Development of High Yielding Glutinous Cytoplasmic Male Sterile Rice ( Oryza sativa L . ) Lines through CRISPR / Cas 9 Based Mutagenesis of Wx and TGW 6 and Proteomic Analysis of Anther

Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T0 mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T1 plants were re-edited but T2 generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC2F1. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD+-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development.


Introduction
The rice (Oryza sativa L.) is an important widely adapted food crop and 20% of the world's dietary energy supply which is feeding more than half of the world's population and 3 billion people uptake rice daily [1,2].Due to the fast-growing population, the global rice consumption is projected to increase from 450 million tons in 2011 to about 490 million tons in 2020 and 40% more rice is needed to be produced by 2050 to meet people's demand for food [3,4].The cytoplasmic male sterility (CMS) is the foundation to exploit the heterosis of hybrid rice which uses a three-line system consisting of a cytoplasmic male sterile (CMS) line (A line), a maintainer (B), and a restorer (R line) for hybrid seed production [5].China is the pioneer of hybrid rice production and with the development of latest breeding tools the yield of rice has been increased more than 20% and newly developed genotypes performing better than conventional verities and now accounts more than half of the annual rice planting area in China [6].The development of new CMS has become the main interest of breeders because very few genotypes exhibit a strong restoration ability as effective restorer for CMS in the development of hybrid rice [7,8].Yield and quality are typical quantitative traits governed by multiple genomic loci, while yield is directly depends on grain weight (GW) which is mainly determined by the synthesis and accumulation of starch in the endosperm of the grain [9,10].To solve this problem, we must resort to new technologies and new genetic improvement strategies.Starch is one of the important indicators for evaluating rice quality and 90% of rice endosperm is starch [11].
Rice waxy gene Wx-encoded granular bound starch synthase I (GBSSI), also known as Waxy protein is the major gene controlling amylose synthesis in endosperm [12].Wx gene differentiates into alleles Wx a and Wx b , indica rice is dominated by Wx a which confers higher amylose content by producing 10-fold higher mRNA and protein level than Wx b while japonica rice is dominated by Wx b with lower amylose content [13,14].Wx exon or intron structural change would affect Wx expression by affecting messenger RNA (mRNA) stability [12,15].Several studies have reported that mutations in the functional site of the Wx gene led to 14.6 to 2.6% reduced amylose content (AC) in rice transgenic lines and hybrids obtained with mutant lines [15][16][17][18][19][20][21][22][23], while Wx overexpression lines showed increased AC by 6-11% [24].
With the development of some new molecular biology techniques such as CRISPR/Cas9 (clustered regulatory interspersed short palindromic repeat/CRISPR associated proteins) a lot of achievements has been made in plants and animals.CRISPR/Cas9 technology is widely used to study the gene function and regarded as the third-generation genome-editing tool established after zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN), based on guided RNA (gRNA) engineered nucleases, which is most applicable due to their simplicity, efficiency, and versatility [40,41].CRISPR/Cas9 make a double-stranded break (DSB) in the target DNA which is subsequently repaired by natural repair mechanism of homologous recombination (HR) precise pathway or non-homologous end joining (NHEJ) [42], which creates random insertions and deletions and results in targeted gene knockouts or gene replacement [40,43,44].CRISPR/Cas9 is the most advanced genome editing tool in plant biology [45,46] and has been widely used in animals, yeast, human non-human cell lines [42,47,48], as well as in the model species A. thaliana and N. benthamiana [43,49], as well as crops such as rice [50][51][52], wheat [53], maize [54], potato [55], and tomato [56].
Conventional plant breeding techniques are effective but laborious and time consuming, therefore we used CRISPR/Cas9-mediated gene editing to introduce a loss-of-function mutations into the Wx and TGW6 genes associated with lower AC and increase yield in rice maintainer line 209B.Our results show that mutations in the Wx and TGW6 gene produce decreased AC and enhanced yield in rice CMS line offering an effective strategy of accelerating the hybrid rice breeding program.Through one generation of hybridization and two generations of backcrossing with mutant maintainer lines as the male parent and 209A as female parent, the glutinous cytoplasmic male sterile lines (CMS) were successfully achieved.The protein of CMS line pollen and mutant maintainer line were separated by two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and differentially expressed spots were analyzed.This study gave new insights into the mechanism of CMS and maintainer lines and demonstrated the power of proteomic in plant biology.Present study showed that the CRISPR/Cas9 technology provides the tool set to fasten the rice breeding program to achieve desired agronomic characters and improved yield.

Rice Material, CRISPR/Cas9, and gRNA Vectors
The cytoplasmic male sterile line 209A and its maintainer line 209B developed by Professor Li Rongbai, were collected from Rice Research Institute of Guangxi University.The maintainer line 209B was used for genetic transformation to which have the characteristics of resistance to drought and blast with compact plant type.Plants were grown in the experimental field of Guangxi University during normal rice growing season and maintained regularly.The Cas9 vector pYLCRISPR/Cas9-MT(I) and the gRNA vectors (OsU6a, OsU6b, OsU6c, and OsU3m) (Figure 1) were provided by Professor Liu Yaoguang, South China Agricultural University, Guangzhou, China.Through one generation of hybridization and two generations of backcrossing with mutant maintainer lines as the male parent and 209A as female parent, the glutinous cytoplasmic male sterile lines (CMS) were successfully achieved.The protein of CMS line pollen and mutant maintainer line were separated by two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and differentially expressed spots were analyzed.This study gave new insights into the mechanism of CMS and maintainer lines and demonstrated the power of proteomic in plant biology.Present study showed that the CRISPR/Cas9 technology provides the tool set to fasten the rice breeding program to achieve desired agronomic characters and improved yield.

