**3. Botany of radish**

Radish (*R. sativus* L.) is an entomophilous flower classified as an allogamous plant [35]. Regular flowering appears from three florets on the tip of each branch of the panicle, and each flower is effective in producing a pod up to 1–3 inches long and consists of 1–6 seeds. The radish flowers open in the morning with fresh corolla and remain until the next day [36]. Kremer also reported that pollen receptivity of the flower limits up to few hours a day. Its flowers are 1.5–2 cm in width, whitish to pinkish and purplish colour with purple veins and have four erected sepals and clawed petals, six stamens and 3–4 cm long style [37, 38]. Siliqua or seedpod, a type of seed capsule of radish, is 1.5 cm wide and 3–7 cm long, consisting of 6–12 seeds/pod with a long conical seedless beak [13]. The inflorescence of radish is a typical elongated, erected, an oblong raceme of Cruciferae. The main objective of the investigation was the cross-pollination of radish by [39] found that the 'Icicle' and 'Scarlet Globe' cvs were self-incompatible pollinated with the help of honeybees [40]. The studies indicated that the seed yield is greatly influenced by the number of honeybees striking the radish flowers. Radchenko [41] also reported that honeybees were the main pollinators of radish flowers, approximately 77–99% in total, increasing the crop yield by 22% and enhancing the seed quality. Therefore, radish is considered almost entirely insect-pollinated. During fruit maturation, seeds' colour is somewhat yellow and turns reddish-brown with age. The mature radish leaves are alternate, arranged in a rosette pattern and have a lyrate shape set apart pinnately with an enhanced terminal lobe and minor lateral lobes. A longer root form, including oriental radishes, daikon or mooli and winter radishes, grows up to 60 cm (24 in) long with foliage about 60 cm (24 in) high with a spread of 45 cm (18 in) [42].

### **4. S haplotype**

Radish is a self-incompatible crop exhibiting the high heterosis, the production of F1 hybrids based on self-incompatibility is desired to eliminate laborious hand emasculation in radish [43]. The main aim of a plant breeder is to identify breeding lines of S haplotypes. The plant breeder can avoid cross-influencing of the parental lines [44]. The S haplotype of each parental line needs to show the compatible reaction between parental lines. Therefore, for producing F1 hybrid breeding, it is very important to identify the S haplotypes of parental lines [45]. The abundance of S haplotype determines a specific S haplotype by using traditional methods, including test cross method, pollination, isoelectric focusing, immunoblot analysis and the pollen tube fluorescence analysis [46, 47]. The S alleles are highly variable in S haplotype. Moreover, Nikura and Matsuura identified 37 alleles in radish [48].

Several S haplotypes in *Raphinus sativus* were identified based on polymorphism in SLG, SRK and SCR/SP11 sequence and S haplotypes are numbered as S-1, S-2, S-3, etc. [48]. Although radish belongs to a genus different from Brassica, nucleotide sequences of SP11, SRK and SLG alleles of radish and Brassica are intermingled in phylogenetic trees of SP11, SRK and SLG, respectively, indicating that diversification

of these alleles predates speciation of these genera [44]. SP11, SRK and SLG alleles of some S haplotypes in radish are highly similar to those of some S haplotypes in Brassica, and one S haplotype in radish has been revealed to have the same recognition specificity as that of one S haplotype in Brassica rapa [44]. Comparison of nucleotide sequences of SP11 and SRK alleles and recognition specificities between similar S haplotypes of radish and Brassica may provide valuable information for understanding the molecular structures of SP11 and SRK proteins. However, researchers' numbering of S haplotypes in radish varies, and nucleotide sequence information on S haplotypes is thus confusing [43].

Besides, analysis of SLG and SRK is utilised to identify S haplotype in Raphanus and Brassica by using methods of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) [49, 50]. However, the own limitation of PCR-RFLP, first, is challenging to design a universal primer that can amplify SLG and SRK alleles; second, the presence of multiple genes homologous to the SLG or SRK genes in Brassicaceae plants aggravates PCR amplification of specific SLG or SRK alleles [51–53]. Additional advanced radish cultivars (cultivars with improved yield and higher quality) were also produced by the Ogura CMS method to assist in radish hybrid [54]. This variety shows that bulk selection, mixed mass pedigree selection or bud pollination will take 8–12 years to produce a new variety, new varieties must be produced from other genetic means [55].

### **5. Inter-specific hybridization**

Inter-specific and intra-specific hybridization has a vital role in the genomic study and crop improvement by introducing desirable agronomic characteristics and specific traits such as disease, insects and stress resistance from wild species to cultivated ones [56, 57]. The study indicated that the average podding rate of the cross between radish and turnip (67.03%) was much higher than that of the reciprocal cross between a turnip and radish (55.04%) [57], and it was also reported that the average seed-setting rate and hybrid acquisition rate of the radish and turnip based on cross pattern (e.g. 2.25 and 0% respectively), however, seed production of the F1 hybrids and their F2 progeny was up to 0.4 and 2%, respectively, as compared with wild radish [58]. Therefore, the study indicated a low hybridization affinity between radish and Chinese kale, but incompatibility still prevailed [57].

Similarly, the radish-wild mustard inter-specific hybrid was studied. It was found that production was higher with radish pollen competition, i.e. 42 and three interspecific hybrid seeds per 1000 seeds were observed [59]. Another study indicated that the modified flower culture method is the best method for hybridization between radish and (transgenic) oilseed rape (*Raphanobrassica* hybrids) without labourintensive production in vitro ovule or embryo rescue techniques. This is a potential approach for breeding programmes by introducing useful radish genes, e.g. nematode resistance genes, into oilseed rape [60]. Moreover, clubroot is a common disease of cabbages, cauliflower, radishes, turnips and other plants of the family Brassicaceae caused by *Plasmodiophora brassicae* [61]. Radish is a close relative of the brassica family, and it was found that a synthesised allotetraploid *Brassicoraphanus* (RRCC, 2n = 36) between *R*. *sativus* cv. HQ-04 (2n = 18, RR) and *Brassica oleracea* var. *alboglabra* (L.H Bailey) (2n = 18, CC) proved resistant to multiple clubroot disease pathogen *P*. *brassicae* causing club root disease [62]. However, the spontaneous hybridization event between *Brassica napus* (oilseed rape) and *Raphanus raphanistrum* (wild radish) was screened. It was found that hybrids with wild radish as the seed parent contribute to herbicide resistance belonging to rape. Another study indicated that wild radish in an oilseed rape field produced as many as three interspecific hybrids per 100 plants and was the first ever such report of such a spontaneous event [58].
