Accelerated molecular breeding of novel cytoplasmic male sterility lines in rice using orfH79 or orf290 haplotypes

The identification and development of new cytoplasmic male sterility (CMS) lines in higher plants is Molecular markers assisted selection (MAS) based on CMS-associated genes or mitochondrial-specific chimeric sequences are important for rapid and effective breeding of new CMS lines and hybrids. In our study, the distribution and allele variation of orfH79 and orf290 genes were characterized from 273 wild and cultivated rice in the AA genome species. Based on the alignment of nucleotide and amino acid sequences, four accessions with orfH79 and three accessions with orf290 were screened. Four novel CMS lines carrying orfH79 haplotypes and three novel CMS lines carrying orf290 haplotypes were then developed using multiple backcross generations with a maintainer line under MAS. The breeding process used in our study provides an efficient and feasible approach for selecting new CMS lines. CMS lines selected in our study are important for enriching rice germplasm resources and guaranteeing rice breeding programs.

from Hubei Academy of Agricultural Sciences (Wuhan, China) and 5 CMS lines or maintainer lines related to HL-CMS. All plant materials were planted in the experimental field of the South-Central University for Nationalities in Wuhan, China, from 2015 to 2017.

Isolation of nuclear and mitochondrial DNA
Total nuclear genomic DNA was isolated from green leaves using the modified CTAB method (Zhang et al., 1992). DNA quality and quantity were estimated spectrophotometrically by visualizing under ultraviolet light, using a specific amount of lambda DNA (MBI, USA) on an agarose gel.
Mitochondrial DNA (mtDNA) was isolated by initially harvesting pure mitochondria using etiolated seedlings by differential centrifugation and DNase I processing (Promega, USA), following the modified method of Yi et al. (2002). Mitochondria was then resuspended using lysis buffer, following phenol-chloroform extraction, and mtDNA was isolated using precipitated ethanol.

DNA sequencing and sequence alignment
DNA polymorphism bands were collected from agarose gels using a AxyPrepTM DNA Gel Extraction Kit 50-prep according to the manufacturer's specifications (Axygen, USA). Purified PCR products were cloned using a TA-cloning® kit pCR®2.1 (TakaRa, Japan) and sequenced by Qingke Corporation (Wuhan, China). DNA and amino acid sequences of ORFH79 and ORF290 from the different candidate accessions were aligned using the ClustalX program.

Fertility scoring of hybrid plants
Hybrid plants were obtained by crossing wild or cultivated rice with the Honglian maintainer YTB as the male or female parent. Fertility evaluation was performed according to pollen stainability in a 1% I 2 -KI solution and the seed-setting rate of spikelets. All fertility results were recorded as mean ± SD.

Results
Distribution of orfH79 and orf290 in AA genome rice orfH79 and orf290 distribution in the AA genome of wild and cultivate rice accessions was determined using PCR amplification with orfH79 sequence-specific primers H1 and H2 and orf290 sequencespecific primers O1 and O2, respectively. Specific bands of both orfH79 and orf290 were recorded in 11 rice accessions, similar to those in YTA and CGA belonging to Honglian CMS lines; one accession IRRI81 was from cultivated rice and 10 accessions were from wild rice. The 10 wild rice accessions belonged to three species: two were from Oryza glumaepatula (W1 and W2), five were from Oryza nivara (W3, W4, W7, W9 and W11), and three were from Oryza rufipogon (W14, W15 and W18). The 11 rice accessions came from Cambodia, China, India, Sri Lanka, Suriname, Brazil, Bangladesh, the Philippines and Laos. Eight accessions deriving from Oryza nivara and Oryza rufipogon (cultivated rice IRRI81 and wild rice W5, W6, W8, W10, W12, W16 and W17) contained only specific bands of orfH79 (or orf79). Only one accession in wild rice (W18) only contained a specific band of orf290 (Fig. 1). 13 accessions of cultivated rice (Z593, Z595, Z597, Z604, Z612, Z613, Z615, Z616, Z620, Z624, Z740, Z743 and Z747) contained a specific band of orf290 without a specific band of orfH79 (Fig. 2). All plant materials containing either orfH79 or orf290 in our study are shown in Table 1.

