Cytological Observations and Bulked-Segregant Analysis Coupled Global Genome Sequencing Reveal Two Genes Associated with Pollen Fertility in Tetraploid Rice

Abstract

Neo-tetraploid rice with high fertility is a useful germplasm for polyploid rice breeding, which was developed from the crossing of different autotetraploid rice lines. However, little information is available on the molecular mechanism underlying the fertility of neo-tetraploid rice. Here, two contrasting populations of tetraploid rice, including one with high fertility (hereafter referred to as JG) and another with low fertility (hereafter referred to as JD), were generated by crossing Huaduo 3 (H3), a high fertility neo-tetraploid rice that was developed by crossing Jackson-4x with 96025-4x, and Huajingxian74-4x (T452), a low fertility autotetraploid rice parent. Cytological, global genome sequencing-based bulked-segregant (BSA-seq) and CRISPR/Cas9 technology were employed to study the genes associated with pollen fertility in neo-tetraploid rice. The embryo sacs of JG and JD lines were normal; however, pollen fertility was low in JD, which led to scarce fertilization and low seed setting. Cytological observations displayed low pollen fertility (25.1%) and approximately 31.3 and 27.2% chromosome lagging at metaphase I and II, and 28.8 and 24.8% chromosome straggling at anaphase I and II in JD, respectively. BSA-seq of F2–3 generations and RNA-seq of F4 generation detected a common fragment, i.e., 18,915,234–19,500,000, at chromosome 7, which was comprised of 78 genes associated with fertility. Among 78 genes, 9 genes had been known to be involved in meiosis and pollen development. Two mutants ny1 (LOC_Os07g32406) and ny2 (LOC_Os07g32040) were generated by CRISPR/Cas9 knockout in neo-tetraploid rice, and which exhibited low pollen fertility and abnormal chromosome behavior. Our study revealed that two unknown genes, LOC_Os07g32406 (NY1) and LOC_Os07g32040 (NY2) play an important role in pollen development of neo-tetraploid rice and provides a new perspective about the genetic mechanisms of fertility in polyploid rice.

An uncharacterized protein NY1 targets EAT1 to regulate anther tapetum development in polyploid rice

“…Knock-out mutant of these genes resulted in abnormal meiosis process and pollen sterility. Similar trend of abnormal meiosis process and pollen sterility was also observed in ny1 mutant [23]. Taken together, these results suggest that the down-regulation of important tapetal development and meiosis-related genes play crucial role and might be a major reason for pollen sterility in ny1 mutant.…”

“…In our previous study, NY1 showed abnormal pollen fertility and high frequency of chromosome behavior abnormalities. As a consequence, seed set rate in the mutant were markedly reduced compared to H1 (the wild type) [ 23 ]. In order to explore the reproductive roles of NY1 , reciprocal crosses were made between ny1 and H1.…”

“…Neo-tetraploid rice lines exhibited high yield potential, unique structural classification and a novel specific allele associated with heat tolerance [ 20 , 21 ]. Previous studies detected at least four genes, including MOF1 , OsMND1 , NY1 and NY2 that may associate with pollen fertility in tetraploid rice [ 18 , 22 , 23 ]. However, the molecular mechanism of these genes underlying pollen fertility process remained largely unknown in tetraploid rice.…”

Comparative cytological and transcriptome analyses of ny2 mutant delayed degeneration of tapetal cells and promotes abnormal microspore development in neo-tetraploid rice

“…After years of work, neo-tetraploid rice and tetraploid PMeS lines have recently been generated with high and stable seed setting characteristics ( Chen et al., 2022 ). These rice materials are useful resources for the study of fertility-related molecular mechanisms and also the breeding application of tetraploid rice ( Cai et al., 2007 ; He et al., 2011 ; Guo et al., 2017 ; Bei et al., 2019 ; Chen et al., 2019 ; Xiong et al., 2019 ; Ghaleb et al., 2020 ; Kamara et al., 2021 ; Liu et al., 2022 ).…”

Thermo-Sensitive Genic Male Sterile Lines of Neo-Tetraploid Rice Developed through Gene Editing Technology Revealed High Levels of Hybrid Vigor

“…In order to solve the problem of low fertility of autotetraploid rice, our research team developed a new type of tetraploid rice (known as “neo-tetraploid rice”), including Huaduo 1 to Huaduo 5 and Huaduo 8 [ 4 , 21 , 22 , 23 , 24 , 25 ]. The neo-tetraploid rice showed normal fertility and strong yield heterosis when crossed with low fertility autotetraploid rice lines [ 4 , 23 , 24 ]. Comparative genomic analysis revealed polyploid rice as an independent group with unique classification and containing novel allele, such as HSP101-1 [ 25 ].…”