Transferring Clubroot Resistance From Chinese Cabbage (Brassica Rapa L.ssp.pekinensis.) To Abbage (Brassica Oleracea) By Distant Hybridization

Clubroot is a worldwide disease caused by Plasmodiophora brassicae.It has been the main disease of cruciferous vegetables through soil spread.Cabbage?Brassica oleracea?is one of the main vegetable crops in China.In recent years,due to the rapid development of clubroot,production and quality of cabbage has been significantly reduced.Breeding resistant cultivars is one of the most effective controlling measures.However,the lack of clubroot resistance resources in cabbage has led to a slow progress in the breeding of cabbage.There are lots of resistant resources in the Chinese cabbage?Brassica rapa L.ssp.pekinensis?,and the research on resistance gene loci is also developed deeply.Distant hybridization can be used to transfer the resistant genes from Chinese cabbage to cabbage by embryo rescue technique,which can improve the cabbage resistance,and this will lay a solid foundation for the clubroot resistance breeding of cabbage.In this study,interspecific hybridization and two backcrosses were carried out between Chinese cabbage and cabbage.The embryo rescue technique was used to obtain new materials for hybridization between cabbage and Chinese cabbage.Morphological identification,cytological identification,pollen fertility identification,identification of artificial indoor clubroot disease resistance and molecular marker identification of the obtained hybrid materials F₁,BC₁ and BC₂ were carried out,the main findings were as follows:1.The clubroot resistance of 20 Chinese cabbage materials were tested by naturally induced inoculation in the field.The results showed that 1 material was immune against clubroot,5 materials were highly resistant,12 materials were resistant and 2 materials were susceptible.6 materials with better economic traits and strong resistance were selected for artificial inoculation identification in the controlled condition.The results of the two identifications were combined to select two materials with strong resistance and stability.CR12 and CRYX were used as the source of resistance to distant hybridization.2.Choosing 43 published molecular markers closely linked to clubroot resistance genes,which were used to test in 4 clubroot resistance materials?CR12,CRYC,CR-Diwang26,Jingchun CR3?,and 4 susceptible materials?Jingfeng No.1,Xiakui,D6,and329?,results showed the SSR marker B0902 could amplify a specific band of 250 bp in4 resistant materials,and a specific band of 200 bp in 4 susceptible materials.Therefor,the marker B0902 can be used for molecular marker-assisted selection of clubroot resistance.3.This study was conducted with five cabbage cultivars D6,329,R,f416,309as the female parent,two Chinese cabbage cultivars CR12,CRYX as the male parent.Five interspecific hybrids were obtained by using bud stage pollination combined with embryo rescue technique.They were YX?1??D6×CRYX?,YX?2??329×CRYX?,YX?3??R×CRYX?,YX?4??f416×CRYX?,CR12?5??309×CR12?.By comparing the compatibility of these crosses,the frequency of pods per flower for D6×CRYX was 87.92%,and frequency of immature embryos survived/buds pollinated was 0.97%,higher than others.Morphological identification of five hybrid F₁ generations revealed that CR12?5?was a false hybrid,and the remaining four F₁ were true hybrids,and the number of chromosomes was 19.Anthers of four true hybrids were small,no pollen,no fertility.F₁were identified by artificial indoor inoculation,and three lines YX?2?,YX?3?,YX?4?were resistant to 4 and 11 races,and YX?1?were resistant to 11 race,and the susceptible to 4race.At the same time,combined with the identification of molecular marker B0902,the specific bands of the parents could been amplified in the F₁ generation materials.4.In this study,colchicine was used to treat F₁ lines.Compared with the normal materials,the doubled materials had larger leaves and flower buds,and its fertility was also restored.The chromosome number of the doubled materials?YX?1?Q,YX?2?Q,YX?3?Q,YX?4?Q?was 38.A total of 20 hybrid combinations were set by using hybrid F₁and heterotetraploid materials as the female parents and 5 cabbages cultivars as the male parents.5 cross combinations cloud obtain progenies,and a total of 12 plants were obtained.The number of frequency of immature embryos survived/buds pollinated of YX?1?Q×329 was 2.99%,and higher than other combinations.The progenies were true hybrids by morphology identification and cytology identification.The number of chromosomes was 22-28,and the pollen was fertile.BC₁ were identified by artificial indoor inoculation,and YX?1?Q-2-1,YX?1?Q-3-1,YX?1?Q-3-2,YX?1?Q-5-1,YX?1?Q-5-2,YX?1?Q-5-3,YX?1?Q-5-4 were resistant to 11 race,and YX?3?Q-1-1,YX?3?Q-5-1,YX?3?Q-5-2,YX?3?Q-5-3,YX?3?Q-5-4 were resistant to the 4 race.Molecular marker-assisted identification showed that YX?3?Q-1-1,YX?3?Q-5-1,YX?3?Q-5-2,YX?3?Q-5-3,YX?3?Q-5-4 could amplify the parental specific bands.YX?1?Q-2-1,YX?1?Q-3-1,YX?1?Q-3-2,YX?1?Q-5-1,YX?1?Q-5-2,YX?1?Q-5-3,YX?1?Q-5-4 losed the resistance band of the female YX?1?Q,but the specific band of YX?1?Q was amplified.5.A total of 60 backcross combinations were set by using 12 BC₁ lines of the hybrid progeniesas the female parents and cabbage as male parents.4 cross combinations,YX?3?Q-5-2×D6,YX?3?Q-5-2×R,YX?3?Q-5-2×f416,YX?3?Q-5-2×329 obtained backcross progenies,and number of plants was 1,2,1,2 respectively.The best affinity combination in this experiment was YX?3?Q-5-2×R.The backcross progenies were identified to be true hybrids by morphology identification,and the number of chromosomes was 18 or 20,and pollen was fertile.BC₂ were identified by artificial indoor inoculation,The backcross progenies,YX?3?Q-5-2-1-1,YX?3?Q-5-2-3-1,YX?3?Q-5-2-5-1,YX?3?Q-5-2-5-2,were resistant to 4 and 11 races,however,YX?3?Q-5-2-2-1,YX?3?Q-5-2-2-2 were susceptible to 4 and 11 races,which was consistent with the molecular molecular marker-assisted identification.In summary,a total of 5 hybrids F₁ were obtained in this experiment,which were crossed by cabbage×Chinese cabbage.Morphological identification and cytological identification results showed that CR12?5?was a false hybrid,and the other four F₁ were true hybrids,and the somatic chromosome number was 19,and there was not pollen.Hybrids were identified by artificial inoculation and molecular markers identification,three lines,YX?2?,YX?3?,YX?4?,were resistant to 4 and 11 races,and YX?1?were resistant to 11 race and susceptible to 4 race.The resistant hybrid F₁ was backcrossed with cabbage to obtain 12 BC₁ lines,which were identified to be true hybrids,and the numbers of chromosomes were 22-28.Pollen of hybrids had not fertility.The results of clubroot resistance identification were that 7 BC₁ hybrids were resistant to 11 race,and5 BC₁ hybrids were resistant to the 4 race.The clubroot resistant BC₁ were used as the female parent to backcross with cabbage,and 6 BC₂ hybrids were obtained.They were identified to be true hybrids by morphology identification and cytology identification,and the number of chromosomes was 18 or 20,and the pollen of hybrids were fertile.Artificial inoculation combined with molecular marker-assisted identification results showed that 4 BC₂ generations were resistant to 4 and 11 races,and 2 BC₂ generations were susceptible to 4 and 11 races.