Alignment of DNA and amino acid sequences
Due to the very high similarity between orfH79 and orf79, it is impossible to distinguish between them using band size of the PCR products. PCR amplification products with primers H1 and H2 from mtDNA of YTA, BYA, IBBI81, W5, W6, W8, W10, W12 and W17 were retrieved, cloned and sequenced. All sequence alignments indicated that the four accessions of W6, W8, W12 and W17 were identical to orfH79 sequences in YTA or CGA in the HL-CMS lines. IRRI81, W5, W10 and W16 accessions also had a high level of similarity, with IRRI81, W5 and W10 being identical to orf79 sequences in SJA or BYA in the Boro-II-CMS lines. W16 differed in only one base in the nucleotide site 147 from A to T compared with the orf79 sequence in SJA or BYA. Amino acid sequences differed at sites 48aa (Leu to Met), 49aa (Asp to Glu) and 60aa (Tyr to His) between ORFH79 in YTA and ORF79 in SJA. Amino acid sequences in W6, W8, W12 and W17 were identical to the ORFH79 sequence in YTA or CGA, but they were not identical among IRRI81, W5, W10, W16 and SJA or BYA (Fig. 3a). These results suggest that YTA, CGA, W6, W8, W12 and W17 shared the same mitotype holding orfH79 and SJA, BYA, IRRI81, W5, W10 and W16 shared the similar mitotype holding orf79.
Of the 14 cultivated rice accessions containing orf290, 11 nucleotide sites were different. Three of the 14 accessions (Z597, Z615 and W18) are identical to nucleotide sequences in YTA. Compared with the YTA nucleotide sequence, up to five nucleotide differences were identified in the other 11 accessions.

Southern blotting
To further assay the specificity mitotype and copy number of orfH79, mitochondrial genomic DNA of four accessions with only specific bands of orfH79 and the control accessions YTA, YTB and BYA were digested with EcoR I and hybridized using a orfH79 probe. Only one band was detected around 300 bp for W6, W8, W12 and W17 as YTA, suggesting that orfH79 is a single copy gene in these mitochondrial genomes. In order to verify the absence of the mitotype with orf290, all of the over accessions were digested with EcoR V and Xho I and hybridized with a orf290 probe. Results for southern blotting indicated that neither the sequence nor homologous sequence of orf290 was detected in the mitochondrial genomes of W6, W8, W12 and W17, and a special band was recorded for orf290 in YTA around 1700 bp (Fig. 4).
The same method was used to evaluate the mitotype and copy number of orf290 and exclude the existence of gene orfH79 in accessions of W18, Z593, Z595, Z597, Z604, Z612, Z613, Z615, Z616, Z620, Z624, Z740, Z743 and Z747. Although results from this analysis indicated that homologous sequences of orfH79 were not present, the special band at 1700 bp of orf290 was detected in these mitochondrial genomes (Fig. 5).

