Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of related genes in neo-tetraploid rice

: Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells （ PMCs ） abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 1483 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 433 were annotated as pollen fertility-related genes, and 240 (~55.4%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( CSA , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS5 as a candidate gene and analysed its phenotype in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in phenotypic charts, pollen development process and expression level in H1 and its TMS5 knockout lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, the present study provides a new useful germplasm for polyploidy rice breeding. from two of neo-tetraploid rice variation and a higher percentage of fertility than of fertility for high fertility in fertility and greater heterosis of the autotetraploid rice there are to understanding the high fertility mechanism of neo-tetraploid rice. To the regulatory mechanism of high fertility in neo-tetraploid rice, we combined the re-sequencing and RNA-sequencing analysis to analyse the genomic and transcriptional variations between neo-tetraploid rice and its two parents. Using the transcriptome analysis profile, we identified a total of 1483 DEGs that showed at least a 2-fold change in neo-tetraploid rice compared with its two parents. Gene ontology (GO) analysis was conducted to annotate the up- and downregulated genes that were differentially expressed in neo-tetraploid rice and its parents in this study. The GO enrichment classification indicated that differentially expressed genes in the cellular component, molecular function and biological process categories showed significant variations. Notably, upregulated genes were mainly involved in the response process, pollen development and transcription regulator activity.


Background
Polyploidy is one of motivation in biological evolution, and it prevalently occurs in the plant evolution process (Doyle et al. 2008). Approximately 70% of plants have experienced at least one polyploidy during their evolutionary history (Masterson 1994). Several advantages, including greater variation, high biomass yield and resistance to insect pest and diseases, were found in polyploidy species when compared with their original species (Bingham et al. 1994;Marhold and Lihová , 2006). Two categories of polyploidy plants, including the autopolyploidy and allopolyploidy species, usually exist in nature (Comai, 2005). In contrast to the higher attraction of allopolyploidy plants, very few know the real appearance of autotetraploid plants in nature despite potential weaknesses, such as meiotic instability and reduced fertility. Increasing evidence indicates that the real appearance of autotetraploid plants in nature might be significantly underestimated (Parisod et al. 2010).
Autotetraploid rice is a useful germplasm derived from diploid rice by chromosome doubling. In comparison with corresponding rice, stronger biological vigour and heterosis were found in autotetraploid rice (Shahid et al, 2010;He et al, 2011;Wu et al. 2013); however, low pollen fertility is the major hindrance of its utilization Chen et al. 2018). Pollen abnormalities appear to be the major obstacles for a normal seed set (He et al. 2011;Wu et al. 2014). Several previous studies have focused on the causes of low pollen fertility in autotetraploid rice, and these results were mainly focused on the abnormal pollen development process (He et al. 2011;Wu et al. 2014;Wu et al. 2015;Wu et al. 2017;Li et al. 2018;Wu et al. 2019). Previously, our group has reported three neo-tetraploid rice materials that could overcome the sterility of autotetraploid rice and produce high heterosis (Guo et al. 2017;Chen et al. 2018;Bei et al. 2019). However, little is known regarding the complex regulatory mechanisms of heterosis and fertility in neo-tetraploid rice.
High-throughput technologies, such as whole-genome re-sequencing and transcriptome analysis, can provide useful insight for detecting genetic variation in rice. Using whole-genome re-sequencing, a high number of sequence polymorphisms, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (Indels), can be detected (Huang et al. 2013;Varshney et al. 2009). SNPs and Indels within a genome affect gene expression and can alter gene function. Therefore, detecting genomic polymorphisms relevant to functional changes is important for elucidating phenotypic differences. Extensive genome-wide studies using high-throughput technologies to identify SNPs and Indels have identified phenotypic variations and variation in gene expression and function in rice. In autotetraploid rice, very few studies have focused on the relationship between the genetic variations within the genome and pollen fertility (Guo et al. 2017;Li et al. 2018;Bei et al. 2019).
Neo-tetraploid rice is thought to be one effective way to overcome the low fertility of autotetraploid rice; therefore, understanding the mechanism of high fertility in neo-tetraploid rice is important. In this study, we developed a new neo-tetraploid rice, named Huaduo1 (H1), which was registered for PVP (Protection for New Varieties of Plants) in China in 2016. We used cytological analysis, whole-genome re-sequencing and RNA-sequencing analysis to analyse the mechanism of high pollen fertility in neo-tetraploid rice with respect to that of its two parents. Cytological analysis was used to compare the phenotypic differences between the neo-tetraploid rice and its parents.
Whole-genome re-sequencing and transcriptional analysis were used to discover that a large number of differentially expressed genes resulted in high fertility in neo-tetraploid rice. Further, to analyse the relationship between the pollen fertility genes and genome-wide variation in neo-tetraploid rice and its parents, we selected a known genes, TMS5, to verify our hypothesis. The results of this study may help to understand the molecular mechanism of high fertility in neo-tetraploid rice.

