The Creation And Genetic Background Analysis Of Ogura CMS Broccoli Fertility-Restored Materials

Ogura CMS male sterility is the most widely used type of sterile line in the seed production of broccoli,but its application also severely restricts the reuse of excellent materials.Breaking through the limitation of Ogura CMS technology and obtaining excellent fertility-restored materials are of great significance to improve the breeding level and resource collection of broccoli in China.In this study,Chinese kale 14Y12,an intermediate material containing fertility-restored gene Rfo,was used as parent,and the Rfo gene was introduced into six varieties of Ogura CMS broccoli through backcrossing.Meanwhile,microspore culture and genetic transformation techniques were used to obtain fertility-restored DH lines and Rfo-positive individual.The main findings are as follows:1.Rfo-positive individual were obtained by backcrossing and its genetic background was analyzed.Fertility-restored lines 18B592 and 19B711 were obtained by crossing the intermediate material of Chinese kale 14Y12 with broccoli 17B1253 and 18QB693.F₁generation was obtained by crossing18B592-1 with 18-NH（excellent cold resistance）,18-YX（Yanxiu）,18BS-1,18BS36,18T2B2 and18B1023.19B711-1 was interbred with 19Q Yacui 91,19Q Xiyingmen and 19Q King 1 to obtain F_1-2generation.BC₁generation was obtained by backcrossing 18B592-1 with F₁generation Rfo-positive individuals.Cytological observation showed that there were significant differences in pollen viability of F₁generation Rfo-positive individuals,and the variation range was from 50%to 70%.There were abnormal phenomena of unequal division and the chromosome loss in anaphase of meiosis.Ploidy identification showed that 11 of the F₁generation Rfo-positive individuals were diploid,accounting for78.57%of the total,and 3 individuals were heteroploidy between triploid and tetraploid,accounting for21.43%of the total.The genetic background analysis showed that at the genetic similarity coefficient of0.50,the intermediate material of Chinese kale 14Y12,broccoli 17B1253,and F₁generation breeding individuals could be divided into two categories,and the genetic similarity coefficient between F₁generation breeding individuals and the intermediate material of Chinese kale 14Y12 was 0.24 to 0.88.The genetic similarity coefficient between 19B809-8 and broccoli was 0.76,which can be used as the parent material for the fertility-restored of Ogura CMS broccoli.The Rfo-positive individual of BC₁generation had normal floral organs,high pollen vigor（greater than 70%）,and the transmission rate of Rfo gene was 14.29%～55.56%.Comprehensive agronomic traits,cytology and genetic background,with a genetic similarity coefficient of 0.60,the 48 Rfo-positive individuals of the offspring of Rfo backcrossing could be divided into five categories.The genetic similarity coefficient between 20B826-2and 18B592-1 was 0.66,and the genetic similarity coefficient with broccoli B6 was 0.52.The individual type is similar to broccoli,which provides direct recovery material for the offspring of Ogura CMS broccoli.2.The microspore culture method created fertility-restored materials and analyzed of their genetic background.The broccoli 18QB693,the fertility-restored line 19B711 and the F₁generation Rfo-positive individuals were cultured for microspores.The microspore Rfo-positive individuals showed significant genetic separation in leaf type,flower bud size and style traits,with diploid and tetraploid traits.The important fertility-restored materials of B693-2,B693-3 and B711-1-5 were obtained,and the microspore Ogura CMS fertility-restored technology system was constructed.The genetic background analysis showed that the genetic similarity coefficient of microspore B693-2,B693-3 and broccoli20B724-1 was 0.73,which was closest to broccoli genetic background.The genetic similarity coefficient between microspore individual B711-1-5-1 and Chinese kale was 0.63,which was close to the genetic background of Chinese kale.Therefore,microspore individual B711-1-5-1 could be used for the innovation and utilization of excellent Ogura CMS broccoli resources..3.Rfo-positive individual was obtained by transgenic technology approaches.Individual 18-NH and 18-YX of Ogura CMS broccoli were used to transformation.The Rfo gene of radish was inserted into PBWA（V）BII vector backbone to construct the p BWA（V）BII-Rfo expression vector.The recombinant plasmid was introduced into Agrobacterium tumefaciens strain EHA105 by freezing-thaw method.The transgenic individuals were obtained by Agrobacterium-mediated method.After identification,46 Rfo-positive individuals were obtained.After planting,33 individuals currently showed no pollen and were false-positive individuals.The remaining transgenic-positive individuals need further observation and identification.