Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice

doi:10.1016/j.rsci.2024.06.001

Abstract

Abstract:

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F_5:6 generation plants derived from inter-subspecific crosses (Nipponbare × KDML105) with low (LS) and high seed-setting rates (HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4‒5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes (DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS- specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase gene (CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall (OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion (Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties.

Key words: bulked segregant RNA-Seq, flavonoid, inter-subspecific cross, pectin, pollinated pistil, prezygotic barrier, spikelet fertility

Kanokwan Kaewmungkun, Keasinee Tongmark, Sriprapai Chakhonkaen, Numphet Sangarwut, Theerachai Thanananta, Amorntip Muangprom. Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice[J]. Rice Science, 2024, 31(5): 556-571.

Figures/Tables 9

Fig. 1. Rice plants with high (HS) and low seed-setting rates (LS) in F5 population, as well as their parents Nipponbare and KDML105. Data are Mean ± SD (n = 48), and different lowercase letters above the bars show significant differences at P < 0.05.

Fig. 2. Microscopic analysis of anther morphology in high (HS) and low seed- setting rate (LS) lines at anthesis in F5 generation. A and B, Morphology of six mature anthers from HS (A) and LS (B) lines. Scale bars are 1 mm, and the field diameters are 6.7×. C and D, Light microscope images of an anther from HS (C) and LS (D) lines. Scale bars are 1 mm, and the field diameters are 30×. E and F, Scanning electron microscope images of an anther from HS (E) and LS (F) lines. Scale bar are 500 µm, and the field diameters are 50×. G and H, Morphology of dehisced anthers from HS (G) and LS (H) lines. Scale bars are 500 µm, and the field diameters are 50×. I and J, Dehiscence for pollen dispersal at the apical of anthers from HS (I) and LS (J) lines. Scale bars are 100 µm, and the field diameters are 300×.

Fig. 3. Filled grains and pollen fertility in high (HS) and low seed-setting rate (LS) rice lines. A and B, Filled grains in a plant (A) and a panicle (B) in HS and LS rice lines. Scale bars are 1 cm. The red arrows represent a zoom from the red square. C, Pollen stained with I2-KI solution in HS and LS rice lines. Scale bars are 200 μm. D, Pollen fertility rate of HS and LS rice lines.

Fig. 4. Pollen-stigma adhesion, pollen grain germination, and pollen tube growth of high (HS) and low seed- setting rate (LS) rice lines in vivo. A and B, Aniline blue staining of HS (A) and LS (B) pistils at 1‒2 h before flowering (BF). C and D, Aniline blue staining of HS (C) and LS (D) pistils at 1‒2 h after flowering with the pollinated state. E and F, Aniline blue staining of HS (E) and LS (F) pistils at 4‒5 h after pollination. Scale bars are 100 μm. The white arrows indicate pollen-stigma adhesion, and the red arrows indicate pollen tubes.

Fig. 5. Co-expression, hierarchical cluster analysis, and significant differentially expressed genes (DEGs) specific to high (HS) and low seed-setting rate (LS) rice lines. A, Venn diagram presenting the number of genes uniquely expressed in each sample, with overlapping regions indicating the number of genes expressed in both samples, based on a 0.5 FPKM (fragments per kilobase of transcript per million reads) threshold. B, Hierarchical clustering of genes specifically expressed in HS and LS samples based on FPKM values. Green indicates lower expression, while red indicates higher expression. Columns represent individual experiments, and rows represent genes. C, The number of up-regulated and down-regulated genes specific to HS and LS rice lines. Green and red represent up-regulated and down-regulated DEGs, respectively, and the numbers on the columns indicate the count of DEGs.

Table 1. Eleven high seed-setting rate (HS)-specific differentially expressed genes assigned to metabolic pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.

Gene ID	Gene	Gene annotation	KEGG orthology accession	Log₂(Fold change)
Os06g0133000	Wx	Granule-bound starch synthase, synthesis of amylose in endosperm	K13679	2.32
Os09g0457400	Amy3A	Alpha-amylase isozyme 3A precursor (1,4-alpha-d-glucan glucanohydrolase)	K01176	2.07
Os06g0185100		Similar to estradiol 17-beta dehydrogenase 8	K00059	1.94
Os11g0701100		Similar to class III chitinase homologue	K01183	1.83
Os02g0467600		Similar to cinnamate 4-hydroxylase CYP73	K00487	1.82
Os03g0382100		Thiolase-like, subgroup domain containing protein	K15397	1.45
Os02g0134400		Fumarate reductase/succinate dehydrogenase flavoprotein, C-terminal domain containing protein	K00278	-1.07
Os03g0670200		Similar to YJR013Wp (Fragment)	K05284	-1.20
Os03g0368900		Haem peroxidase family protein	K00430	-1.20
Os04g0573100	HTH1	Putative glucose-methanol-choline oxidoreductase, cutin biosynthesis, anther development, and pollen fertility	K07729	-1.25
Os03g0321800		Similar to transcription factor WRKY55	K23735	-1.46

Fig. 6. Expression analysis of high (HS)- and low seed-setting rate (LS)-specific differentially expressed genes (DEGs) before and after pollination by qRT-PCR. A‒C, Relative expression levels of Os02g0467600 (A), Os12g0563700 (B), and Os06g0583900 (C). The relative expression values were normalized to the rice UBQ5 gene (OsUBQ5). The results were obtained from two biological replicates. Error bars indicate standard deviation.

Table 2. Highest fold enrichment of low seed-setting rate (LS)-specific differentially expressed genes (DEGs) in pentose and glucuronate interconversion and flavonoid biosynthesis pathways.

Gene ID	Gene	Gene annotation	KEGG orthology accession	Log₂(Fold change)
Pentose and glucuronate interconversion pathway
Os03g0300500	OsPME10	Pectin methylesterase 10	K01051	1.43
Os12g0563700	OsPME35	Pectin lyase fold domain-containing protein	K01051	1.13
Os02g0214400	OsPLL3	Pectate lyase-like protein	K01728	1.21
Os06g0583900	OsPLL9	Pectate lyase	K01728	1.30
Flavonoid biosynthesis pathway
Os02g0767300		Flavonol synthase/flavanone 3-hydroxylase	K05278	-1.34
Os04g0101400		Flavone synthase II, biosynthesis of tricin O-linked conjugate	K23180	-1.73

Fig. 7. Physical maps showing co-localization of dominant enrichment genes specific to high (HS) and low seed-setting rates (LS) with previously identified QTLs/genes involved in spikelet fertility. The previously identified QTLs and functional genes involved in spikelet fertility, pollen growth, and yield components are labeled in blue, and the dominant enrichment differentially expressed genes specific to HS and LS are labeled in black and red, respectively.

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