Breeding procedure of neo-tetraploid rice Huaduo1
A hybrid of two autotetraploid rice plants, Jackson-4x (T45-4x) and 96025-4x (T44-4x), was generated in 2004, and the F1 hybrid plants were harvested and continuously self-crossed until F5 in 2007. One line with more than 80% seed setting was found in that year, and a neo-tetraploid rice line, Huaduo1 (H1), was developed in 2009 and registered for PVP in China in 2016 (Fig. 1). H1 displayed significant differences in agronomic traits compared with its parent, which included high pollen fertility (95.62%) and seed setting (80%) ( Fig. 1; Tables 1 and 2). Moreover, H1 also showed significant heterosis in yield-related traits, including number of filled seeds, 1000 seed weight and seed setting (Additional file 1: Table S1).
To evaluate the ability to overcome the sterility of hybrids in H1, we developed hybrids with gene interactions in pollen sterility loci, Sa, Sb and Sc using H1 crossed with Taichung 65-4x and its pollen sterility isogenic lines. All of the hybrids had a high seed setting (>80%) with gene interactions in pollen sterility loci, Sa, Sb and Sc (Additional file 1: Table S1), suggesting that H1 may have neutral pollen fertility genes that could overcome the sterility of hybrids. All of these results indicate that H1 5 shows greater genetic variation compared with its two parents.

Meiosis in neo-tetraploid rice compared with its two parents
As for the important role of meiosis in autotetraploid rice, we focused on the chromosomal behaviour of PMCs in H1 and its parents. Similar meiotic processes and stage divisions were found in neo-tetraploid rice and their parents, which is consistent with our previous study (Fig. 2). A total of six key meiosis stages, including Metaphase І, Anaphase І, Telophase І, Metaphase II, Anaphase II and Telophase II, were observed, and the percentage of abnormalities is summarized ( Fig. 3; Additional file 2: Table S2). H1 showed a lower percentage of abnormal PMCs than those of its two parents (Fig. 3).
Additionally, chromosome configurations between H1 and its two parents were significantly different (Fig. 2). Higher percentages of quadrivalent and bivalent configurations were frequently observed in H1 relative to those in its two parents (Additional file 4: Table S4). However, chromosome configurations in T45-4x and T44-4x exhibited a much more complicated pairing style, such as univalent, trivalent and other types of multivalent (Additional file 4: Table S4). All of these results indicate that H1 had a higher percentage of normal cells and more stable chromosome configurations than those of their parents during meiosis.

