Genetic characterization of a new radish introgression line carrying the restorer gene for Ogura CMS in Brassica napus

Creating a homologous restorer line for Ogura cytoplasmic male sterility (Ogu-CMS) in Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed production. Previously, an independent development of a new Ogu-CMS restorer line (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n = 56) and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was characterized in this study. The results showed that the fertility restoration gene in CLR6430 presented a distorted segregation in different segregating populations. However, the majority of somatic cells from roots had a regular chromosome number (2n = 38) and no radish signal covered a whole chromosome was detected using GISH. Thirty-two specific markers derived from the introgressed radish fragments were developed based on the re-sequencing results. Unique radish insertions and differences between CLR6430 and R2000 were also identified through both radish-derived markers and PCR product sequences. Further investigations on the genetic behaviors, interactions between the fertility restoration and other traits and specific molecular markers to the introgression in CLR6430 were also conducted in this study. These results should provide the evidence of nucleotide differences between CLR6430 and R2000, and the specific markers will be helpful for breeding new Ogura restore lines in future.

Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed 21 production. Previously, an independent development of a new Ogu-CMS restorer line 22 (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n=56) 23 and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was 24 characterized in this study. The results showed that the fertility restoration gene in CLR6430 25 presented a distorted segregation in segregating population. However, the majority of somatic 26 cells from roots had a regular chromosome number (2n=38) and no radish signal covered a 27 whole chromosome was detected using GISH. Thirty-two specific markers derived from the 28 introgresssed radish fragments were developed based on the re-sequencing results. Unique 29 radish insertion and differences between CLR6430 and R2000 were also identified through 30 both radish-derived markers and PCR product sequences. Further investigations on the 31 genetic behaviors, interactions between the fertility restoration and other traits and specific 32 molecular markers to the introgression in CLR6430 were also conducted in this study. These 33 results should provide the evidence of nucleotide differences between CLR6430 and R2000, 34 and the specific markers will be helpful for breeding new Ogura restore lines in future. 35

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Cytoplasmic male sterility (CMS) is a widespread inherited trait in plants, which is controlled 40 by an incompatibility between the nucleus and mitochondrial genes and has been widely used 41 for F1 seed production. Some CMS systems have been identified in B. napus, such as Polima 42 CMS (pol CMS) (1), Ogura CMS (ogu CMS) (2), the common B. napus CMS (nap CMS) (3), 43 and a novel type introgressed from Chinese woad (inap CMS) (4). Ogu CMS was first 44 discovered in Japanese radish (Raphanus sativus) from an unknown cultivar (Ogura 1968), 45 and the sterility of ogu CMS was stable in various climate conditions (5), which has been 46 utilized more and more widely. 47 The application of Ogu CMS has been difficult as the restorer gene (Rf) only existed in some 48 Europe and Japanese wild radish, but not in B. napus nor any B. rapa varieties. Therefore, the 49 Rf gene had to be introduced from radish into oilseed rape through intergeneric hybridization. 50 Homologous recombination between R and A and C genome were achieved through 51 polyploidy cross (6), gamma irradiation of donor pollen (7), or asymmetric protoplast fusion 52 (8). However, along with the introduction of the Rfo gene into B. napus, extra segments of 53 radish genome was introgressed into B. napus which brought undesirable traits for breeders, 54 such as elevated glucosinolate level and poor seed set (the number of seed per silique) (9). 55 The early version of Brassica Ogu restorer lines developed by INRA identified a close 56 linkage between glucosinolate content and the Rfo gene (10), suggesting more difficulties in 57 breeding double low varieties. Therefore, following research was dedicated for the 58 improvement of the restorer line. Delourme (11) reported a low glucosinolate (9-18 µmol/g) 59 Ogu-CMS restorer line in B. napus assisted by RAPD markers, however the Rfo loci was 60 heterozygous and not heritable to siblings. Successful break-through of low glucosinolate 61 Ogu-CMS restorer line was obtained through gamma radiation when crossing with B. napus 62 of low glucosinolate content, and named as R2000 (7, 11). Other independent breeding of B. 63 napus Ogu restorer line using radiation or other methods were reported by Pioneer Hi-bred 64 Company and Syngenta Biotechnology Company (12-14), with shortened radish fragment in 65 new generations. 66 The Rfo gene, orf687, encoding a 687-amino-acid protein with multiple pentatricopeptide 67 repeats, alters the expression of the sterile gene orf138 at post-transcriptional level (15, 16). 68 The gene has been localized physically and mapped in the distal region on linkage group N19 69 in Pioneer-derived Ogu-CMS restorer line, which corresponds to the C genome in B. napus 70 (17). Moreover, apart from the identified Rfo gene (15), other alleles were identified by Wang,71 Zhang (18) and Wang,Zhang (19), and was 1.6 cM distant from Rfo locus in Ogu-CMS 72 radish. Further investigations of these alleles revealed a heterozygous type (RsRf3-1/RsRf3-2) 73 encoding PPR proteins were responsible for the fertility of male-sterile radish, and had higher 74 expression and RNA polymerase II occupancy compared with their homozygous alleles 75 (RsRf3-1/RsRf3-1 or RsRf3-2/RsRf3-2) (20). However, those new identified alleles were 76 studied only in radish. Therefore, associations between these alleles and the Rfo gene and 77 their independent functions when transferring into B. napus remain unclear. 78 The introgression of the Rfo gene from radish into rapeseed also introduced chromosomes or Previously we reported a newly-developed Ogu-CMS restorer line (CLR650) from crossing 92 between Raphanobrassica (AACCRR, 2n=56) and B. napus using grafting to overcome the 93 incompatible obstacle between these two parents (24). Further analysis of the updated version 94 of material (CLR6430) developed using the restorer CLR650 through backcross with 95 double-low line and self-pollination was conducted in this study to investigate the 96 chromosomal behaviors and fertility ability. Resequencing was also applied to CLR6430 to 97 explore the detailed information during the introgression from radish fragment into B. napus, 98 enabling more precise designs for marker-assisted breeding for the new Ogu-CMS restorer 99 lines application. 100 101