Rice Material, CRISPR/Cas9, and gRNA Vectors
The cytoplasmic male sterile line 209A and its maintainer line 209B developed by Professor Li Rongbai, were collected from Rice Research Institute of Guangxi University.The maintainer line 209B was used for genetic transformation to which have the characteristics of resistance to drought and blast with compact plant type.Plants were grown in the experimental field of Guangxi University during normal rice growing season and maintained regularly.The Cas9 vector pYLCRISPR/Cas9-MT(I) and the gRNA vectors (OsU6a, OsU6b, OsU6c, and OsU3m) (Figure 1) were provided by Professor Liu Yaoguang, South China Agricultural University, Guangzhou, China.

Vector Construction
The selected binary plasmids were isolated from E. coli (Escherichia coli) TOP10F' strains according to the previous established methods [57,58] with some modifications (Figure S2).The target site containing sequence primers WxgRT1/OsU6aWxT1 and WxgRT2/OsU6bWxT2 TGgRT1/OsU6cTGT1 and TGgRT2/OsU3TGT2, (Table S2) were combined by annealing, and then the target site sequence-containing chimeric primers were cloned into the sgRNA expression cassettes pYLsgRNA-U6a, pYLsgRNA-U6b, pYLsgRNAU6c and pYLsgRNA-U3m at a BsaI site (Figure S3).The integrated sgRNA expression cassettes were then amplified by nested polymerase chain reaction (PCR) using U-F/Reverse adapter primers and gR-R for the first round, and the corresponding site-specific primers Pps-R/Pgs-2, Pps-2/Pgs-3, Pps-3/Pgs-4, and Pps-4/Pgs-L (Table S2) for the overlapping PCR (Figure S4) to ligate four-target-sgRNAs expression cassettes into the pYLCRISPR/Cas9Pubi-H vector and ligation product was transformed into DH5α competent cells according to the established protocol [20] with some modifications (Figure S5).The monoclonal inoculation culture was picked and were amplified by using SPL1 and SP-R primers (Table S2) and the clones confirming the product length ware sent to Beijing Genomics Institute (BGI) for sequencing.

Agrobacterium-Mediated Transformation of Rice Callus
The constructed plasmid was transformed into Agrobacterium EHA105 by electroporation according to the established method [59], and positive clones were used for rice callus transformation and transformed plants were obtained by hygromycin screening.

Identification of T-DNA Free Mutant Lines and Cross Section Analysis of Grain Endosperm
The genomic DNA of T 1 and T 2 generations was extracted and amplified by using Cas9-F and Cas9-R specific primers (Table S2).The amplified product was subjected to 1% agarose gel to check the T-DNA free mutant plants.Scanning electron microscopy was used to observe the cross section of the mutant and its wild type (WT) mature grain according to the previous established method [60].

Expression Analysis
Total RNA was extracted from WT and T 1 mutant plants by using TaKaRa MiniBEST Plant RNA Extraction Kit according to manufacturer instructions.The specific primers for Wx gene W-F/W-R and TG-F/TG-R were used for TGW6 (Table S2).The rice OsActin gene was used as internal control and 20 µL reaction was prepared with 2 µL cDNA, 0.4 µL each of the forward and reverse primers, 10 µL, Synergy Brands, Incorporated (SYBR) Green Master Mix, 7.2 µL ddH 2 O. PCR Amplification procedure was as followed, 30s at 94 • C, 5s at 94 • C, and 30s at 60 • C with 45 cycles.The relative expression of genes was calculated from three biological replicates per sample according to the 2 -∆∆Ct method [61].

Determination of AC, SC, GC, and GT
The AC of T 0 , T 1 , and T 2 generations were measured after 3 months of harvesting.The total AC and SC were measured by using Megazyme Amylose Assay Kit (KAMYL), Guangzhou, China and Total Starch Assay Kit (AA/AMG), Guangzhou, China.The GC was evaluated for random five samples [62] and alkali digestion test was used to estimate GT [63].

Phenotyping
The data was recorded in five plants per line for GW (g) in T 0 , T 1 , and T 2 generation while the data for main agronomic traits was recorded in T 2 generation, such as plant height (PH) (cm), number of panicles (NOP), flag leaf length (FLL) (cm), flag leaf width (FLW) (cm), panicle length (PL) (cm), grains per spike (GPS), and seed setting rate (SSR) (%), as described previously [28].

Backcrossing and Observation of Pollen and Spikelet Fertility
The mutant maintainer lines T2-4-2, T2-7-1, T2-14-4, T2-19-3, T2-23-5, and 209A sterile line were crossed with two CMS lines MX-G1 and MX-G2 and during next season the 12 testcrosses along with respective male parents were transplanted and fertility test was performed at flowering stage.The pollen and spikelet fertility test were done by 1% (m/v) I 2 -KI solution at flowering stage to evaluate the fertility restoration ability of restorer lines.The young spikelets were collected in the early morning to determine pollen fertility and kept in the jar about 2 h for opening the spikelets.The pollen was stained with KI solution and observed under a compound microscope.The stained pollens with round shape and well developed were considered as viable and irregular shaped and non-stained pollens were counted as sterile pollens.The criteria for classifying the parental lines as maintainers and restorers were followed as proposed previously [64].Subsequent backcrosses were made between mutant lines as male and 209A as female to develop glutinous CMS lines.

Pollen Protein Analysis
Anthers of CMS line and maintainer line were taken from upper part of one panicle inside in the spikelet and located in the middle of panicles were collected and protein was extracted according to previous established method [65].The protein separation was performed through SDS-PAGE gel electrophoresis [66] and proteins were identified by peptide mass fingerprinting (PMF) [67].The analysis was performed with mass to charge ratio (m/z) formula to identify the monoisotopic masses and search in the NCBInr database using MASCOT (Matrix Science) software.