Genome-wide alterations reveal the genetic variation of pollen fertility genes in neo-tetraploid rice and its parents
To reveal the cause of the higher pollen fertility in neo-tetraploid rice, we used re-sequencing analysis to detect whole genome variation between the neo-tetraploid rice and its two parents. A total of 68.20 GB of high-quality clean reads were obtained from H1 and its parents using the Illumina sequencing platform. Large numbers of high-quality reads varying from 214 to 251 million were obtained in these tetraploid lines (Additional file 5: Table S5). From this study, nearly 87.87% of the reads mapped to the japonica rice genome (Nipponbare), which covered ~85% of the total genome for each cultivar (Additional file 5: Table S5).
Genome-wide alterations in neo-tetraploid rice compared with its two parents were analysed across the 12 rice chromosomes. Two differential comparison groups, including H1 vs T44 and H1 vs T45, were analysed in this study. In the H1 vs T44 group, the largest number of SNPs was detected on chr4, whereas the smallest number of SNPs was detected on chr5. The highest number of SNPs in the H1 vs T45 group was detected on chr11, whereas smallest number of SNPs in H1 was found on chr10 ( Fig. 4a). Greater variation was also detected in the insertions/deletions (Indels) of 12 different chromosomes. In the H1 vs T44 group, the largest number of Indels was detected on chr4, and chr9 had the smallest number. The highest number of Indels in the H1 vs T45 group was detected on chr11, and chr10 had the smallest number of SNPs in H1 (Fig. 4b).
Two types of genetic variation, including non-synonymous SNP mutations and Indels of the CDS region, were further conducted in this study for their important role in influencing the gene expression of relevant proteins. Using Snp Eff software, a total of 26423 SNPs involved in 8296 genes were predicted to have differences between neo-tetraploid rice and its two parents (Additional file 6: Table   S6). These genetic variations primarily belong to non-synonymous mutations, which could lead to variations in the gene-coding region (Fig. 5). Based on the indel analysis, two types of Indels containing insertions and deletions were also detected and analysed in this study. A total of 896 Indels involving 584 genes were detected in neo-tetraploid rice compared with its two parents (Additional file 6: Table S6). All of these results indicate that significant variation exists in genomic sequences between neo-tetraploid rice and its two parents.
To reveal the genetic variations, including the non-synonymous SNP mutations and Indels of the CDS that play important roles in the neo-tetraploid rice, we further combined these genetic variations with pollen fertility-related genes, such as pollen fertility candidate QTLs and pollen fertility-related genes. From this study, a total of 29 fertility candidate QTLs and 113 pollen fertility-related genes were found to exhibit genetic variation in neo-tetraploid rice compared with its parents (Additional file 7: Table S7). To validate this genetic variation data in neo-tetraploid rice, we selected nine pollen fertility genes and used Sanger sequencing to detect the genetic variation between the neo-tetraploid rice and its two parents. From this study, genetic variation between the neo-tetraploid rice and its two parents was also consistent with the re-sequencing analysis (Additional file 8: Table S8).

Transcriptome analysis reveals that significant variation exists in neo-tetraploid rice compared with its two parents in meiosis
We further combined the transcriptome profiling with re-sequencing data to detect possible variations in gene expression that could result in the high pollen fertility seen in meiosis. Three comparison groups were used to identify the possible DEGs between the neo-tetraploid rice and its parents. Group I is termed "genome variation of re-sequencing data between the neo-tetraploid rice and its parents", referring to possible genetic variation for the non-synonymous SNP mutations and Indels of the CDS region, which might influence the gene expression of relevant proteins in neo-tetraploid rice. Group II is termed "differential gene expression of transcriptome data between the neo-tetraploid rice and its parents", referring to genes that were differentially expressed in the neo-tetraploid rice and its parents in meiosis. Group III is related to the genes found to demonstrate genetic variation in the genome and that are also differentially expressed at the transcriptional level ( Fig. 6).
Using these comparison groups, we have identified a total of 1483 genes that are differentially expressed (2-fold at P value<0.05) between the neo-tetraploid rice and its parents in meiosis (Fig. 6a).
Among these DEGs, 786 genes are upregulated in the neo-tetraploid rice relative to its parents, and 697 genes are downregulated ( Fig. 6b; Additional file 9: Table S9). We then categorized both the up-and downregulated genes using Cluster 3.0 software and obtained an overview of the transcriptome relationships ( Fig. 6c).
Gene ontology (GO) analysis was conducted to annotate the up-and downregulated genes that were differentially expressed between the neo-tetraploid rice and its parents (Fig. 7). The GO enrichment classification suggested that the genes from the cellular component, molecular function and biological process categories showed significant variation. In the biological process category, eight prominent functional gene classes, including cell division, response to auxin, reproductive process, reproduction, pollination, pollen exine formation, pollen-pistil interaction and recognition of pollen, were over-represented in the upregulated gene classes. Additionally, cell cycle, purine nucleobase biosynthetic process and regulation of protein dephosphorylation were over-represented in the downregulated gene classes (Fig. 7a). In the cellular component category, one prominent functional gene class, the vacuolar membrane, was over-represented in the upregulated gene classes, and three prominent functional gene classes, apoplast, chloroplast thylakoid membrane and microtubule-associated complex, were over-represented in the downregulated gene classes (Fig. 7b). In the molecular function category, four prominent functional gene classes, transcription regulator activity, 8 DNA binding transcription regulator activity, obsolete transcription regulator activity, and cation binding, were over-represented in the upregulated gene classes, and seven prominent functional gene classes, including protein kinase binding, peptidase inhibitor activity, xyloglucan xyloglucosyl transferase activity, hydrolase activity, peptide receptor activity, transmembrane signalling receptor activity and motor activity, were over-represented in the downregulated genes (Fig. 7c). All of these results indicate that the upregulated genes were mainly involved in response processing and transcription regulator activity-related genes. Alternatively, the downregulated genes were mainly involved in the external encapsulating structure and cell wall-related genes.
Pollen fertility-related genes are primarily upregulated in neo-tetraploid rice compared with its two parents RNA-sequencing and re-sequencing analysis were used to reveal the cause of the higher pollen fertility in the neo-tetraploid rice; thus, we focused on the pollen fertility-related genes that were differentially expressed in neo-tetraploid rice compared with its two parents. We compared our DEGs detected from neo-tetraploid rice and its two parents with the large number of rice pollen fertility-related genes (Fujita et al. 2010;Deveshwar et al. 2011;Yant et al. 2013;Wright et al. 2015;Wu et al. 2014).
Of these DEGs, 433 genes were annotated as pollen fertility genes when combined with other analysis results (Fig. 8). Notably, 240 of the pollen fertility genes were shown to be upregulated in neo-tetraploid rice compared with its two parents ( Fig. 9; Additional file 10: Table S10). Predicted protein-protein interaction analysis was used to further evaluate the relationship of these pollen fertility genes. From this study, a stronger interaction network and co-expression were detected in the neo-tetraploid rice (Additional file 11: Figure S1). Moreover, several pollen fertility genes, such as LOC_Os01g16810, LOC_Os02g12290, LOC_Os04g53760, LOC_Os04g37960, LOC_Os09g27620, LOC_Os03g12414, LOC_Os06g49840, LOC_Os07g39220, LOC_Os07g30240 and LOC_Os10g35180, were detected and upregulated in the neo-tetraploid rice. It is noted that three genes, CSA (LOC_Os01g16810), TMS5 (LOC_Os02g12290) and TMS9-1 (LOC_Os09g27620), are pollen fertility-related genes that are also responsive to the environment. CSA (LOC_Os01g16810) is a MYB family transcription factor encoding a MYB protein domain that plays an important role in the pollen development process under the conditions of a short day. TMS5 (LOC_Os02g12290) is a nuclear ribonuclease Z that processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (UbL40) genes into multiple fragments, which could result in pollen sterility under high temperature. TMS9-1 is a transcript factor containing a PHD-finger domain that controls pollen sterility under high temperature.
To verify the expression profiles of pollen fertility genes in neo-tetraploid rice and its two parents, nine representative pollen fertility-related genes were selected and submitted to quantitative real-time reverse transcription PCR (qRT-PCR) analysis. From this study, nine genes, including LOC_Os01g16810, LOC_Os04g53760, LOC_Os04g37960, LOC_Os09g27620, LOC_Os03g12414, LOC_Os06g49840, LOC_Os07g39220, LOC_Os07g30240 and LOC_Os10g35180, were consistent with the transcriptome analysis. These results showed that expression levels of the nine genes were consistent with the transcriptome analysis, indicating the reliability and accuracy of RNA-sequencing results (Additional file 12: Figure S2).

