Studies On Hau Cytoplasmic Male Sterility In Brassica Juncea

Rapeseed is one of the major oilseed crops.Utilization of rapeseed heterosis is widely applied.Cytoplasmic male sterility(CMS) is an important approach to exploit heterosis in this crop.The hau CMS(00-6-102A) was originally found as a spontaneous male sterile mutant in B.juncea in the experimental field of HAU in 1999.Stable CMS line was obtained by backcrossing.We adopted an integrated approach to classify the five types of CMS systems at morphological,cellular,genetic and molecular levels and confirmed that the hau CMS is different from others.Fertility mechanism of hau CMS was primarily confirmed by analysis of the Realtime-PCR,Northern blot and mitochondrial DNA Polymorphism As the sterility of hau CMS lines was stable and complete,this CMS was introgressed to B.napus and Cruciferous vegetables by hybridization.At the same time, the restorer lines were being screened.1.After twelve generations of backerossings with B.juncea 00-6-102B,a stable sterile B.juncea line(hau CMS) was established.The anthers in CMS plants were replaced by thickened petal-like structures and pollen grains were not detected. The"sterility degree" of 3,000 flowers was 100%and the "sterility rate" of 300 plants was also 100%in Lanzhou and Wuhan,indicating the stability of hau CMS in two different environments.This CMS was transferred to B.napus through interspecific hybridization. The resultant B.napus CMS was also absolute and stable when 6w-301 and 6w-307 were used as maintainers.2.Out of 320 materials of different origin,14 known resteror and maintainer lines in B.napus were selected for test-crossing with hau,pol,ogu,nap and tour CMS.The hau CMS line 6W-301A has the same nucleus genetic background as its B.napus maintainer line 6W-301 after eight backcross generations(BC8).The results of test-crossing with restorer lines indicated that fertility in the hau CMS was not restored by restorer lines from pol,ogu,nap and tour CMS systems.Therefore we concluded that hau CMS was a distinct type of cytoplasmic male sterility.3.Cytological observation of hau CMS anther development indicated the transverse section were thicker with increased cellular layers than normal petals and had no pollen sacs.The stamen primordium deviated from the normal polarization and formed petal primordium,which developed into petal-type structures at the stamen location.These characteristics differed from other CMS systems reported so far.The pollen abortion stage was earlier than other CMS types.The anther development of maintainer 00-6-102B was normal,and each anther had four normal pollen sacs in papilionaceous shape.The A-lines of pol,nap,tour,and ogu CMS systems formed anthers,but were devoid of functional pollen.4.The polymorphism of mitochondrial DNA was detected with mitochondrial probes atpl,orf222,atp6,atp9,cob,coxâ…¡,Orf222-nad5-orf139,coxâ… ,atpa and Orf263-atp6.Out of 40 mitochondrial probe/enzyme combinations,16 combinations exhibited polymorphism such as atp6/Hindâ…¢,atp6/EcoRI,atp6/EcoRV,atp9/BamHI,atp9/EcoRI and atp1/BamHI.Hau CMS 00-6-102A showed different band patterns from the other four CMS lines and five probe/enzyme combinations alone distinguished the five CMS systems from each other.These results indicated that the mitoehondrial DNA of hau CMS 00-6-102A was different from those of pol,nap,ogu and tour CMS.5.By analysis of the Realtime PCR,Northern blot and mitochondrial DNA Polymorphism,six genes were possibly associated with fertility mechanism of hau CMS. Furthermore,PCR-walking and sequence analysis were conducted.The results showed that a new open reading frame 'ORF288' probably associated with hau CMS was located at the downstream of atp6 gene.6.This CMS was introgressed to Cruciferous vegetables(such as B.juncea var.capitata Hort.ex Li,B.juncea var.multiceps Tsen et Lee,B.juncea Czern.et Coss.var.tsatsaiMao,B.rapa Linn.var.purpurea Bailey and B.rapa var.pekinensis ) by hybridization and backcross.The sterility of obtained CMS lines was stable and complete,in contrast to other four CMS types.7.Test crossing with 320 materials of different origins including B.juncea,B.napus, B.carinata and interspecific hybrids indicated material completely restoring the fertility of hau CMS was not found so far.The individuals obtained from test-crossing with two materials were partially fertile.