NBS-LRR genes (blue) in the same region. c Gene structure and synteny analysis of JnNBS-LRR126 and 11 J. regia NBS-LRR genes on chromosome3. Sequence comparison between J. nigra chromosome 3 and J. regia chromosome 3: the upper figure shows, in grey, extensive regions of collinearity >100 kb; the middle figure shows the region from 13 665 230 bp to 136 741 633 bp on the chromosome 3 of J. nigra that includes 11 genes (NBS-LRR) deleted in J. regia; the bottom figure shows NBS-LRR gene collinearity on the chromosome 3 between J. regia (from 13.54 Mb to 13.83 Mb) and J. nigra (from 13.94 Mb to 14.02 Mb). e–f The morphology of five tissue/organs (leaf, female flower, twig, stem, and fruit) and expression heat map of JnNBS-LRR126, JnNBS-LRR80, JnNBS-LRR157, JnNBS-LRR221, and JnNBS-LRR303 in those five tissues. Expression levels of those five NBS-LRR genes during three development stages of J. nigra husks, shell, and embryo. (H = husk; S = shell; E = embryo; for details of samples, see Table S10).