Statistical Analysis
The data were analyzed using SPSS 16.0 Statistical Software Program.The graphs were developed by GraphPad Prism (version 7.0, GraphPad Software Inc., San Diego, CA, USA).

gRNA Design and Vector Construction
The 20bp long target sites were chosen in Wx and TGW6 codon region and predictive cleavage site for the TGW6 was 5pb upstream from initiation codon (ATG) and 72bp downstream of Wx.The sgRNA expression cassette was generated by overlapping PCR (Figure 3a) and ligating of the target adaptors to the BsaI-digested sgRNA intermediate plasmid and amplified products were successfully assembled in to pYLCRISPR/Cas9-MT(I) binary plasmid with Golden Gate ligation method (Ma et al. 2015).The ligation product was transformed to E. coli and positive clones were verified.The CRISPR/Cas9 binary plasmid was constructed and sequencing peaks confirmed that four targets were assembled in the plasmid successfully (Figure 3d,e).

T0 Genotyping and Off-Target Analysis
Total 55 positive mutant plants were obtained, and the DNA of 25 plants was extracted by CTAB method to analyze the mutations in target sites.The sequencing results showed that for Target1 of Wx gene there were 10 homozygous mutant plants, 5 heterozygous, 7 bi-allelic, and 1 chimeric and 2 were WT; while, for the Target2 of Wx gene, there were 4 homozygous mutant plants, 6 heterozygous, 10 bi-allelic and, 1 chimeric and 4 were WT.The mutation frequency for Target1 of TGW6 gene was as; 9 homozygous, 7 heterozygous, 6 bi-allelic, 1 chimeric, and 2 WT and for Target2 of TGW6 there were 5 homozygous, 8 heterozygous, 9 bi-allelic, 1 chimeric and 2 WT (Table 1).Five plants with good

T 0 Genotyping and Off-Target Analysis
Total 55 positive mutant plants were obtained, and the DNA of 25 plants was extracted by CTAB method to analyze the mutations in target sites.The sequencing results showed that for Target1 of Wx gene there were 10 homozygous mutant plants, 5 heterozygous, 7 bi-allelic, and 1 chimeric and 2 were WT; while, for the Target2 of Wx gene, there were 4 homozygous mutant plants, 6 heterozygous, 10 bi-allelic and, 1 chimeric and 4 were WT.The mutation frequency for Target1 of TGW6 gene was as; 9 homozygous, 7 heterozygous, 6 bi-allelic, 1 chimeric, and 2 WT and for Target2 of TGW6 there were 5 homozygous, 8 heterozygous, 9 bi-allelic, 1 chimeric and 2 WT (Table 1).Five plants with good phenotype (4,7,14,19,23) were selected and identified with deletions of DNA fragments between distinct target sites (Figure 4a,b).In two of these homozygous mutant plants (4 and 7) were found with large fragment deletion at target sites of both genes.The mutant plant (14) with bi-alleic mutations in both genes was also found.The DSBs occurred either specifically in the upstream of the PAM (Figure 4), or at imprecise sites thus eliminating the genomic sequence beyond the PAMs.However, we analyzed the limited number of amplicons per transformant so maybe the actual number of mutations might be higher.In 25 of the plants the WT sequences of both genes were also detected, suggesting that Cas9 was inactive in these events.The average mutation types for both genes were 32% bi-allelic, 28% homozygous, 26% heterozygous, 4% chimeric, and 10.0% were WT (Table 1).The mutation rate for both genes was 90% and based on allele mutation types, for Wx gene 80% (4/5) of the mutations were simultaneous nucleotide deletions and insertions, 20% (1/5) of the mutations were only deletions with no insertions, and there were no mutations with only insertions (Figure 4a).The allelic mutation types for TGW6 were 60% deletions, 40.0%simultaneous deletions and insertions and there was no mutation with only insertions (Figure 4b).As for the deletion mutations, the 40% mutations were large fragment deletions ranging from −11 to −120, while 60% were short (≤ 10 bp) deletions ranging from −1 to −6 and as for the insertion mutations, 90% (9/10) were 1 bp insertions and 10% (1/5) were +2 insertions (Figure 4).Comparison of WT and mutant's deduced amino acid sequences revealed that mutations resulted in changed conserved amino acid sequences (Figure 5).
The off-target predictions by CRISPR-P tool were analyzed and three off-target sites were selected for each target and examined by PCR based sanger sequencing in T 0 generation.The results showed that there were no off-target effects in the selected putative loci and targeted mutation were easily detected (Table S3).

Expression Level of Target Genes in WT and Mutant Lines
qPCR was used to detect the relative expression of TGW6 and Wx gene of T 0 plants.The expression of WT was not altered, and the expression of mutant plants was substantially downregulated in the Wx and TGW6 mutants compared with WT (P < 0.01, Figure 6) indicating that mutations have successfully affected the target genes expression.The homozygous mutant lines (4,7) with large fragment deletions showed lower expression level of Wx and TGW6 gene.

Expression Level of Target Genes in WT and Mutant Lines
qPCR was used to detect the relative expression of TGW6 and Wx gene of T0 plants.The expression of WT was not altered, and the expression of mutant plants was substantially downregulated in the Wx and TGW6 mutants compared with WT (P < 0.01, Figure 6) indicating that mutations have successfully affected the target genes expression.The homozygous mutant lines (4,7) with large fragment deletions showed lower expression level of Wx and TGW6 gene.

Screening of T-DNA Free T1 Generation and Seed Cross-Section Analysis
We addressed the genetically modified (GM) related regulations and issue of social acceptance of GM foods and to avoid public controversy by removing the transgenes from CRISPR/Cas9-edited waxy rice lines by self-pollination in the T1 generation which allowed to produce non-GM lines containing the desired mutations.The DNA of T1 and T2 generations was extracted to investigate the possibility of obtaining rice lines harboring the desired modifications in target genes but without transferred DNA (T-DNA), the Cas9 gene specific primers Cas9-F and Cas9-R were used and amplified by PCR and T-DNA negative lines were selected for sequencing of the target regions.Notably, 13 T1 plants were failed to generate the Cas9-specific amplicon (Figure 7a).Similarly, the PCR assay also failed to detect the Cas9 specific amplicon in the same 13 mutant lines of T2 generation (Figure 7b).These results showed that T-DNA-free plants carrying the desired gene modifications can be acquired through genetic segregation in later generations.
Scanning electron microscopy of cross section of grain endosperm revealed that CRISPR/Cas9 mutant line showed opaque appearance compared to WT endosperm.The starch granules in the cross-section of WT grain were packed like polyhedral structure but in the mutant line a greater number of small and irregularly arranged starch granule structures were observed (Figure 7d,e,f,g), indicating a change in the mature grain of mutant line.The change in structure may cause the scattering of the light as it passes and resulted in opaque appearance.