Plant material 103
Using Raphanobrassica (AACCRR, 2n=56) as the donor of Ogu CMS restorer resource, we 104 successfully obtained the hybrids of Brassica with Raphanobrassica through grafting to 105 overcome the incompatible obstacle between these two parents. After 15 years with high 106 intensive selection, a stable homozygous restorer line CLR650 of Ogu CMS in B. napus was 107 bred which can restore the male fertility of progenies from self-pollination and testcross (24). The photographs of well-separated chromosome slides were taken under the fluorescence 138 microscopy following the methods describing by Ge and Li (25). 139

Collection of re-sequencing sample, DNA extraction and sequencing 140
Seeds from CLR6430 line (46608, double confirmed with restorer gene) were sown in pots 141 and grow to seedling stages (with 3-4 leaves) before fresh leaf samples were taken for whole 142 genome sequencing (Shanghai OE Biotech. Ltd, China). 143 The DNA was extracted using DNeasy plant mini kit (Qiagen, Hilden, Germany). The quality 144 and concentration of DNA were tested before digested into fragments. 145 The library was prepared using TruSeq DNA LT Sample Prep kit (Illumina, SanDiego, CA, 146 USA) with pair-end method. 147

Analysis of markers in the radish introgression 181
The specific markers were used to compare CLR6430 with the known rapeseed restorer 182 R2000 (7). Parts of PCR amplification products were sequenced by Sanger sequencing, and 183 the consensus sequences amplified from the CLR6430 and R2000 were aligned using 184 DNAman ver3.0 (Lynnon BioSoft, Quebec, Canada). 185 187 Ogu CMS A-lines 20QA and SC3QA were crossed with the restorer line CLR6430 (20QA× 188 CLR6430 and SC3QA×CLR6430) and then selfed for F2 and F3. Fertility and sterility ratio of 189 tested plants in both F2 and F3 population were closely to 1:2 (Table 1) 207 Chromosome numbers were determined in more than 30 plants of CLR6430. Majority of 208 somatic cells (>82%) in roots had 2n=38, ranging around 38-40 (Figure 1). The radish 209 introgression was detected using GISH with radish genomic probe and B. napus genome as 210 block (Figure 2 a and 228 In total, we obtained 134.338 Gb of pair-end sequencing data with a high-quality base ratio of 229 98.2%, and 71.6% of the reads aligned to the reference with an average coverage of more than 230 50X (Table 3). While de novo assembling using SOAPdenovo2, all reads were assembled into 231 568,120 scaffolds and 641,453 contigs (sequence reads >500 bp were summarized). 232 Specific molecular marker development 236 The re-sequencing mapping and alignment results revealed homologous reads derived from 237 R9 in radish assembled into R9 of "reference genome" and the average coverage of the region 238 between 66.13 (Rsa1.0_00994.1) and 138.53cM (Rsa1.0_00176.1) was over 115, much 239 higher than the other regions in R9 group, indicating a high possibility of introgressed 240 fragment from radish into CLR6430. The sequences of this region were then used for 241 developing specific molecular markers in segregating population (Figure 3). Considering the 242 conservation and variability of SSR markers between the two species, sequences from 243 introgressed fragment were used to develop SSR markers for marker-assisted breeding. 244

Re-sequencing of CLR6430 and its genomic assembly
Thirty-two specific SSR markers were developed and verified with corresponding fertility 245 condition in field. Detailed information of specific SSR markers (Table 4) and sequence of 246 amplified PCR products from Table 4 were presented in Additional Table 1.

