Development Of A Complete Set Of Brassica Napus-Isatis Indigotica Monosomic Alien Addition Lines And A Novel CMS In B. Napus

Monosomic alien addition lines (MAALs) are valuable for elucidating the genome structure and transferring the useful genes and traits in plant breeding. Isatis indigotica Fort.(2n=14, II)(Chinese woad) which belongs to the Isatideae tribe of the Brassicaceae family has been widely cultivated as a medicinal and dye plant in China,and shows resistance to tobacco mosaic virus (TMV) and the fungus Sclerotinia sclerotiorum causing stem-rot in rapeseed. So the plant can be used as valuable gerplasm for genetic improvementfor B. napus. Herein, the intertribal somatic hybrid (2n=52, AACCII) between B. napus cv. Huashuang3(2n=38, AACC) and I. indigotica produced previously was backcrossed recurrently to parental B. napus, and MAALs were isolated from backcross progenies by phenotype, cytological method,and SSR markers, together with male sterile plants of carpelloid stamens obtained. The main results were described as follows:1. Development of a complete set of MAALs. Among BC3and BC4progenies derived from the somatic hybrid between B. napus cv. Huashuang3(2n=38, AACC) and I. indigotica,32MAALs were isolated by somatic chromosome counting and GISH (genomic in situ hybridization) analysis. Based on their phenotype,5S and45S rDNA loci, and chromosome-specific SSR markers, these MAALs were classified into seven groups corresponding to potential seven types of MAALs carrying one of seven I. indigotica chromosomes (Ma-Mg). One of MAALs (Me) could be distinguishable by expressing the brown anthers of I. indigotica, other two (Mf and Mg) hosted the chromosome with5S and45S rDNA locus respectively, but the remaining4MAALs were identifiable by SSR markers.The simultaneous detection of the same SSR maker in different MAALs revealed the paralogs on the chromosomes involved. Chromosome c shared two markers with e, and similarly d and f shared two markers, suggesting a significant level of homoeology between c and e and between d and f, but lower or no homoeology between chromosome b and others. Using these MAALs,184SSR markers and four genes were located on I. indigotica chromosomes. HCDPK and IiMYB wereon chromosome a. IiSDDRl locates on chromosome b. liPAL relates to chromosome c, d, e and f, showing that the gene had four homologous copies in/. indigotica. All MAALs but Me were male sterile by expressing male sterile and homeotic conversion of stamens into carpelloid structures. The PCR analysis revealed that the somatic hybrid and MAALs contained a mixed mitochondrial composition with both B. napus and I. indigotica mtDNA. The normal development of stamens in the MAAL (Me) indicated that the alien chromosome e carried the gene(s) for fertility restoration.Homoeologous pairings between chromosomes d, f, g of I. indigotica and chromosomes of B. napus were revealed in the pollen mother cells (PMCs) at diakinesis of respective MAALs. Chromosome e in the background of B. napus interfered normal pairing of A and C genome, for some trivalents were observed in Me. At meiosisanaphase I, the woad chromosomes inPMCs of MAALsmoved to one polar ordivided precociously, or lagged behind in some PMCs. In some PMCs, chromosomes of B. napus separated unequallyas18:20,17:21,18:19, etc. Female transmission rate of woad chromosome in MAALs was18.19%-67.74%, while the male transmission rate was higher (66.70%-72.30%, except Mf,13.10%). The higher content of eicosenoic acid and erucic acid in the seeds of Mg suggested that the gene FAE1located on woad chromosome g.2. Production of new CMS lines with carpelloid stamens. By recurrent backcrossing the somatic hybrid between B. napus cv. Huashuang3and I. indigotica as female with parental B. napus, male sterile plants which expressed carpelloid stamens were obtained. AFLP analysis showing none DNA fragment of I. indigoticaorigin, butrecombinantmtDNA revealed in the mitochondrial genome of the lines. The lines produced the sterile hybrid after pollination by B. napus, showing one type of cytoplasmic male sterility (CMS). Thetetradynamous stamens of CMS developed into carpelloid structures and the two shorter stamens were only filaments without anthers. These feminized stamens contained stigmatoid structures at their tips, adhered ovuleat bottom, and two from same side usually fused together. Histological analysis of the CMS showed that the abnormal development at the sites of the petals and stamens started in the young floral buds (stages4and5), and ovules appeared in the carpelloid structure at stage9. The B. napus plants (2n=38) with normal stamens and high pollen fertility were identified in the selfed progenies of the Me, which probably carried the gene(s) for fertility restoration on the added woad chromosome. The CMS line and fertility-restoration line were promising for hybrid breeding in future.