Screening of T-DNA Free T 1 Generation and Seed Cross-Section Analysis
We addressed the genetically modified (GM) related regulations and issue of social acceptance of GM foods and to avoid public controversy by removing the transgenes from CRISPR/Cas9-edited waxy rice lines by self-pollination in the T 1 generation which allowed to produce non-GM lines containing the desired mutations.The DNA of T 1 and T 2 generations was extracted to investigate the possibility of obtaining rice lines harboring the desired modifications in target genes but without transferred DNA (T-DNA), the Cas9 gene specific primers Cas9-F and Cas9-R were used and amplified by PCR and T-DNA negative lines were selected for sequencing of the target regions.Notably, 13 T 1 plants were failed to generate the Cas9-specific amplicon (Figure 7a).Similarly, the PCR assay also failed to detect the Cas9 specific amplicon in the same 13 mutant lines of T 2 generation (Figure 7b).These results showed that T-DNA-free plants carrying the desired gene modifications can be acquired through genetic segregation in later generations.
Scanning electron microscopy of cross section of grain endosperm revealed that CRISPR/Cas9 mutant line showed opaque appearance compared to WT endosperm.The starch granules in the cross-section of WT grain were packed like polyhedral structure but in the mutant line a greater number of small and irregularly arranged starch granule structures were observed (Figure 7d-g), indicating a change in the mature grain of mutant line.The change in structure may cause the scattering of the light as it passes and resulted in opaque appearance.

Transmission of Mutations in T1 and T2 Generations
The sequencing results showed that mutations in T0 generation were not stable in some mutant plants but T2 generation was completely stable.The mutant plants of 7-1, 14-4, and 19-3 lines in T1 generation exhibited insertions and deletions in the Wx target regions, while 4-2 and 23-5 showed consistent mutations in T0 and T1 (Figure 8a; Figure 4a).The mutant plants of 4-2, 7-1, and 23-5 showed insertions and deletions at TGW6 target sites in T1 generation, while mutations in 23-5 and 14-4 were similar in T0 and T1 generation (Figure 8b; Figure 4b).The transmission of mutations from T1 to T2 generation were investigated and sequencing results of T2 plants showed that the mutations were consistent with the T1 generation without any insertions or deletions, indicating that the T2 generation has been stabilized (Figure 8a,b; Figure 4a,b ).

Transmission of Mutations in T 1 and T 2 Generations
The sequencing results showed that mutations in T 0 generation were not stable in some mutant plants but T 2 generation was completely stable.The mutant plants of 7-1, 14-4, and 19-3 lines in T 1 generation exhibited insertions and deletions in the Wx target regions, while 4-2 and 23-5 showed consistent mutations in T 0 and T 1 (Figure 8a; Figure 4a).The mutant plants of 4-2, 7-1, and 23-5 showed insertions and deletions at TGW6 target sites in T 1 generation, while mutations in 23-5 and 14-4 were similar in T 0 and T 1 generation (Figure 8b; Figure 4b).The transmission of mutations from T 1 to T 2 generation were investigated and sequencing results of T 2 plants showed that the mutations were consistent with the T 1 generation without any insertions or deletions, indicating that the T 2 generation has been stabilized (Figure 8a,b; Figure 4a,b).

AC, GC, GT, and SC
AC of T0, T1, and T2 generations were determined while the GC and GT was recorded for T2 generation.The AC of mutant lines were significantly decreased (P < 0.01) as 18.2% to 1.7%.The homozygous mutant lines with long fragment deletion (4, 7) showed more decreased AC than heterozygous and bi-allelic mutants (Table 2).Grains of mutant plants were white and fully opaque in contrast with the typical non-waxy WT (Figure 7c).There was no effect on other grain quality traits, as result showed that total SC was unchanged in mutant and WT plants (Table 2).Another trait related to eating and cooking, GT, was also greatly decreased in the mutant plants and GC was increased as compared to the WT (Table 2).

AC, GC, GT, and SC
AC of T 0 , T 1 , and T 2 generations were determined while the GC and GT was recorded for T 2 generation.The AC of mutant lines were significantly decreased (P < 0.01) as 18.2% to 1.7%.The homozygous mutant lines with long fragment deletion (4, 7) showed more decreased AC than heterozygous and bi-allelic mutants (Table 2).Grains of mutant plants were white and fully opaque in contrast with the typical non-waxy WT (Figure 7c).There was no effect on other grain quality traits, as result showed that total SC was unchanged in mutant and WT plants (Table 2).Another trait related to eating and cooking, GT, was also greatly decreased in the mutant plants and GC was increased as compared to the WT (Table 2).

Yield and Yield Contributing Traits
The results of the GW in T 0 , T 1 , and T 2 generation was recorded (Table 3), and results showed that the mutant plants significantly increased the GW (>5%).As expected, the GW of mutant line (T2-4-2) was 24.0 g maximum, whereas T2-7-1 presented a GW of 23.1 g (Table 3).The GW of T2-14-4, T2-19-3, and T2-23-5 was 23.4 g, 23.1 g and 23.7 g respectively, which was greater than the value of 21.1 g recorded in WT (Table 3).However, there were no significant differences detected in the other main agronomic traits between mutant lines and WT, including the PH, NOP, FLL, FLW, PL, GPS, and SSR (Table 4).
To reduce the breeding cycles to develop glutinous rice lines, the developed mutant glutinous maintainer lines (as the male parent) were used to hybridize with CMS line 209A (as female parent) to produce F 1 hybrids, and then the F 1 hybrids were backcrossed with mutant lines.Molecular marker-assisted selection (MAS) was used to select the homozygous plants in the BC 1 F 1 and CMS plants were selected and again backcrossed with mutant lines and two homozygous BC 2 F 1 CMS lines (GX-B1, GX-B6) were obtained with no genetic segregation and increased yield and waxy-grain phenotype (Figure 7c), which will be used for the further breeding of hybrid glutinous rice.