Pollen fertility-related gene knock-out causes pollen abortion in neo-tetraploid rice
To verify candidate genes for high pollen fertility in neo-tetraploid rice, we used CRISPR/cas9 technology to conduct the study. There is no evidence that CRISPR/cas9 technology has previously been used for analysing gene function in neo-tetraploid rice. Therefore, we selected one of the important upregulated genes, named TMS5, as the candidate gene to verify our results as to the number of pollen sterility-related genes found to be upregulated in neo-tetraploid rice. In the present work, we obtained at least 20 independently regenerated transgenic lines after the transformation. The mutant lines were grown in the field, and the T2 mutants were sequenced. We selected one homozygous mutant, which we named nt-tms5-1, and sequencing results indicate that it carried the sequence deletion that was predicted to lead to an amino acid change (Additional file 13: Figure S3).
As TMS5 is a male sterility gene that is responsive to temperature, we further grew the plants in August at an LD and under high temperature conditions to verify their pollen fertility. Both nt-tms5-1 and its wild type showed marked differences in anther, panicle and pollens (Fig. 10). Pollen fertility in TMS5 knock-out lines (nt-tms5-1) showed higher pollen sterility, and its value was notably reduced compared with that of its wild type (Fig. 10). The statistical analysis results demonstrated that the pollen fertility value of nt-tms5-1 was much lower than that of its wild type. Moreover, WE-CLSM analysis of anther in nt-tms5-1 verified that the number of normal pollen was obviously decreased compared with that of the wild type (Fig. 10).
Anther development was investigated further for nt-tms5-1 and its wild type H1. The results indicate that the anther development process of nt-tms5-1 was similar to that of its wild type (Fig. 10).
Anther development in nt-tms5-1 was primarily divided into eight differential stages: pollen mother cell formation, meiosis, the early, middle, and late microspore stages, and the early bicellular, late bicellular and mature pollen stages. In the wild type, a four-layer anther wall (from the outside to the inside: epidermis, endothecium layer, middle layer, and tapetum) was generated at the pollen mother cell formation stage. No obvious defects were found between the WT and nt-tms5-1 anthers in the formation of PMCs. During pollen mother cell (PMC) meiosis, the PMCs underwent meiosis, and normally formed dyads with cell plates in the WT and nt-tms5-1 were clearly observed (Fig. 10).
Subsequently, dyads also formed in nt-tms5-1, and no abnormalities were found in this stage (Fig. 10).
Thereafter, the microspores of WT underwent vacuolation and mitosis to form mature pollen with spherical or elliptical shapes (Fig. 10). In contrast, the microspores of nt-tms5-1 degraded further after the late microspore stage and completely disappeared at the mature pollen stage, which resulted in an empty anther locule (Fig. 10). All of these results suggest that the lack of TMS5 also causes the defects in the microspores as well abnormal pollen in the neo-tetraploid rice during the pollen development.
To further investigate how TMS5 works in regulating the pollen development process of neo-tetraploid rice, 10 genes were selected to analyse their gene expression levels in nt-tms5-1 and its wild type (Fig. 11a). We conducted the qRT-PCR analysis to detect the nine transcripts of these genes in both nt-tms5-1 and its wild type (Fig. 11b). In this study, the expression levels of the nine genes were shown to be differentially expressed between nt-tms5-1 and its wild type. For example, four genes, LOC_Os04g50600, LOC_Os04g32930, LOC_Os03g40110, and LOC_Os04g59600, were downregulated in the meiosis stage in nt-tms5-1 compared to their levels in the wild type. Five genes, including LOC_Os06g43690, LOC_Os03g13160, LOC_Os03g05720, LOC_Os09g30486 and LOC_Os04g46310, were upregulated in meiosis in nt-tms5-1 compared to those of the wild type. These results verify that TMS5 showed the stronger effect in neo-tetraploid rice and that knockout of this gene could result in low pollen fertility in neo-tetraploid rice.