Comparison of the introgression between CLR6430 and R2000 with markers 254
The above-mentioned 32 markers were used to characterize the CLR6430 in comparison 255 with R2000. The minimum size of the introgressed fragment in CLR6430 was estimated at 256 72.14 CM (between Rsa1.0_00994.1 and Rsa1.0_00071.1). Comparison to CLR6430, R2000 257 had five continuously lost markers (CLR9-20, CLR9-21, CLR9-26, CLR9-27, CLR9-30,) 258 (Table 5), estimating the introgressed region about 48.48 CM (between Rsa1.0_00994.1 and 259 Rsa1.0_00045.1). And the size of fragments amplified by primers pairs CLR9-1, CLR9-6, 260 CLR9-7, CLR9-10 and CLR9-31from CLR6430 and R2000 were different. Besides, the 261 amplified PCR sequences by the new developed SSR markers in this study revealed 262 significant differences between CLR6430 and R2000, presenting various sequence insertion, 263 deletion and replacements (Figure 4). 264 Table 5. Specific marker comparison for CLR6430, R2000 and radish in this study. "+" and 265 "-" were used to describe the results as positive or negative. "Baiyuchun" was the name of a 266 radish cultivar. 267 The radish-derived male sterility has the advantage of complete and stable sterility in B. radish genome has also been available (31-33) and useful for the development of specific 294 markers for breeding purposes. In our study, CLR6430 was re-sequenced and aligned against 295 a combined reference genome, which was the whole B. napus genome plus an additional 296 chromosome from radish that contained the Rfo gene. The high coverage sequences 297 assembled into R9 in the reference genome indicating the most likely homologous regions 298 from radish introgressd into B. napus, which is the region between 66.13 (Rsa1.0_00994.1) 299 and 138.53cM (Rsa1.0_00176.1) Therefore, there is about 72.4 cM of radish genome 300 integrated into CLR6430. And the following design of SSR markers for CLR6430 was also 301 based on the re-sequencing and alignment results, providing solid and more precise 302 information for later improvement and utilization of CLR6430. 303

Radish genome
Secondly, creation of an ideal restorer line for Ogu-CMS B. napus remains difficult. 304 BAC-FISH results (Figure 2) has demonstrated restorer gene from Radish located in A 305 genome rather than N19 from C genome as previously reported (7, 21, 23), therefore, the 306 introgression of radish genome into Brassica napus is different. After obtaining homozygous 307 (RfRf) instead of heterozygous (Rfrf) in the loci of the restorer gene, we still have to break the 308 linkages with some deleterious traits such as high glucosinolate content and 309 self-incompatibility. R2000, the widely accepted Ogu-CMS restorer line developed by INRA,310 has been utilized for three-line system production of hybrid rapeseed. 311 Meanwhile, the comparison between CLR6430 and R2000 in this study has shown significant 312 differences, revealing the different origin. CLR6430 has improved performances in terms of 313 glucosinolate content and selfing ability compared to its earlier version CLR650, while the 314 introgressed fragment from radish remain long and require further improvement. In this case, 315 developed markers for R2000 or other reported Ogu-CMS restorer were not applicable for 316 CLR6430, hence self-designed markers based on resequencing results are meaningful for 317 further selection and improvement for CLR6430. 318 Besides, segregation distortion was found in CLR6430 in selfing and backcrossing 319 populations. Distorted segregation is the deviation of the observed genotypic ratios from the 320 expected frequencies based on Mendelian's laws of inheritance, which is considered as an 321 evolutionary force and associated with genetic factors involved in reproduction and fitness 322 The radish-derived male sterility has the advantage of complete and stable sterility in B. 332 napus and was favored by breeders in commercial production (36). As one of the most 333 important rapeseed market, China has rarely utilized this Ogu-CMS system in hybrid seed 334 production, mainly due to the lack of self-developed restorer line and patent restrictions 335 applied from other restorer sources such as R2000 and SRF developed by INRA and Pioneer 336 Hibred, respectively. Making good use of CLR6430 could be the breakthrough for future 337 application of Ogu-CMS system in this market. 338 In summary, the DNA sequence surrounding the Rfo in CLR6430 is different from that of 339 R2000, and resequencing-based Rfo-related markers presented a more efficient method for the 340 improvement of CLR6430. However, the glucosinolate content remains higher than market 341 level, and marker-assisted improvement still at early stage for seed settings and other 342 agronomic traits. Moreover, the genetic mechanisms underlying the observed segregation 343 distortion for Rfo in the inbreeding and backcross offspring requires further investigation. 344 Further studies are required to locate and reduce the size of the introgression in CLR6430 for 345 better breeding outcomes.