Pollen Protein Identification
A total 25 spots in both genotypes were exercised and ultimately 16 spots were successfully identified, and proteins associated with pollen development (Table 6).The important proteins Putative acetyl-CoA synthetase and isoamylase were upregulated in both lines which clearly showed that these proteins have important role in CMS and control of AC in rice (Table 6).* Molecular weight (Mr) and isoelectric point (pI) of matched proteins, + Indicates that the protein spot is present, − Indicates that the protein spot is absent, ++ Indicates more than a 2-fold increase.UDP: uridine diphosphate; NAD + : nicotinamide adenine dinucleotide.
The identified proteins have various biological functions based on known functions from known functions from the European Bioinformatics Institute (EMBL-EBI) and literature.The protein spots related to GX-B1 were cellular protein catabolic process, RNA-binding, oxidoreductase activity, calcium-dependent phospholipid binding, nucleoside metabolic process, photosystem I assembly, acetyl-CoA biosynthetic process from acetate and amylopectin and their beta-limit dextrins (highly expressed).The protein spots of 4-2A were related to malate metabolic process (highly expressed), NAD + binding, nucleoside metabolic process, RNA binding, starch biosynthetic process, acetyl-CoA biosynthetic process from acetate (highly expressed) and amylopectin and their beta-limit dextrins.