orf290
CMS is a maternally inherited trait that, when induced by one or more mitochondrial chimeric genes, contains partial fragments of mitochondrial function genes. Nuclear-cytoplasmic incompatibility, showing a CMS phenotype is the result of the combination of a nuclear genome lacking Rf genes and a mitochondrial genome containing CMS-inducing mitotypes. In order to develop new CMS lines only holding orfH79 or orf290, interspecies crosses were performed using four accessions (W6, W8, W12 and W17) only carrying orfH79 and three accessions (W18, Z597 and Z615) only carrying orf290 in the mitochondrial genome as maternal parents with YTB as the male parent. Fertility analysis of the backcross offspring indicated that the percentage of stainable pollen grains of F1 hybrids were significantly low, and over 50% of abortive pollen grains were spherical. The seed-setting rates of bagged spikelets were also noticeably reduced. Backcrosses were then undertaken repeatedly between the offspring of low-fertility as maternal parents and YTB as recurrent male parents (Fig. 6a).
The fertility of backcross progeny decreased with an increase of backcross generations (Table 3), and fertility of backcross offspring BC7F1 was almost close to complete sterility (Table 3). On the other hand, another interspecies cross was performed using YTB of artificial emasculation as maternal parents with four wild rice accessions (W6, W8, W12 and W17) and three accessions (W18, Z597 and Z615) as male parents. The offspring plants as female parents crossed with four wild rice as recurrent male parents (Fig. 6b). Compared with YTB, the fertility of these combinations was still very high (Table 3). For these results, the following conclusions can be drawn: (i) W6, W8, W12 and W17 shared the same orfH79 mitotype and W18, Z597 and Z615 shared the same orf290 mitotype; (ii) the fertility of offspring between YTB as male parent and YTB as maternal parent was completely different; (iii) inconsistent fertility between reciprocal and positive crosses indicates that low fertility belongs to cytoplasmic inheritance, and low fertility may be caused by the cytoplasmic male sterility gene. Discussion CMS genes are usually chimeric genes derived from the rearrangement between the fragment of mitochondrial functional genes and other unknown sequences (Hanson and Bentolila 2004). To date, several types of CMS-associated genes or sequences have been reported in rice, including orfH79 and orf290 for CMS-HL (Yi et al. 2002;Yang et al., 2018), orf79 for CMS-BT and CMS-LD (Wang et al. 2006;Kazama et al. 2016), orf307 for CMS-CW (Fujii et al. 2010), orf113 for CMS-RT98 (Igarashi et al. 2013), WA352c and WA352a for CMS-WA (Tang et al. 2017), and orf352 for CMS-RT102 (Okazaki et al. 2013).
The same CMS related gene has been recorded to exist in different CMS types, such as orf79 for CMS-BT and CMS-LD, and WA352a (orf352) for CMS-WA and CMS-RT102. Two CMS related genes or sequences have also been isolated from the same CMS line, for example orfH79 and orf290 for CMS-HL and WA352c and WA352a for CMS-WA. All of these genes or specific sequences can be used as molecular markers in the breeding of CMS lines.
The identification of CMS cytoplasm and the development of new CMS lines in higher plants mainly depends on traditional test-cross breeding and molecular markers based on mitochondrial genome sequences. Due to over dependence on breeders' experience and uncertainty of field judgment, it is difficult to create a new sterile line in a short time period using traditional breeding methods. Due to its fast, efficient, cost-effective and accurate characteristics, molecular marker assisted selection (MAS) has been widely used in crop breeding. Based on the report of distinguish different cytoplasmic using molecular markers to backgrounds in radish, rapeseed and rice (Kim et al., 2007;Zhao et al., 2010;Fang et al., 2015), specific sequences or ORFs in mitochondrial genomes can assist in the selection of new strains or varieties of crop. Of the mitotype-specific sequences (MSS) tested, 14 MSS were related to CMS, including nine MSS specific to sporophytic CMS, three specific to gametophytic CMS, and two shared by all types of CMS. Many mitotypes in wild rice can be differentiated and new CMSs can be developed using MSS molecular markers (Xie et al. 2014). Previous studies documented four completely sterile alloplasmic CMS lines developed from wild rice by successive recurrent backcrossing of sterile plants from a BC1F1 population with the HL maintainer YTB, respectively.
These alloplasmic CMS lines, carrying different orf(H)79 haplotypes, displayed various fertilityrestoring models through test-crossing (Li et al., 2008). Using the same method, sterile CMS lines were developed from wild rice accessions carrying L-sp1, a CMS-associated mitochondrial sequence (Tan et al., 2015). Recently, based on the sequence of L-sp1, a novel CMS-related mitochondrial chimeric gene (orf290) which can induce CMS in the binucleate pollen stage of rice was cloned and confirmed from HL-CMS line YTA (Yang et al., 2018). In our study, we developed several new CMS lines via backcross (using the same male parent YTB) from accessions containing either orfH79 or orf290 using molecular marker assistance.
It has been suggested that CMS-associated mitotypes have a parallel evolutionary relationship with Rf-candidate-related nucleotypes within plant species, and that different Rf alleles interact with CMS in a gene-for-gene manner (Taylor et al., 2001;Van Damme et al., 2004;Tan et al., 2011). In our study, two new haplotype CMS lines were bred with either orfH79 or orf290, having shared similar maintainer lines. Fertility of these lines could be restored to different degrees by independently restoring either Rf5 or Rf6 (Unpublished data). Our results indicate that the corresponding relationship between Rf genes and CMS-associated genes is not a simple one-to-one relationship.

Availability of Data and Materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.

Ethics Approval and Consent to Participate
Not applicable.

Consent for Publication
Not applicable.  Figure 1 The distribution of orfH79 and orf290 in wild rice in this study. A: orfH79; B: orf290

Figure 2
The distribution of orfH79 and orf290 in cultivated rice in this study. A: orf290; B: orfH79 Breeding process of CMS lines and identification of pollen fertility