Transcriptome Analysis of Floral Buds Deciphered an Irregular Course of Meiosis in Polyploid Brassica rapa

Abstract

Polyploidy is a fundamental process in plant evolution. Understanding the polyploidy-associated effects on plant reproduction is essential for polyploid breeding program. In the present study, our cytological analysis firstly demonstrated that an overall course of meiosis was apparently distorted in the synthetic polyploid Brassica rapa in comparison with its diploid progenitor. To elucidate genetic basis of this irregular meiosis at a molecular level, the comparative RNA-seq analysis was further used to investigate differential genetic regulation of developing floral buds identified at meiosis between autotetraploid and diploid B. rapa. In total, compared to its diploid counterparts, among all 40,927 expressed genes revealed, 4,601 differentially expressed genes (DEGs) were identified in the floral buds of autotetraploid B. rapa, among which 288 DEGs annotated were involved in meiosis. Notably, DMC1 identified as one previously known meiosis-specific gene involved in inter-homologous chromosome dependent repair of DNA double stranded breaks (DSBs), was significantly down-regulated in autotetraploid B. rapa, which presumably contributed to abnormal progression during meiosis I. Although certain DEGs associated with RNA helicase, cell cycling, and somatic DNA repair were up-regulated after genome duplication, genes associated with meiotic DSB repair were significantly down-regulated. Furthermore, the expression of randomly selected DEGs by RNA-seq analysis was confirmed by quantitative real-time PCR analysis in both B. rapa and Arabidopsis thaliana. Our results firstly account for adverse effects of polyploidy on an entire course of meiosis at both cytological and transcriptomic levels, and allow for a comprehensive understanding of the uniformity and differences in the transcriptome of floral buds at meiosis between diploid and polyploid B. rapa as well.

Keywords: Brassica rapa; RNA-seq analysis; floral buds; meiosis; polyploidy; transcriptome.

Figure 1

Abnormal chromosome behavior in the synthetic autotetraploid B. rapa. Normal chromosomal behavior displayed in the diploid B. rapa from early prophase to anaphase II (A–D), and normal chromosome morphology through early prophase and metaphase I was also observed in autotetraploids (E–G). However, a subset of cells consisting of multivalents and univalents (arrows) chaotically dispersed at metaphase I (H). Homologous chromosomes segregate equally during anaphase I forming two polar groups of chromosomes (I). Unequal segregation of chromosomes in the ratio of 19: 21 at anaphase I (J), Normal alignment of chromosomes in cell at metaphase II (K), but cells at anaphases II consist of unequally segregated chromosomes (L). Dotted oval consist of 19 chromosomes while solid oval has 21 chromosomes. Bar = 10 μm.

Figure 2

Statistical analysis of abnormal behavior of chromosomes during meiosis in autotetraploid B. rapa.

Figure 3

Comparison of the FPKM values among the biological replicates. Pearson correlation and scatter plot matrix of log₂ normalized expression of the Brassica rapa expressed genes in autotetraploids (T04, T05, T06) and diploids (T01, T02, T03). The pink solid line is the identity line. Panels (A,B) were about correlation between biological replicates, the (C) showed correlation between the autotetraploid and diploid FPKM-values.

Figure 4

Distribution and expression analysis of differentially expressed genes. (A) Percentage of DEGs up-and down-regulated. (B) MA plot of RNA-seq data obtained from diploid and autotetraploid. Y-axis representing log₂ fold change of all expressed genes vs. average expression normalized to log₂ scale for each gene. (C) Heatmap analysis of a subset of DEGs across three biological replicates in autotetraploid and diploid B. rapa. Expressed genes are FPKM normalized log₁₀ transformed values. Nine cluster are shown (K1–K9) from the subset of expressed genes.

Figure 5

Histogram representing clusters of orthologous genes (COG). Out of 4,602 transcript, 2,615 sequences were clustered in the various COG groups. The corresponding graph represents concentrated functional clusters with number and percentage of transcripts in parenthesis. The asterisks (^*) represent functional clusters overrepresented for putative meiosis-related genes.

Figure 6

Gene ontology classification of novel DEGs. A total of 142 DEGs were classified into three groups of biological process, cellular component and molecular function, according to gene ontology.

Figure 7

DEGs enriched by KEGG pathways in autotetraploid B. rapa.

Figure 8

Distribution and expression analysis of meiosis-related genes in COG classification. (A) A number of up- and down-regulated meiosis-related genes clustered into 4 COG categories. (B) Overrepresented genes enriched in Replication, repair, and recombination, (C) Analysis of enriched genes in Chromatin structure and dynamics, (D) Overrepresented genes enriched Cell cycle control, cell division, chromosome partitioning (E) Z: Cytoskeleton. The log₁₀-transformed FPKM-values range from 2 to −2.

Figure 9

qRT-PCR validation of expression of selected meiosis-related genes. The relative expression was normalized by the comparison with β-actin expression. Notably, the expression of DMC1 was significantly down-regulated. Note: the expression level of other selected genes was also analyzed (see Figure S4).