Discussion
CRISPR/Cas9 is an emerging genome editing technology developed in past few years with high specificity and editing efficiency.Relative to ZFNs [68] and TALENs [69,70], CRISPR/Cas9 is simple and flexible and only one gRNA and one nuclease (Cas9) are needed to achieve the mutations in the DNA sequence of the target gene.Current research focuses on the development of CRISPR/Cas9 technology and specific gene knockouts.
Breeding for consumer-preferred grain yield and quality have thus become a major goal for breeding programs and in the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties for food security considerations.The improved living standards and fast economic growth are shifting public attention toward quality characteristics such as, nutrition, flavor, appearance, and cooking which are linked to starch physical properties.With the development of latest gene editing technologies such as CRISPR/Cas9, many yield related quantitative trait loci (QTLs) has been edited and their functions have been explored in different verities [37].In the rice grain endosperm, starch is the major component consisting of a linear polysaccharide amylose which determines the cooking and eating quality of rice.Cooking of high AC (25-33%) verities results in separated, dry and firm rice grains, becoming hard after cooling while glutinous rice with low AC (5-20%) is especially sticky and soft when cooked [71].The improvement of maintainer line in hybrid rice breeding system is most inevitable to achieve target traits.In China, the indica hybrids considered low quality owing to high AC that makes them hard and dry when cooked.The breeding for low AC and improved grain quality and yield is a major objective of breeders.
In this study the CRISPR/Cas9 construct with 20-nt target sequence for the sgRNA was carefully designed with high GC content and low off-target score and the Wx and TGW6 gene with expectation to produce a null mutation were edited.The schematic representation of whole procedure of generation and analysis of targeted mutated plants was described in Figure S6.The goal of this study was to develop a high yielding CMS rice line with low amylose content to facilitate the hybrid rice breeding program and proteins from anthers of maintainer and CMS line were also identified.We sequenced the Wx and TGW6 gene and confirmed that 209B contains both genes (Figure S7).Four targets were designed in the exon regions of Wx and TGW6 gene, the corresponding promoters were OsU6a, OsU6b, OsU6c, and OsU3m and Agrobacterium tumefaciens based transformations was successfully achieved with the CRISPR/Cas9 cassette and mutations in the target regions were analyzed by sanger sequencing by decoding it using online DSDecodeM tool.The results of this study indicate that the CRISPR/Cas9 gene editing technology can successfully edit rice targeting DNA sequences with high efficiency and multiple mutations can be generated at the same target site, and base deletion or insertion occurs before the target site PAM.
The total mutation frequency was up to 90%, wherein homozygous mutations were about 28%, which indicate that the CRISPR/Cas9 editing facilitates homozygous mutations in the T 0 generation (Table 1).The previous studies showed that the CRISPR/Cas9 induced the homozygous mutations in T 0 generation and mutations mainly take place in transformed calli cells [72].The expression level of targeted genes was lower in mutant lines than WT (Figure 6).The off-target mutations were not detected for all targets (Table S3).The comparison of T 0 and T 1 generations showed that the mutation frequency of homozygotes was stably inherited regardless of whether T-DNA is present.The conserved amino acid sequence was totally changed in mutant plants and mutant plants showed divergence to WT in amino acid sequence alignment (Figure 5).
The glutinous rice lines were obtained, and all mutant lines seeds showed low AC decreased from 18.2% to 1.7 % and homozygous mutant lines showed less percentage of AC than heterozygous and bi-allelic mutants (Table 2).It is reported that the Wx gene also affect the GC and GT of rice [22,73] and our results showed an increase in GC from 58.65 mm to 138.62 mm and decrease in GT from 5.67 ASV to 3.12 ASV, while there was no effect on total SC (Table 2).The GW was increased from 21.1 g to 240.8 g (Table 3), while there was no effect on PH, NOP, FLL, FLW, PL, GPS, and SSR (Table 4).The cross-section analysis by electron microscope showed that endosperm of mutant grains was shrunken corresponding to their WT.The T-DNA free lines were obtained to address the social values of laws about genetically modified (GM) foods by selecting the transgene free lines by self-pollination in the T 1 and T 2 generations (Figure 7).Our results showed that the T 1 mutant lines were re-edited while mutations were inherited and stable in T 2 generation (Figure 8).The T 0 lines are frequently difficult to predict which suggests that the mutations in T 0 generations are not stable but the mutations in T 1 generations transmitted stably to later generations.These results are consistent with previous reports that the editing site of the T 1 generation mutant plant target sequence may also have a sequence recognized by the gRNA target, resulting in re-editing, which makes the T 1 generation unpredicted which can stabilize in later generations [74].Together, these results clearly demonstrate that CRISPR/Cas9-induced gene mutations can be stably transmitted to subsequent generations.
The shape of pollen grains and staining patterns in male sterility inducing cytoplasm and sterility maintaining nuclear genes are influenced by the pollen abortion stage related to nuclear stage [75].Mutant maintainer lines were assessed for pollen fertility status and results showed that pollen fertility rate was randomly distributed and six genotypes were found sterile having pollen fertility 0-9%, two genotypes PS with 10-29% pollen fertility, two genotypes were recorded PF which is 12.5% of total and four genotypes were identified as CF and two were F as these genotypes had above 80% pollen and spikelet fertility which is 37.50% of the total genotypes (Table 5).The developed maintainer lines were crossed with CMS line to develop F 1 and after subsequent backcrossing glutinous CMS line was achieved.
The CMS lines has been widely used in hybrid rice production, but the molecular mechanism of CMS remains poor understood.The protein identification tool is a powerful tool to study anther development and pollen production in plants [76][77][78][79].The CMS is different plant species are cause by a specific ORF containing chimeric genes in mitochondrial genome [80] with rare similarity but sharing same male gamete abortion phenomenon [81].The mitochondrial amplification events suggest an increased demand for energy during pollen development [82] but lowered ATP production was also observed in some CMS flowers [81,83].In this study the proteins identified in maintainer and CMS line helped to understand the molecular mechanism of rice male sterility.Sixteen proteins were identified between sterile and maintainer anthers (Table 6).The identified proteins have potential roles in anther and pollen development and may help to clarify the mechanism of male sterility in rice.
The proteins of CMS line and GX4-2 maintainer mutant line anthers were separated by two-dimensional electrophoresis and SDS-PAGE as the second.The silver stained proteins were analyzed using Image Master 2D software.The identified proteins were, 20S proteasome beta 4 subunit, putative RNA-binding protein, Putative berberine bridge enzyme (BBE), putative mitochondrial NAD + -dependent malic enzyme, Putative calcium-binding protein annexin, UDP-glucuronic acid decarboxylase, putative phosphoribosyl pyrophosphate synthase, putative RNA binding protein (RBP), H + -transporting two-sector ATPase alpha chain-rice mitochondria, glucose-1-phosphate adenylyltransferase large subunit 3, putative membrane-associated salt-inducible protein, putative leucine-rich repeat protein, putative acetyl-CoA synthetase (ACOS), putative lipoamide dehydrogenase, Isoamylase (fragrant), and DNA binding protein (Table 6).These proteins are closely associated with metabolism, protein biosynthesis, transcription, signal transduction and many other activities which are important in cell activities and essential to pollen development.
Dysfunctions of mitochondria in the pollen caused CMS in plants and several other mitochondrion regions have been identified associated with CMS [84].ATP synthase β-subunit helps to fulfill the demand of energy for respiratory function and cellular energy to develop male gametophyte also observed in mitochondria [85], and defective β-subunit resulted non-functional pollens and abnormal anther development [84].The 20S proteasome is the proteolytic complex actively involved in removing abnormal proteins with several biological functions [86], while RBP is involved to regulate transcriptional and post-transcriptional levels to control the gene expression.Plants respond to pathogen infection with rapid reprogramming of gene expression and loss of function of RBP showed enhanced resistance to pathogens [77].Biochemical and biological function of BBE are unexplored [87].Plant annexins regulate diverse aspects of plant development, stress responses and growth [88].ACOS played role in plastids and in several metabolic pathways [89] and has significant role in anther development [90,91].The ACOS in anther prevent the conversion of pyruvate into acetyl-CoA which leads to pollen sterility.The degeneration and formation of various tissues during pollen development needs high energy for key biosynthetic intermediates.Isoamylase in combination with pullulanase plays a predominant role in amylopectin synthesis and also essential for the construction of the amylopectin multiple-cluster structure by removing the excessive branches to avoid interference with the formation of double helices of the cluster chains of amylopectin and crystallization of starch in the endosperm.These proteins or enzymes are involved in multiple physiological and biochemical reactions such as carbon metabolism and starch synthesis, as well as signal transduction and protein expression regulation [92].
In short, the increase yield and reduction of AC are valuable parameters in crop breeding and CRISPR/Cas9 is excellent technology to achieve targeted mutations in genes.In this study the rice maintainer line and new CMS lines were developed with increased yield and improved quality while maintaining all agronomic traits.We also took precautionary approach and produced T-DNA-free plants to avoid foreign bacterial DNA integration and bypass GMO rules.The most likely off-target effects were analyzed and Cas9 free plants were selected for food safety assessments and it was ensured that the other plant traits were not affected.In our work, we improved existing traits by directly rewriting the plant genetic code without any cutting and pasting genes from animals or bacteria into rice plants.Our study provides some insights to study the gene functions and generation of new rice CMS lines with increased yield and improved quality without compromising on nutritional value to facilitate the hybrid breeding programs of rice to develop elite crop verities.This study is the first example to develop rice CMS lines with increased yield and low AC and the protein identification in mutant rice maintainer and CMS line which will be the source material for further breeding of hybrid glutinous rice verities in short period.The identified proteins in anther of maintainer and CMS lines provide the insights to the actual mechanism underlying in sterility of rice lines.The study showed the genetic mutations are not only helpful to improve the plant characteristics, they also help in understanding the mechanisms underlying the biochemical behavior changes in cell of the plants.

