Genome-wide analysis of DNA polymorphisms, the methylome and transcriptome revealed that multiple factors are associated with low pollen fertility in autotetraploid rice

<div><p>Autotetraploid rice is a useful germplasm with high biomass production; however, low fertility is the main barrier in commercial utilization. In our previous study, differential expression of meiosis-related miRNAs was found to be involved in the pollen sterility of autotetraploid rice. However, genome-wide DNA variations and methylomes associated with low fertility of autotetraploid rice are still poorly understood. Here, we measured both global DNA variations and the methylome and compared them with the transcriptome during pollen development in autotetraploid rice by high-throughput sequencing. A total of 34416 SNPs, 6993 InDels, 1003 SVs and 25 CNVs were detected, and 11367 and 41117 differentially methylated regions showed hypermethylation and hypomethylation in 02428-4x. In total, 1110 genes displayed differentially expression in 02428-4x during meiosis, of these six harbored CNVs, including four upregulated genes with gain CNVs, such as <i>LOC_Os11g38620</i>. We identified 122 genes by comparing with the previous data that might be associated with low fertility during pollen development in 02428-4x. Of the 122 gens, 98 were displayed methylation and differential expression, including <i>OsMADS98</i>, <i>CYP703A3</i> and <i>OsABCG26</i>. The downregulation of these three genes were confirmed by qPCR during meiosis of 02428-4x, which played pivotal roles in pollen fertility. These results indicate that the low fertility of autotetraploid rice is not only caused by the differential expression of genes involved in pollen development, but also by sequence variation and differential methylation, suggesting that the reason for pollen sterility in autotetraploid rice is complex and might be affected by multiple factors.</p></div

Differentially expressed genes associated with copy number variations in autotetraploid rice.

<p>Differentially expressed genes associated with copy number variations in autotetraploid rice.</p

Semi-thin sections showing anther development in 02428-4x.

<p>(A) Premeiotic interphase, (B-H) meiosis stage, (I-J) single microspore stage, (K, L) mature pollen. Ep, En, ML and Ta indicate the epidermis, endothecium, middle layer and tapetum, respectively. The red arrow indicates the degenerated middle layer. Bars = 50 μm.</p

Distribution of methylated transposable element genes in 02428-4x.

<p>Distribution of methylated transposable element genes in 02428-4x.</p

Cytological observations of pollen morphology.

<p>(A) Anther phenotypes of diploid (left) and autotetraploid (right) rice. (B and C) 1% I₂-KI staining of the pollen grains of diploid and autotetraploid rice. Bar = 200 μm.</p

Abnormal anther locule during anther development of autotetraploid rice.

<p>(A) Premeiotic interphase, (B-C) meiosis stage, (G) single microspore stage, (H) mature pollen, (B) double vascular bundle (VB). (D, E, F, I, J) abnormal anther locule of each stage. The red arrow indicates the abnormal anther locule. Bars = 50 μm.</p

A hypothesis model of low seed set in autotetraploid rice.

<p>Blue line indicated the action for the next step. Black line means it cause bad effect to pollen and embryo sac fertility. Red line indicated that the low seed set may cause by multiple factors.</p

Abnormal tapetum during anther development in autotetraploid rice.

<p>(A, B) Giant tapetal cells (A) and prevacuolation of giant tapetal cells (B) during premeiotic interphase. (C-E) Redundant tapetal cells (C) and tapetum degenerating along with the middle layer (D, E) during meiosis. (F, G) Unbalanced tapetum size after meiosis. (H, I) Tapetum cytoplasm that is not deeply stained at the microspore stage. (J, K) Degeneration of the tapetum (J) and abnormal proliferation (K) during the microspore stage. (L) Arrested microspores and larger endothecium in the mature pollen stage. En, ML and Ta indicate the endothecium, middle layer and tapetum, respectively. The red arrow indicates the abnormalities. Bars = 50 μm.</p

Protein-protein interactions of candidate meiosis-related genes in autotetraploid rice.

<p>Group 1 genes: genes related to DNA polymorphisms and that exhibit differential expression patterns. Group 2 genes: genes related to DNA methylation and that exhibit differential expression patterns. Group 3 genes: genes related to DNA polymorphisms and DNA methylation.</p

The homozygous DNA polymorphisms between autotetraploid (02428-4x) and diploid (02428-2x) rice.

<p>The homozygous DNA polymorphisms between autotetraploid (02428-4x) and diploid (02428-2x) rice.</p