Development And Study Of Brassica Napus-isatis Indigotica Addition Lines With Clubroot Resistance

The medicinal plant Isatis indigotica Fort.（2n=14,II）（Radix isatidis）with antiviral and antibacterial effects is an excellent germplasm for genetic improvement of rapeseed.In our group,the somatic hybrid（2n=52,AACCII）between Brassica napus L.（2n=38,AACC）and I.indigotica was backcrossed continuously with B.napus,and a complete set of B.napus-I.indigotica monosomic additional lines（Ma～Mg）was obtained,which were called Banlangen rapeseed.Mg was found to show high resistance to both flu virus and SARS-Co V-2.In this study,in regards to the popular occurrence of the disease clubroot caused by Plasmodiophora brassicae,Mg was crossed with two genotypes（H5R、409R）resistant to clubroot carrying the Pb Ba8.1 and CRb2 locus,respectively.The progenies of two generations were selected by molecular marker-assisted selection,cytological identification,agronomic traits and resistance identification,to obtain Mg carrying the CRb2 locus and clubroot resistance.The main results are as follows:1.Molecular marker identification of additional progenies.By SSR markers specific to addition chromosome,a total of 280 additional plants were screened out among 488 F₂from Mg×CRb2 parent,accounting for 57.38%of the total number of plants.Among1206 F₃ plants,911 additional plants were obtained（75.54%）.In the cross Mg×Pb Ba8.1parent,among 60 F₂ plants,10 additional plants were obtained（16.7%）.Among 4 F₃plants,4 additional plants were obtained（26.67%）.In the 200 F₂progenies by the cross（Mg×409R）×（Mg×H5R）,99 additional plants were obtained（49.5%）.Among 60 F₃plants,46 additional plants were obtained（76.67%）.2.Molecular marker screening of additional plants with clubroot resistance loci.By molecular marker of CRb2 locus,among 280 additional F₂ plants,83 ones with the locus were screened out,accounting for 29.64%of the total number.Similarly,755 F₃plants with locus were selected among 911 additional ones（82.88%）.In the cross Mg×Pb Ba8.1parent,2 F₂plants with locus were selected among 10 additional ones（20%）.One F₃plants with locus were selected among 4 additional ones（25%）.In the F₂plants from the cross（Mg×409R）×（Mg×H5R）,45 F₂plants with Pb Ba8.1 locus（47.87%）,9 F₂plants with CRb2 locus（9.57%）.4 F₂plants with both CRb2 and Pb Ba8.1 locus were selected among 99 additional ones（4.26%）.In the F₃population of 56 plants,24 F₃plants with Pb Ba8.1 locus（51.61%）,32 F₃plants with CRb2 locus（68.81%）were identified,of which 13 F₃plants（13.34%）with both CRb2 and Pb Ba8.1 locus were included.3.Cytological analysis.The additional plants carrying the CRb2 locus had 2n=39,40 for somatic chromosomes,which were paired as 19II+1I,20II,respectively.Chromosomal laggards appeared in anaphase I,II.One additional chromosome from I.indigotica was identified by genomic in situ hybridization（GISH）in monosomic addition,and the chromosomes were segregated as 19:20 at anaphase I.4.Morphology and fertility.For plant morphology,the additional plants carrying the CRb2 locus was shorter than the parent H3,had the larger stem,more branches,and the more compact plant type.They showed 63%～88%pollen fertility and higher rate of the seed-set than that of Mg.5.Identification of clubroot resistance.By inoculating the physiological race 4 of P.brassicae,the parent HS3 were highly susceptible,H5R and 409R showed immunity.Three additional lines carrying the CRb2 locus all showed high resistance.In summary,the antiviral addition lines with high resistance to clubroot were selected,which was valuable for their production in large scale.