Transposable element insertion: a hidden major source of domesticated phenotypic variation in Brassica rapa

Abstract

Transposable element (TE) is prevalent in plant genomes. However, studies on their impact on phenotypic evolution in crop plants are relatively rare, because systematically identifying TE insertions within a species has been a challenge. Here, we present a novel approach for uncovering TE insertion polymorphisms (TIPs) using pan-genome analysis combined with population-scale resequencing, and we adopt this pipeline to retrieve TIPs in a Brassica rapa germplasm collection. We found that 23% of genes within the reference Chiifu-401-42 genome harbored TIPs. TIPs tended to have large transcriptional effects, including modifying gene expression levels and altering gene structure by introducing new introns. Among 524 diverse accessions, TIPs broadly influenced genes related to traits and acted a crucial role in the domestication of B. rapa morphotypes. As examples, four specific TIP-containing genes were found to be candidates that potentially involved in various climatic conditions, promoting the formation of diverse vegetable crops in B. rapa. Our work reveals the hitherto hidden TIPs implicated in agronomic traits and highlights their widespread utility in studies of crop domestication.

Keywords: Brassica rapa; crop domestication; intraspecific diversification; pan-genome; transposable element insertion.

Figure 1

Characteristics of transposable elements (TEs) in B. rapa genomes. (a), The TE content in each of the 20 B. rapa genomes. The phylogenetic tree was constructed using single‐copy genes within the 20 B. rapa genomes, and the accession “JZS” (Brassica oleracea) was used as an outgroup. DNA transposons were classified into Helitron and other five major superfamilies (Feschotte and Pritham, 2007) DTM (Mutator), DTA (hAT), DTC (CACTA), DTH (PIF/Harbinger), and DTT (Tc1/Mariner). (b) The ratio of nonsynonymous SNPs to synonymous SNPs in genes with and without TE insertions in introns. (c) Relative transcription levels of genes with and without TE insertions in introns. Multiple comparisons were performed using the Student‐Newman–Keuls test with a = 0.01 (the same as presented in Figures 4 and 6).

Figure 2

The pipeline for identifying transposable element (TE) insertion polymorphisms (TIPs) in B. rapa genomes by a combination of pan‐genomic and population resequencing strategies. (a) Identification of insertions and deletions in the B. rapa pan‐genome. Each of the 20 B. rapa genomes is used as the reference for comprehensively identifying insertions and deletions. (b) Construction of the TE insertion dataset. Each insertion or deletion sequence was mapped onto the B. rapa TE library to determine TE insertions in B. rapa. TE insertions in the pan‐genome were divided into TE insertions in the “aligned regions” and “unaligned regions.” (c) Determination of TIPs in 524 B. rapa genomes. Short reads were mapped to the TE sequence and flanking sequences for genotyping. Based on the mapping patterns, we genotyped each TE insertion and calculated polymorphic TE insertions. “G_1,” “G_2,”‡, “G_n” represent each re‐sequenced B. rapa genome. “CC” and “GG” indicate the two genotypes with and without TE insertion, respectively, in the corresponding resequenced genome.

Figure 3

The distributions of transposable element (TE) insertions in the B. rapa genome. (a) Chromosomal distributions of genes (i) and TEs (ii) as well as TIP‐containing genes (iii) across the 10 chromosomes of the B. rapa Chiifu genome. Number of SNPs (iv) and InDels (v) in a sliding window of 100 kb. (b) The number of genes with TE insertions in different B. rapa genomes. (c) The number of genes with TE insertions in the genic regions after combinations of individuals. The different combinations were randomly selected from the 20 B. rapa genomes. (d, e) The numbers (d) and the ratios (e) of different TE insertions in the genic regions. Genic regions include 2 kb upstream and downstream of the gene body.

Figure 4

Transcriptional impacts of transposable element (TE) insertions. (a) Comparisons of genes with and without TE insertions in the B. rapa Chiifu genome. The ratio (number) of genes with significant changes in CDS length. Significant changes (down/up change >20%) in CDS length were calculated in all syntenic genes as well as genes with TIPs in coding and intron regions. Variations in CDS length (b) and number of coding regions per gene (c) between syntenic genes with and without TE insertions. We calculated syntenic gene pairs between a Chinese cabbage genome line (CCB) and a turnip rape line (OIB) and identified TIPs between syntenic gene pairs. (d) An example of a TE insertion acting as a new intron and being related to the changes in gene expression. The boxplot shows comparisons of expression levels of the BraA07g030180.3.1C with and without the LTR/Copia element insertion.

Figure 5

Transposable element insertion polymorphisms (TIPs) associated with B. rapa morphotype domestication and selective development. (a) Phylogenetic tree of 524 B. rapa accessions using TIPs. Pictures placed beside each clade show typical morphotypes for the corresponding groups. (b) The genetic components calculated for the 524 B. rapa accessions using TIPs. (c) PCA plot of 524 B. rapa accessions using TIPs. (d) Phylogenetic tree of 192 Chinese cabbages using TIPs. The turnip was employed as the outgroup. The three ecotypes of spring, summer, and autumn Chinese cabbage were empirically divided by Su et al. (2018).

Figure 6

Transposable element (TE) insertion polymorphisms (TIPs) played a crucial role in the domestication of morphotypes in B. rapa. (a) Genomic signatures of selection in genomes of Chinese cabbage using whole‐genome TIPs. We calculated the frequency of each TE insertion in the heading and non‐heading B. rapa populations and conducted Fisher’s exact test for each TIP. After that, each P‐value was normalized by ‐log10 and z‐score. (b) The genotypes for the top 50 selection signatures in the heading and non‐heading populations in B. rapa. The phylogenetic tree of 524 B. rapa accessions was reported in our previous B. rapa pan‐genome analysis. CC indicates that the genotype in the corresponding accession was consistent with the reference genome, and GG indicates that the genotype in the accession was different from the reference genome, while missing loci (NN) and heterozygous loci (CG) are filled with gray and brown. (c–e) Comparison of selection pressure between TIPs and non‐synonymous SNPs in TIP‐containing candidate genes during the domestication of Chinese cabbage (c), turnip (d), and caixin (e) morphotypes. Accessions from the target morphotypes (Chinese cabbage, turnip, and caixin) are respectively set as derived groups, and all accessions except the target morphotypes were set as control groups. The red number on the right side of each picture shows the number of accessions of the target morphotype.

Figure 7

The TIP‐containing gene BrMYB18.1 was under strong selection during the domestication of Chinese cabbage. (a) The Copia element inserted in coding regions of BrMYB18.1 in the Chinese cabbage genome. (b) The distribution of the insertion of the Copia element in 524 B. rapa genomes. The tree was constructed using whole‐genome SNPs, reported in our previous pan‐genome analysis (Cai et al., 2021). The blue and red stars indicate the corresponding accessions with and without the transposable element (TE) insertion, respectively. (c) The distribution of haplotypes in the BrMYB18.1 gene region in 524 genomes. Homozygous sites of AA, CC, GG, and TT are filled using different colors as described in the figure, while missing loci (NN) and heterozygous loci (Hetero) are not filled with color. (d) The expression patterns of the BrMYB18.1 gene in the Chinese cabbage leaves. L1–L9 represented different leaves from inner to outer at the Chinese cabbage heading stage. R1–R5 represented different regions of each leaf as described by Guo et al. (2021).