Development And Cytology Of Disomic Alien Addition Lines Between Brassica Napus And Isatis Indigotica

Alien chromosomes addition lines, substitution lines and translocation lines developed through interspecific hybridizations can be used to study the relationships between different genomes, gene localization and expression and to introduce beneficial traits and genes of relative species into crops, etc. The Chinese woad(Isatis indigotica Fort., 2n=14, II), the Isatideae tribe of Brassicaceae family, is widely grown as medicinal plants in our country, and is also an important germplasm resource of improving rapeseed resistance. In the previous study, somatic hybrids(2n=52, AACCII) between Brassica napus L.(2n=38, AACC) cv. “Huashuang 3” and I. indigotica as the female parent were backcrossed successively to “Huashuang 3”and the progenies were analyzed, which led to the establishment of a complete set of monosomic alien addition lines(2n=39, AACC+1Ia-g), with each of 7 woad chromosomes added to the genome of B. napus. But these addition lines were male sterile due to carpellody development of stamens caused by the recombinant cytoplasm genome, except for the one with the chromosome(e). So it is difficult to obtain their dosomic alien addition lines, bringing troubles to their preservation and research. In this study, in order to convert the recombination cytoplasm of these addition lines to the normal cytoplasm of B. napus and to eliminate the harmful cytoplasmic effect on the development of flowers organs and male sterility,“Huashuang 3” was pollinated by the set of monosomic alien addition lines and the monosomic alien addition lines identified in the offsprings were selfed to generate dosomic alien addition lines(2n=40, AACC+1IIa-g, DAALs). The analyses on their morphology, molecular markers and cytology were made and the main results were as follows:1. Breeding and identification of DAALs with normal rapeseed cytoplasm.From the offsprings of “Huashuang 3”(the female parent) and the set of monosomic alien addition lines(the male parent) with low pollen fertility, monosomic alien addition lines(2n=39) were identified for five addition lines with the woad chromosomes a, b, d, e, g. Their flowers developed normally, and pollen fertility was obviously improved than those addition lines with recombination cytoplasm.Producing number of seeds, from them, identifing DAALs plants(2n=40,AACC+1IIa,b,d,e,g) attached a pair of I. indigotica chromosomes through cytological and molecular markers.2. Cytological behaviors and pollen sterility of DAALsConvention cytological and GISH investigations showed that thirty-eight chromosomes from rapeseed and two from woad were paired as 20 bivalents in the pollen mother cells(PMCs) at diakinesis and segregated equally as 20:20, and that the normal tetrads and four daughter nuclei were produced, while the laggards occurred with very low rates. The pollen stainability was low(66.40%) for DAAL with the chromosome b, but was higher for the other four DAALs(>90%).3. Transmission rate of additional woad chromosomes in DAALs.With I. indigotica centromeric-specific primers Cen1 R and Cen1 F for amplifications of leaf DNA, amplification products were produced from I. indigotica,and the selfing progenies of 5 DAALs, but not from “Huashuang 3”. With SSR primers specific for each woad chromosomes, amplification bands were obtained for I.indigotica and more than 90% offsprings, except for “Huashuang 3”, suggesting that the woad chromosomes in DAALs inherited relatively stably and had high transmission rate.4. Morphology and fatty acid composition of DAALs.DAALs had some differences in leaf color and shape, plant type and branching habit, color and smell of flower. Although DAALs’ silique lengths differed and were shorter than those of “Huashuang 3”, the rates of seed-setting of some DAALs were similar to “Huashuang 3”. The fatty acid compositions of selfed seeds from these DAALs were similar to those of “Huashuang 3”, while DAAL with the chromosome g showed the lower content of oleic acid but higher content of eicosenoic and erucic acid, even higher than the content of I. indigotica.