Conclusions
The CRISPR/Cas9 technology induces fastest changes to plant genome than other molecular approaches and mutations passed to the next generations without any rewriting or emendations.Different types of mutations were achieved for both genes and a mutant library was generated which laid an important material basis for further high-yield and stable hybrid breeding of rice.This study provides an important theoretical and practical significance and reference for the rapid creation of excellent rice germplasm with important application value such as rice quality, and male sterility, and is expected to provide a safe and efficient new way for rice germplasm resources innovation.This study applied a proteomic approach to identify the regulating proteins of a CMS and mutant maintainer rice line and it is concluded that pollen development in different genetic material is associated with the differential expression of several proteins.These results collectively suggested that the knowledge of these parameters in rice breeding may be further applied as criteria to develop rice CMS lines.The new germplasm with important application value was obtained which laid an important material basis for further breeding program to facilitate the rice breeding to improve yield and quality.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4395/8/12/290/s1, Figure S1: Schematic representation of secondary structures of both sgRNAs used in this experiment.(a) structure both sgRNA's for Wx targets and (b) structure of both sgRNA's for TGW6 targets.The stem loop sgRNA secondary structure was predicted by online tool (http://crispr.hzau.edu.cn/cgi-bin/CRISPR/CRISPR); Figure S2: Isolation of the binary plasmids and sgRNA intermediate plasmids; Figure S3: Sequences of the sgRNA vectors and those of the expression cassettes; Figure S4: sgRNA expression cassette procedure by overlapping PCR containing a target sequence.The chimeric primers with target sequence strands are given in additional file 3. The first PCR is carried out in two separated reactions with U-F/U#T#-and gRT#+/gR-R primer pair, U# indicates a given promoter, and T#+ and T#-indicate forward and reverse strands of a target sequence; Figure S5: Illustration for transformation of E. coli; Figure S6: Schematic diagram of the procedure for CRISPR/Cas9 based generation of mutant plants and analysis of target regions.The targets were selected using CRISPR-GE online web-based tool and expression cassette was constructed by using overlapping PCR and inserted into a binary vector.Agrobacterium mediated transformation was performed and T 0 plants were regenerated and sequencing was performed, and later generations were produced by self-pollination and genotyping was performed by using target specific primers in T 1 and T 2 generations.The phenotypic data of mutant and wild type plants were recorded and further analyzed.Pollen fertility analysis and protein identification was also performed; Figure S7: Sequence alignment of the (a) Wx and (b) TGW6 gene in reference genome and 209B maintainer line.The SNPs between reference genome and 209B are indicated in red box; Table S1: Efficiency score and positions of four targets; Table S2: List of primers used in the study; Table S3: Detection of mutations on the putative off-target sites; Table S4: Pollen fertility status of F 1 lines.

Figure 1 .
Figure 1.Maps of pYLCRISPR/Cas9-MT(I) and pYL-U3/U6a-b-gRNA ve ctors.(a) The binary vector with Cas9p drive n by maize ubiquitin promoter (PUbi).The ke y sequences and re striction sites for Golde n Gate ligation are shown.The expression of the sgRNA scaffold is drive n by the rice U6a/U3 small nucle ar RNA promote rs; the expression of hygromycin (HPT) is drive n by 2 CaMV35S promote rs.NLS: nucle ar localization signal; Tnos: ge ne terminator; LB and RB: le ft borde r and right borde r, re spectively.(b) The physical map of the sgRNA inte rme diate plasmids.U3/U6 promoters from rice use d for pre paration of multiple sgRNA expression cassettes in single binary constructs.(c) BsaI site s (1, 2) in the sgRNA plasmids and the ir sequence information.The se BsaI-cutting (small arrows) site s of the plasmids make s compatible site s for ligation by ge ne rating distinct non-

Figure 1 .
Figure 1.Maps of pYLCRISPR/Cas9-MT(I) and pYL-U3/U6a-b-gRNA vectors.(a) The binary vector with Cas9p driven by maize ubiquitin promoter (P Ubi ).The key sequences and restriction sites for Golden Gate ligation are shown.The expression of the sgRNA scaffold is driven by the rice U6a/U3 small nuclear RNA promoters; the expression of hygromycin (HPT) is driven by 2 CaMV35S promoters.NLS: nuclear localization signal; Tnos: gene terminator; LB and RB: left border and right border, respectively.(b) The physical map of the sgRNA intermediate plasmids.U3/U6 promoters from rice used for preparation of multiple sgRNA expression cassettes in single binary constructs.(c) BsaI sites (1, 2) in the sgRNA plasmids and their sequence information.These BsaI-cutting (small arrows) sites of the plasmids makes compatible sites for ligation by generating distinct non-palindromic ends to the U3/U6 promoters and a common end to the sgRNA sequence.Modified from Ma et al. (2015a) [57].

Figure 2 .
Figure 2. Schematic diagram of gene structures and target sites of the gRNA in the genes locus.(a) Position of two targets in Wx gene and (b) both targets in TGW6 gene locus.

Figure 4 .
Figure 4. Nucle otide sequences at the target site in the 5 T0 mutant rice plants.(a) Mutations induced at Wx and (b) TGW6 targe t site s.The recovered mutated alle les are shown be low the wild-type (WT) sequence.The target sites nucleotides are shown in black capital le tte rs and black dashes.The red capital le tte rs indicate inserted nucle otide s and the apostrophe followe d to red capital le tters indicates inserted nucle otides are not shown in.The Protospacer Adjacent Motif (PAM) site nucle otides are shown in ye llow background le tte rs.The re d dashes indicate the de leted nucle otide s. − and + indicate de le tion and inse rtion of the indicated number of nucleotides, respectively −/+ indicates simultaneous de le tion and inse rtion of the indicate d numbe r of nucle otide s; GW: grain we ight.

Figure 5 .
Figure 5. Amino acid se quence alignment for WT and five transformants in T0 ge ne ration.(a) Amino acid sequences showing the alignme nt about Wx ge ne, and (b) TGW6 mutant plants and WT.The deleted amino acids are shown by black hyphe ns, the translation was te rminated e arlie r in mutants 4 and 7. Highly conse rved and partially conserved amino-acid se quences are indicated with aste risks (*) and dash (-) signs, re spectively.

