*2.3. Male Sterility and Fertility Restoration*

Broccoli displays obvious heterosis and most commercial broccoli varieties are F1 hybrids. The production of broccoli F1 hybrids depends on self-incompatibility before the early 21st century and now nearly completely depends on male sterility-based breeding systems [11,12,59]. Male sterility comprises cytoplasmic male sterility (CMS) and genic male sterility (GMS) [60]. Among them, Ogura CMS, with the advantages of complete male gamete abortion, maternal inheritance and easy transfer, is now the most widely studied and applied male sterility source in broccoli seed production [60,61]. Ogura CMS is a natural mutation found in radish populations [62], which is caused by a mitochondrial gene named *orf138*, and can be fully restored by the nuclear gene *RFO* (*PPR-B*) [63,64].

Researchers have made efforts to introduce the CMS source to *B. oleracea* by distant hybridization and/or protoplast fusion, but the initially created CMSR1 and CMSR2 contain too much radish cytoplasm, displaying undesirable characteristics, including yellowing at low temperature, deformed flower shape and poor seed setting, which cannot be used in seed production [65–67]. Until the late 1990s, the American Asgrow company applied the method of asymmetric protoplast fusion to reduce the proportion of radish mitochondria, creating CMSR3 with normal fertility and pistil structure; this CMS has been transferred to many elite parent lines, playing a dominant role in the seed production of *B. oleracea* crops [67,68]. During the creation and transfer processes of Ogura CMS, specific *orf138* PCR markers were developed for MAS [69]. Additional mitochondrial markers were developed to distinguish the CMS types; detected by these six *orf138*-related and two simple sequence

repeat markers in 2016, Shu et al. divided 39 CMS broccoli accessions into five groups, and observed that CMSR3 constituted 79.49% of the CMS accessions from China [67].

In addition to the Ogura CMS, GMS resources and GMS-based seed production systems were reported as promising alternatives [60,70–73]. A special dominant genic male sterility (DGMS) resource, 79–399–3, which arose in cabbage populations in China, has been successfully and widely applied in cabbage hybrid seed production [61,72]. The DGMS-based breeding system has been established in *B. oleracea* crops, including cabbage, broccoli and kohlrabi [61,72]. Compared with the Ogura CMS, the DGMS-based breeding system displayed advantages of much higher seed quality and yield [61]. However, its utilization is limited in broccoli, largely because homozygous DGMS plants must be preserved and reproduced by tissue culture, which is not effective for large-scale hybrid seed production [61]. Despite these disadvantages, this DGMS-based system has been preserved as an alternative for broccoli hybrid seed production. In recent decades, dozens of broccoli DGMS lines have been created, and several markers have been developed for MAS for the rapid creation of DGMS lines [61,74]. Shu et al. developed generic SSR markers linked to the male-sterile gene, with the marker scaffold10312a showed the highest accuracy of ≥96.43% [74]. By distinguishing the amplified products polyacrylamide gel, these markers were successfully used for identification of male and sterile plants in broccoli breeding lines DGMs8554, DGMs93219 and DGMs94174; enabled DGMS plants selection in the seedling stage. Han et al. developed a high-throughput kompetitive allele specific PCR (KASP) marker K6 with high accuracy and no genetic background bias applicable to all *B. oleracea* crops, including broccoli [61]. This marker was based on allele specific fluorescence on an Applied Biosystems Viia 7 real-time PCR system for high-throughput detection. In the DGMS-based breeding system, this marker was used for identifying homozygous DGMS plants from selfing progenies of heterozygous plants as an alternative to test crossing, which requires at least two years and additional labor in tissue culture [61]. These DGMS-specific markers enable effective selection in breeding programs.

On the other hand, there is increasing demand for the reutilization of CMS resources in *B. oleracea* crops. The Ogura CMS restorer gene *RFO* (*PPR-B*) was introduced from radish to rapeseed and recently to *B. oleracea* crops [75,76]. Liu et al. applied strategies of interspecific hybridization and backcrossing and introduced the *RFO* gene from rapeseed to broccoli. The foreground Rfo-specific markers BnRFO-AS2F/BnRFO-AS2F and BnRFO-AS2F/BnRFO-NEW-R, were used for detecting *Rfo*-positive interspecific hybrids; and 28 background SSR markers were used for detecting true intergeneric hybrids and assessing the genetic backgrounds of *Rfo*-positive interspecific hybrids. By evaluating polymorphism loci of the 28 background markers, the BC2 *Rfo*-positive individuals were found closer to the broccoli's genetic background [76].