Significant phenotypic variation exists in the neo-tetraploid rice compared with its two parents
Autotetraploid rice is a new germplasm resource derived from diploid rice by chromosome doubling.
Abundant advantages, such as a stronger stem, wider leaf and bigger grains, exist in the autotetraploid rice compared to those of its diploid counterparts (Tu et al. 2007;Wu et al. 2013;Wu et al. 2014).
Agronomic traits in autotetraploid rice demonstrate significant potential to improve the rice biomass yield (Tu et al. 2007;Shahid et al. 2011;Wu et al. 2013). However, lower fertility of autotetraploid rice is still an important issue for utilizing its potential vigour. It took us more than twenty years to generate the neo-tetraploid rice, and we found that it was one type of the stable autotetraploid rice lines derived from the progeny of autotetraploid rice (Guo et al. 2017;Bei et al. 2019). In our previous analysis, we proposed that neo-tetraploid rice have higher fertility and hybrid vigour, which could overcome the low fertility of autotetraploid rice (Guo et al. 2017;Bei et al. 2019). Therefore, it is of greater value to evaluate the phenotypic variation of the neo-tetraploid rice compared with its two parents.
In the present work, one of newly developed neo-tetraploid rice lines, named Huaduo 1 (H1), which has been registered for PVP in China, was used. We analysed the phenotypic variation of H1 and detected that three of the seven primary agronomic traits, including the plant height, seed set ratio and 1000-grain weight, varied significantly compared to those of the two parents. Notably, the seed set ratio in the neo-tetraploid rice can reach >80%, which is much higher than that of its two parents.
These obvious phenotypic variations were similar to the other type of neo-tetraploid rice (Bei et al. 2019) and show great potential for the utilization of H1. Additionally, we also evaluated the heterosis and gene interaction effect using Taichung 65-4x and its pollen sterility near-isogenic lines and found that the seed set ratio of the hybrids can reach to more than 80%. These results show the significant potential that H1 may have given the neutral genes for pollen fertility that could overcome the sterility of hybrids. The neutral genes could overcome the hybrid's sterility caused by the multi-pollen sterility loci interactions in autotetraploid rice hybrids Chen et al. 2019).
Pollen fertility was thought to be the important factor for determining production in autotetraploid rice. Abnormal meiotic chromosome behaviour, microtubules or interactions of pollen sterility-related genes were the primary reasons leading to pollen abortion in autotetraploid rice (He et al. 2011;Li et al. 2018). Therefore, we observed the chromosome behaviour of PMCs to detect the genetic variations between neo-tetraploid rice and its parents. The results indicate that the percentage of abnormalities in PMC cells was higher in T44-4x and T45-4x compared with that in the neo-tetraploid rice. For example, the percentages of abnormal cells in T44-4x is 52.14, 15.05, 3.96, 23.10, 52.14 and 12.12% in Metaphase І, Anaphase І, Telophase І, Metaphase II, Anaphase II and Telophase II, respectively. In contrast, the percentages of abnormal cells in H1 were much lower than those of its two parents. Additionally, one interesting phenomenon was found in the configuration of the neo-tetraploid rice at the diakinesis stage. The configuration in neo-tetraploid rice was primarily bivalent and quadrivalent and showed significant differences from its two parents. All of these results indicate that neo-tetraploid rice shows obvious variation and a higher percentage of fertility than its two parents.