Figure 4 .
Figure 4.Nucleotide sequences at the target site in the 5 T 0 mutant rice plants.(a) Mutations induced at Wx and (b) TGW6 target sites.The recovered mutated alleles are shown below the wild-type (WT) sequence.The target sites nucleotides are shown in black capital letters and black dashes.The red capital letters indicate inserted nucleotides and the apostrophe followed to red capital letters indicates inserted nucleotides are not shown in.The Protospacer Adjacent Motif (PAM) site nucleotides are shown in yellow background letters.The red dashes indicate the deleted nucleotides.− and + indicate deletion and insertion of the indicated number of nucleotides, respectively −/+ indicates simultaneous deletion and insertion of the indicated number of nucleotides; GW: grain weight.

Figure 4 .
Figure 4. Nucle otide sequences at the target site in the 5 T0 mutant rice plants.(a) Mutations induced at Wx and (b) TGW6 targe t site s.The recovered mutated alle les are shown be low the wild-type (WT) sequence.The target sites nucleotides are shown in black capital le tte rs and black dashes.The red capital le tte rs indicate inserted nucle otide s and the apostrophe followe d to red capital le tters indicates inserted nucle otides are not shown in.The Protospacer Adjacent Motif (PAM) site nucle otides are shown in ye llow background le tte rs.The re d dashes indicate the de leted nucle otide s. − and + indicate de le tion and inse rtion of the indicated number of nucleotides, respectively −/+ indicates simultaneous de le tion and inse rtion of the indicate d numbe r of nucle otide s; GW: grain we ight.

Figure 5 .
Figure 5. Amino acid se quence alignment for WT and five transformants in T0 ge ne ration.(a) Amino acid sequences showing the alignme nt about Wx ge ne, and (b) TGW6 mutant plants and WT.The deleted amino acids are shown by black hyphe ns, the translation was te rminated e arlie r in mutants 4 and 7. Highly conse rved and partially conserved amino-acid se quences are indicated with aste risks (*) and dash (-) signs, re spectively.

Figure 5 .
Figure 5. Amino acid sequence alignment for WT and five transformants in T 0 generation.(a) Amino acid sequences showing the alignment about Wx gene, and (b) TGW6 mutant plants and WT.The deleted amino acids are shown by black hyphens, the translation was terminated earlier in mutants 4 and 7. Highly conserved and partially conserved amino-acid sequences are indicated with asterisks (*) and dash (-) signs, respectively.

Figure 6 .
Figure 6.Re lative e xpression analysis of target genes (a) Wx and (b) TGW6 in wild type (WT) and mutant plants.Transcripts le vel was determined by Q-RT-PCR with cDNA generated from leaves of four-week-old plants.The e xpre ssion value s of the individual genes we re normalize d by using expression le vel of rice Actin ge ne as an inte rnal standard.The data re presents the mean values of thre e inde pe ndent samples [Me an ± SD (standard de viation)].** indicate s significant diffe rence at P < 0.01 (t te st).

Figure 6 .
Figure 6.Relative expression analysis of target genes (a) Wx and (b) TGW6 in wild type (WT) and mutant plants.Transcripts level was determined by Q-RT-PCR with cDNA generated from leaves of four-week-old plants.The expression values of the individual genes were normalized by using expression level of rice Actin gene as an internal standard.The data represents the mean values of three independent samples [Mean ± SD (standard deviation)].** indicates significant difference at P < 0.01 (t test).

Figure 7 .
Figure 7. PCR-base d ide ntification of T-DNA-fre e rice mutant plants and seed analysis of WT and mutant line (T2-4-2).PCR products amplifie d from the progenies of (a) T1 and (b) T2 mutant lines ge nomic DNA using the Cas9 specific primers Cas9-F and Cas9-R.WT: wild-type , M: DNA mole cular marke r, +: positive control (c) Grain phe notype of WT and mutant line s.Cross-section analysis of endosperm in WT (d,e) and mutant line (f,g).

Figure 7 .
Figure 7. PCR-based identification of T-DNA-free rice mutant plants and seed analysis of WT and mutant line (T2-4-2).PCR products amplified from the progenies of (a) T 1 and (b) T 2 mutant lines genomic DNA using the Cas9 specific primers Cas9-F and Cas9-R.WT: wild-type, M: DNA molecular marker, +: positive control (c) Grain phenotype of WT and mutant lines.Cross-section analysis of endosperm in WT (d,e) and mutant line (f,g).

Figure 8 .
Figure 8. Sequence alignment for transmission of mutations at (a) Wx and (b) TGW6 targe t site s in T1 and subsequent T2 ge nerations.The targeted sequence is shown in capital black le tte rs and the PAM sequence in ye llow background.Insertion is re presented by re d le tters, and de le tion by re d hyphens.

Figure 8 .
Figure 8. Sequence alignment for transmission of mutations at (a) Wx and (b) TGW6 target sites in T 1 and subsequent T 2 generations.The targeted sequence is shown in capital black letters and the PAM sequence in yellow background.Insertion is represented by red letters, and deletion by red hyphens.

Table 1 .
Mutation rate of T 0

generation Mutation Type Gene Target Site Bi-Allelic Homozygous Heterozygous Chimeric WT Total
WT: wild-type.

Table 2 .
Amylose conte nt, GC, and GT of WT and mutant plants.

Table 2 .
Amylose content, GC, and GT of WT and mutant plants.

Table 3 .
1000 grain weight (g) of mutant lines and WT in T 0 , T 1 , and T 2 generations.The data listed in the table are mean ± standard error.* indicate significant difference (P < 0.01).GW: grain weight.

Table 4 .
Main agronomic characters in T 2 generation.
PH: plant height; NOP: number of panicles; FLL: flag leaf length; FLW: flag leaf width; PL: panicle length; GPS: grains per spike; SSR: seed setting rate; The data listed in the table are mean ± standard error.ns indicate non-significant difference.

Table 5 .
Classification of mutant lines based on pollen fertility status.

Table 6 .
Rice pollen protein identified by peptide mass fingerprinting.