Development And Genetic Studies Of A Fertility Restorer Line For Inap CMS In Brassica Napus And B.rapa-nigra Additions

Monosomic alien addition lines（MAALs）are important bridges and tools for transferring the available genes and traits in crop breeding,dissecting the genome of donor species and understanding the evolutionary relationship of plants species.The intertribal somatic hybrid between Brassica napus（2n=38,AACC）and Isatis indigotica（Chinese woad,2n=14,II）,a medicinal plant,produced previously was backcrossed recurrently to parental B.napus.On the one hand,the inap CMS line of B.napus with carpelloid stamens was developed;on the other hand,a complete set of B.napus-I.indigotica MAALs（Ma-Mg）were established and the Rf gene of inap CMS was identified on the chromosome e of I.indigotica in MAAL Me.In the present study,phenotypic,cytological and molecular markers analyses were performed on selfed-progenies of MAAL Me for screening the fertility restorer line of inap CMS with the Rf gene introgressed from I.indigotica to B.napus.Moreover,the triploid hybrid（2n=28,AAB）between B.rapa（2n=20,AA）and B.nigra（2n=16,BB）produced previously was backcrossed recurrently to parental B.rapa,and a complete set of B.rapa-nigra MAALs were isolated.The main results are as follows:1. Development of a fertility restorer line for inap CMS.A total of 625 plants were obtained through microspore culture of fertile plants selected from the selfed progenies of MAAL Me,and 192 plants were doubled haploid（DH）lines.Among them,140 plants exhibited carpelloid stamens,other 52 plants developed flowers with different numbers of stamens,mostly with large tetradynamous stamens but 2 tiny stamens,and only a few with 6 well-developed stamens.F₁hybrids between 52 DH plants and the inap CMS line were produced and were observed for fertility restoration in four locations（Wuhan,Chengdu,Changsha and Xining）together with the 52 DH lines,maintainer Huashuang 3 and inap CMS.Finally,a fertility restorer line was selected and named as RF 39.GISH,woad-centromere markers and chromosome e specific SSR markers analyses indicated there was no existence of intact woad chromosome e in RF 39.However,a few segments from woad chromosome e were detected in RF 39 by AFLP analysis.On the phenotype,brown anthers controlled by woad chromosome e was presented in RF 39,as well as the F₁hybrid of RF 39 and inap CMS,and fertile plants of a F₂population.RF 39shared the similar compositions of glucosinolate（GSLs）with Huashaung 3 in seeds by HPLC（high performance liquid chromatography）,but total content of GSLs was much higher than B.napus,because RF 39 had higher contents of PRO and NAP,the two of three componets in I.indigotica,suggesting that the genes related to their synthesis were introduced.In one F₂population with 820 plants,male fertile and sterile plants showed the 3:1 segregation ratio（χ²=0.08,p<0.01）,indicating that the Rf gene of RF 39 was a single dominant gene.The development of RF 39 made inap CMS promising for rapeseed hybrid breeding in future.2. Development of B.rapa-nigra MAALs.The triploid hybrid（2n=28,AAB）of B.rapa（2n=20,AA,genotype 3H120）and B.nigra（2n=16,BB,cv.Giebra）was pollinated successively by parental B.rapa.The FISH and SSR markers analyses were used for screening MAALs(2n=21,AA+1B_1-8)in BC₂to BC₅generations.For MAALs B1-B5,the B-genome chromosome remained unpaired as a univalent（10ⅡAA+1ⅠB）in 82.93%-89.74%,averagely 85.92%,pollen mother cells at diakinesis,and formed a trivalent（9ⅡAA+1ⅠⅡAAB）in 10.26%-17.07%,averagely14.08%PMCs.Transmission frequency of B1-B5 alien chromosomes through the ovules and pollen were both low.For ovules,the frequencies varied from 2.70%to 20.83%,averagely 13.30%;and for pollen,rates varied from 1.22%to 11.76%,averagely 7.12%.The frequency of female was higher than that of male,except for B2.There existed different transmission frequencies in B1-B5 chromosomes,as B1 and B5 chromosomes were transmitted by female at much higher rates（20.83%,20.35%）than the B2（2.70%）,and the B1 chromosome was transmitted by male at much higher rate（11.76%）than B4（1.22%）.No signal of 45S r DNA was detected on B2 by FISH,however,signal on B4chromosome was detected.Signals of 45S r DNA on B3 chromosome and 5S r DNA on B5chromosome were verified.For phenotype,MAALs plants exhibited some obvious traits from B.nigra or induced by the interaction of A and B genome,such as purple color of B3 MAAL plants,yellow seed coat of MAALs B1,B3 and B5.The development of B.rapa-B.nigra MAALs should be valuable for improving the genome sequence quality of B.nigra,exploiting relationship of two species and exploiting the available traits of B.nigra.