Upregulation of pollen fertility genes plays is critical for high pollen fertility in neo-tetraploid rice
Neo-tetraploid rice exhibit higher fertility and greater heterosis than those of the autotetraploid rice lines (Guo et al. 2017;Bei et al. 2019); however, there are still limitations to understanding the high fertility mechanism of neo-tetraploid rice. To understand the regulatory mechanism of high fertility in neo-tetraploid rice, we combined the re-sequencing and RNA-sequencing analysis to analyse the genomic and transcriptional variations between neo-tetraploid rice and its two parents. Using the transcriptome analysis profile, we identified a total of 1483 DEGs that showed at least a 2-fold change in neo-tetraploid rice compared with its two parents. Gene ontology (GO) analysis was conducted to annotate the up-and downregulated genes that were differentially expressed in neo-tetraploid rice and its parents in this study. The GO enrichment classification indicated that differentially expressed genes in the cellular component, molecular function and biological process categories showed significant variations. Notably, upregulated genes were mainly involved in the response process, pollen development and transcription regulator activity.
Meiosis is an important process, where errors can result in low fertility in autotetraploid rice.
Meiosis-related and meiosis-stage specific genes were frequently detected to be downregulated, and this was thought to be the primary cause of the lower pollen fertility in autotetraploid rice Wu et al. 2017;Wu et al. 2019). Compared with autotetraploid rice, pollen fertility and seed set of neo-tetraploid rice were much higher and exhibited normality (Guo et al. 2017;Bei et al. 2019). We speculated that some important pollen fertility-related genes may be expressed normally again or upregulated in neo-tetraploid rice when compared with autotetraploid rice. Therefore, we focused on differentially expressed genes that were upregulated in neo-tetraploid rice compared with autotetraploid rice.
In the present work, we combined our results with the meiosis-related and meiosis stage-specific genes detected by previous analyses (Fujita et al. 2010;Deveshwar et al. 2011;Hollister et al. 2012;Yant et al. 2013;Wu et al. 2014;Wright et al. 2015;Wu et al. 2015). A total of 240 meiosis stage-specific and meiosis-related genes were detected and found to be upregulated in neo-tetraploid rice compared with its two parents. Notably, 93 meiosis-related or stage-specific genes were detected in Wu et al. (2014), and 22 meiosis-related or stage-specific genes showed a similar tendency as those by genes play an important role in neo-tetraploid rice. Moreover, meiosis-related genes and meiosis stage-specific genes in the neo-tetraploid rice of H1 were found to involve a stronger network based on the predicated protein-protein interaction and co-expression analysis.
Among the meiosis-related genes and meiosis stage-specific genes, ten meiosis-related genes, including LOC_Os01g16810, LOC_Os02g12290, LOC_Os04g53760, LOC_Os04g37960, LOC_Os09g27620, LOC_Os03g12414, LOC_Os06g49840, LOC_Os07g39220, LOC_Os07g30240 and LOC_Os10g35180, encoded the meiosis-related proteins that are mainly involved in the rice pollen development process. Notably, several genes have been functionally analysed or predicted that show a relationship in pollen development. Notably, three genes, CSA (LOC_Os01g16810), TMS5 (LOC_Os02g12290) and TMS9-1 (LOC_Os09g27620), are pollen fertility-related genes that are responsive to temperature. CSA (LOC_Os01g16810) is a MYB family transcription factor that encodes a MYB protein domain, which plays an important role in the pollen development process. TMS5 (LOC_Os02g12290) is a nuclear ribonuclease Z that processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (UbL40) genes into multiple fragments, which could result in pollen sterility under low temperature. All of these results indicate that upregulation of pollen fertility genes plays an important role in the high fertility of neo-tetraploid rice.

