Overcoming Self-Incompatibility And Identification Of The S Haplotypes In Radish

Radish(Raphanus sativus L.)is an important root vegetable originated from China.It is a typical cross-pollinated crop with signifcant heterosis.The use of self-incompatibility(SI)is one of the most important strategies to utilize the heterosis of radish,which has a lot of advantages.In practical production,it is also restricted by the difficulties of self-seeding to preserve and propagate self-incompatible lines of radish.At present,several methods could be used to overcome SI of radish to a certain extent,but due to the great differences among genotypes,those methods have not been widely used.Therefore,how to overcome SI and realize efficient reproduction of SI lines becomes a key issue urgently to be solved in the practice of hybrid seed production of radish.In addition,it may occur to the phenomenon of low seed-setting rate at cross between two elite SI lines,which mainly due to the same S haplotype.Therefore,accurately and rapidly identifying S haplotypes can avoid the blindness of the preparation of hybrid combination,which is very critical for the utilization of radish heterosis.Therefore,taking three radish SI lines as materials,the flowering stigmas were sprayed by different chemical reagents,and their fruiting numbers were counted respectively to screen the best method of overcoming SI.It is the aim to simplify the process of propagation of SI lines in radish.At the same time,S locus genes of 52 high-generation SI lines of radish were cloned and sequenced to identify their S haplotypes,combined with field pollination tests and PCR-RFLP analysis for typing.This result provided references for efficient utilization of SI and identification of S haplotypes.The main results are as follows.1.Taking three high-generation SI lines as test materials,the folowering stigmas were sprayed by different concentrations of GA,KT,6-BA,NaCl and boric acid.It was found that compared with deionized water treatment(CK)and flowering pollination(FP),both the compatibility index and fruiting rate of each treatment generally increased,indicating that the five chemical reagents can overcome SI of radish.Different reagents have different effects on overcoming SI of the three materials,especially NaCl treatment.As the NaCl concentration increased from 2%to 4%,seed setting of ’NAU-XZH’ gradually decreased,but all were higher than bud pollination(BP);compatibility index and fruiting rate of’NAU-YH’ also showed a downward trend,and the effect was lower than BP at 4%;while for ’NAU-DF3’,the effect was not obvious after NaCl treatment,which was similar to CK and FP.In addition,It was found that for composite treatment of NaCl plus boric acid,the effect of each material was not as good as NaCl alone.The comprehensive analysis of fruiting situation of the three materials showed that 100 mg/L GA,100 mg/L KT and 100 mg/L,150 mg/L 6-BA treatment can improve compatibility at the flowering stage.2.Totally 50 radish S haplotypes that are currently publicly reported were sorted and uniformly numbered from "NAU-S1" to "NAU-S50".The nucleotide sequences of the kinase domain of SRK and SLG genes of class Ⅰ and class Ⅱ were isolated from 52 radish genotypes,respectively.Based on the sequence homology,the S haplotypes of 52 genotypes were preliminaryly analyzed,including 44 Class Ⅰ homozygous S haplotypes,2 Class Ⅰ heterozygous S haplotypes,2 Class Ⅱ homozygous S haplotypes,and 4 ClassⅠ/Class Ⅱ heterozygous haplotypes.A total of 26 S haplotypes were identified,including 22 Class Ⅰ S haplotypes and 4 Class Ⅱ S haplotypes.Among them,the frequency of NAU-S16 haplotype of class Ⅰ were the highest,the frequency is 19.64%.It was found that SRK gene of NAU-S15 haplotype and SLG gene sequence of NAU-S27 haplotype of class Ⅰ were recombined,and NAU-S8 haplotype and NAU-S39 of class Ⅱ were the same S haplotype.The S locus genes sequence nucleotide of NAU-S19,S21,S29,S44 haplotypes were successfully isolated.In addition,six new S haplotypes were identified,including five classⅠ and one class Ⅱ haplotypes.4.A field pollination test was carried out on different combinations of part materials,and the accuracy of this method was verified.And a set of standard test system of 24 radish S haplotypes was determined.In summary,50 radish S haplotypes had been reported,NAU-S8 and S39 are the same S haplotype,and the 6 newly identified S haplotypes in this study,so there are 55 S haplotypes in radish.Furthermore,using two restriction enzymes(Hinf Ⅰ and Taq Ⅰ),PCR amplification products of SRK gene of class Ⅰ of 21 S haplotypes were analylzed with PCR-RFLP,and all haplotypes could be distinguished with Hinf Ⅰ.These results would provide a basis for the efficient identification of S haplotypes and ultilization of self-incompatibility in radish.