Environmentally sensitive genic male sterility (EGMS) likely regulates pollen fertility in neo-tetraploid rice
High pollen fertility is a primary characteristic of neo-tetraploid rice; therefore, it is of great value to understand the mechanism of its higher pollen fertility. With the advantage of effective tools such as CRISPR/Cas9 technology, we are given the opportunity to knock out important fertility-related genes.
To date, there is no information regarding the effect of knocking out important fertility genes in neo-tetraploid rice. In the present work, we detected three environmentally sensitive genic male sterile genes, CSA (LOC_Os01g16810), TMS5 (LOC_Os02g12290) and TMS9-1 (LOC_Os09g27620), that were upregulated in neo-tetraploid rice compared with its two parents. We proposed that pollen fertility-related genes may be upregulated or normally expressed in neo-tetraploid rice compared with those of the autotetraploid rice. Therefore, we selected one important gene, TMS5, to verify our speculation in neo-tetraploid rice. TMS5 is an important photoperiod-and thermo-sensitive gene that can lead to the TGMS trait through a loss of RNase Z S1 function (Zhou et al. 2014).
From this study, we obtained a homozygous mutant of TMS5 and found that nt-tms5-1 and its wild type exhibited marked differences in anther, panicle and pollen. Notably, pollen fertility and pollen development experienced differential variation during the pollen development stage. Moreover, we also found that TMS5 also has similar results and exhibited pollen sterility under the conditions of a long day. As TMS5 is an important photoperiod-and thermo-sensitive gene, this method also provides the possibility for boosting the development of excellent pollen sterility lines or revealing the mechanism of high pollen fertility in neo-tetraploid rice. In the future, better yield and quality can also be developed by editing important pollen fertility genes for fertility and disease at the same time in neo-tetraploid rice in the appropriate genetic background.

Conclusions
In the present study, we found that upregulation of pollen fertility genes plays an important role in neo-tetraploid rice. Our results provide strong evidence that the upregulation of pollen fertility genes results in high fertility of neo-tetraploid rice using cytological and transcriptome analysis.
Differentially expressed genes, including 240 upregulated meiosis-related genes, can be used as candidate genes to reveal the mechanism of high pollen fertility in neo-tetraploid rice in the future.

Plant material
Three autotetraploid materials, including the H1 and its two parent (T44-4x and T45-4x), were used in this study. H1 was the high pollen fertility material derived from the hybrid crosses from T44-4x and T45-4x and then self-crossed for more than 15 generations. All of these materials were planted under the natural conditions at the experimental farm of South China Agricultural University (SCAU) and standard practices followed the recommendations for the area.

Analysis of agronomic traits and heterosis
To detect the genetic variation of neo-tetraploid rice and its two parent, total eight agronomic traits were selected to detect the phenotypic variation, i.e. plant height (PH, cm), panicle length (PL, cm), effective panicles number (EPN), panicle length (PL, cm), total number of grains per plant (TGP), 1000-grain weight (GWT, g) and SS = seed set ratio ((SS = number of filled grains/total number of grains) × 100). These traits were detected according to our previous study (Wu et al. 2013).
Heterosis analysis was conducted to evaluate the heterosis level of H1, total 13 differential parents crossed with the H1 and F1 hybrid. High-parent heterosis (HPH) and mid-parent heterosis (MPH) were estimated by the following formula: HPH = (F1 − HP)/HP × 100%, and MPH = (F1 − MP)/MP × 100%, where F1 is the performance of first filial generation (hybrid), HP is the performance of the best parent, and MP is the average performance of two parents.

Pollen fertility, chromosome behavior observation
Pollen fertility of H1 and its two parents was observed according to our previous study with minor modifications ). More than 1000 pollen grains were calculated for pollen fertility under a microscope (Motic BA200).
The meiosis chromosome behaviour experiment was performed according to Wu et al. (2014). To observe the chromosome behaviour in the meiosis process, samples were collected from the shoots of rice plants with -2 to 2 cm between their flag leaf cushion and the second-to-last leaf cushion. Then, the samples were fixed in Carnoy's solution (ethanol:acetic acid, 3:1 v/v) for at least 24 h and washed using 95% and 80% ethanol for ~30 min each. Finally, they were washed and kept in 70% ethanol at 4 C until observation. The meiosis chromosome behaviour and meiosis stage divisions were observed according procedures described by He et al. (2011a) and Wu et al. (2014).

DNA library construction, massive re-sequencing, and validation analysis
Genomic DNA of three materials, H1, T44-4x and T45-4x, were extracted from fresh leaves according to the procedure described by Chen et al. (2018). Sequencing libraries were constructed from genomic DNA of neo-tetraploid rice and its two parents and sequenced on an Illumina HiSeqTM2500 according to the manufacturer's instructions. Whole genome re-sequencing analysis was performed by Biomarker Technologies (Beijing, China) with an average coverage of approximately 45× in each material. The sequencing reads were aligned to the japonica Nipponbare reference genome using BWA software.
Identification of polymorphic sites, including SNPs and indel analyses, between neo-tetraploid rice and its two parents was performed with GATK software tools. SNPs and indel annotations were performed using SnpEff software.
Polymerase chain reaction (PCR) was conducted to verify the re-sequencing results using the genomic DNA of neo-tetraploid rice and its two parents as templates. Important polymorphic DNA of pollen fertility-related genes were selected in this study. Primers were designed using Primer Premier 5.0 software, and the product length ranged from 400 to 800 bp (Additional file 14: Table S11). The whole PCR program was 94 °C for 5 min followed by 35 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 5 min. The PCR products were sequenced, and sequence variations were detected using BioEdit software.

Sample preparation, RNA extraction, RNA-sequencing, and qRT-PCR verification
Anthers from pre-meiotic to prophase I of meiosis stage were confirmed by fluorescence microscope.
All samples (H1, T44-4x and T45-4x) were collected in three biological replicates and stored at -80℃ until RNA extraction. Total RNA of each sample was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The quantity and purity of total RNA were analysis by Real-time qRT-PCR analysis was conducted to examine the expression patterns of neo-tetraploid rice and its two parents. Total twelve candidate genes were selected and used to validate the transcriptome data using the same RNA samples of RNA-sequencing (Additional file 14: Table S11).
Reverse transcription reaction was done was done using the Roche Transcriptor First Strand cDNA Synthesis kit. The qRT-PCRs experiment was performed on the Lightcycler480 system (Roche) using the Advanced SYBR Green Supermix Kit (Bio-RAD). The qRT-PCR cycles were using the following reaction conditions: 95°C for 30s, 40 cycles of 95°C denaturation for 5s and 58°C annealing and extension for 20s. All qRT-PCR reactions were performed in triplicate, and the results were calculated using the 2 -ΔΔCt method. Rice ubiquitin gene used as an internal control to normalize the expression levels. Each PCR reaction repeated three times.

Generation and mutation detection of mutant plants in neo-tetraploid rice
The nt-tms5-1 mutant was derived from H1 using the CRISPR/cas9 technique. We used the CRISPR-Cas9 binary vector pC1300-cas9 designed by Zhou et al. (2016)   Breeding procedure and phenotype of neo-tetraploid rice and its parent. a Breeding procedure of neo-tetraploid rice, Huaduo1 (H1). b Morphologies of whole plant between neo-tetraploid rice, and its two parents. c-e Grain size of H1, and its two parents. f-h pollen grains stained with I2-KI in H1 and its two parents. i-k Comparison of the pollen fertility (i), seed set ratio (j) and plant height (k) between H1 and its two parents.     Genes were divided into three categories: biological process, cellular component, and molecular function. Expression patterns of meiosis genes. b Expression patterns of the 12 known tapetum-related genes. c Expression patterns of the 12 known pollen fertility genes.

Fig. 9
The distribution of up-regulated genes involved in meiosis process and anther specific in neo-tetraploid rice comparative with its two parents. Semi-thin sections of nt-tms5-1 anthers. q, pre-meiotic interphase; r, meiosis stage; s, meiosis stage; t, single microspore stage; u, late bicellular; p, v, mature pollen stage. E, En, M and T indicate the epidermis, endothecium, middle layer and tapetum, respectively. Bars = 50 μm.

Fig. 11
Verification of the predicated TMS5 regulation network during the pollen development process in nt-tms5-1 and its wild type. a Predicted protein-protein interaction network of TMS5 gene. b to j,