Cytology And Heredity Of Novel Brassica Napus Lines With High Oleic Acid Content Derived From B. Napus×Orychophragmus Violaceus Hybrids

Interspecific/intergeneric hybridization is one of the improtant approaches for cropgenetic improvements. Extensive ranges of wild relatives for Brassica crops provide richgermplasm with many useful nuclear and cytoplasmic genes. Brassica napus L. (2n=38,genornes AACC) was introduced into China for the first time from Korea in 1930s andsubsequently from Europe in 1940s with limited genetic variability and thus the wideningof its gene pool through suitable approaches including wide hybridizations is pivotal forfurther genetic improvement. After the development of the double low varieties with thenutritionally desired canola quality (i.e.,＜2% erucic acid in the seed oil and＜30μmolglucosinolate/g oil free meal,～60% oleic acid), further increase of oleic acid andreduction of linolenic and saturated acids are seen as new important target for geneticenhancement of B. napus varieties. Orychophragmus violaceus (L.) O.E.Schulz (2n=24),an ornamental crucifer in China, was found to be a new valuable resource for the seedquality improvement of rapeseed as it possess favorable fatty acid composition (～50%linoleic acid,～5% linolenic acid and～1% erucic acid). Intergeneric hybrids between B.napus cv. 'Oro' and O. violaceus were obtained and the progenies were observed andselected for successive generations. In the present study, new B. napus inbred lines withincreased levels of oleic (～70%, 10% higher than that of B. napus parent) and linoleic(28%) acids and reduced glucosinolate content (＜30μmol/g oil free meal) were obtainedby pedigree selection through F₁₂ generation in the progenies of one F₅ hybrid plant(2n=31) with slight yellow petals and their genomic compositions determined by usingthe methods of (GISH) and amplified fragment length polymorphisms (AFLP) and theinheritance pattern of oleic acid analysed. The main results were as follows:1. Morphology and agronomic characters of new B. napus lines. These stable new B.napus lines with uniform phenotypes were derived from two _F6 plants (F₆-8, F₆-9).Phenotypically, most of these lines were very similar to B. napus cv. 'Oro'. Theyexpressed purple color of O. violaceus on leaf veins, stems, petioles and pods at differentdegrees and branched at lower positions. Moreover, they exhibited other heritablevariations such as yellow, slight yellow, nearly white petals of larger size. More than 60lines investigated had normal pollen fertility and seed set. The plants of these linesusually had higher numbers of branches, pods and larger seeds (＞4g/1,000-seed) thanthose of 'Oro', which contributed to the higher seed yield per plant. 2. Fatty acid composition and glucosinolate content. Inbred lines with increasedlevels of oleic acid (≥70%,～10% higher than that of 'Oro') were obtained by pedigreeselection on progenies (F₉-F₁₂) from F₆-8 and F₆-9. The lines with high linoleic acid(～28%) were selected from progenies of F₆-9 and this trait was stably inherited from F₉ toF₁₂ and the sum of oleic and linoleic acids reached～87% with erucic acid＜1%. Nearly2000 plants of F₁₁ and F₁₂ lines unexpectedly showed wide variations in glucocinolatecontents (3.95-139.58μmol/g oil free meal, but 64.93μmol/g for 'Oro') and 238 (13.05%)had＜30μmol/g. Then the double low lines with≥70% oleic acid were selected.3. Chromosomal and genomic compositions. Majority of somatic cells (＞98%) inovaries and roots had 2n=38 in all plants (F₁₁, F₁₂) and remaining cells had 37 and 39chromosomes in some plants. All plants overwhelmingly produced PMCs with normalchromosome pairing (19 bivalents) at diakinesis and 19:19 segregations at anaphaseâ… (AI). One or two more darkly stained chromosomes were lagged between or included inpolar groups or daughter nuclei at anaphase and telophaseâ… /â…¡. PMCs at diakinesis,metaphaseâ… (Mâ… ), Aâ… /telophaseâ… (Tâ… ), Mâ…¡and Aâ…¡/Tâ…¡from one plant of the 21 F₁₁ orF₁₂ lines with high oleic or linoleic acids were subjected to GISH analysis, together withovary or root tip cells from selfed seeds from one plant of F₁₂ lines. Signals of O.violaceus genomic DNA probe with variable sizes and intensities were located mainly atterminal and centromeric parts of some mitotic chromosomes and meiotic bivalents atdiakinesis or chromosomes at Aâ… , but no fully labeled chromosomes/bivalents wereobserved. In some Tâ… /Mâ…¡PMCs, one large patch of chromatin was intensively labeledand separated spatially in some nucleic. It might well be that O. violaceus introgressionwas too small that it could not be detected on metaphase chromosomes.Five-five F₁₂ plants of 51 lines with higher oleic acid (＞70%) and one plant of theline with high linoleic acid (28%) were subjected to AFLP analysis. By using 10 pairs ofprimers randomly selected, the polymorphic bands were revealed and no same profileswere produced by any two plants. The number and percentage of O. violaceus-specificbands, deleted bands in 'Oro' and novel bands for two parents were 85 (15.98%), 59(11.09%), and 53 (9.96%), respectively. For the variations in number, sizes and intensitiesof GISH signals among the cells on the chromosome preparations from one plant, it wasquite difficult to correlate the GISH signals with the number of the O. violaceus-specificAFLP bands for each line. In line with high linoleic acid and one darkly stainedchromosome encountered in its PMCs, the number of O. violaceus-specific bands washigher than those in the majority of the 20 lines with higher oleic acid (17/20). The origin of the novel and deleted bands of B. napus with high frequency accompanied by theintrogression of the O. violaceus fragments in these lines might be related to the fragmentrecombination and sequence elimination which occurred during several rounds of genomefusions and separations and genomic remodeling and stabilization. These changes shouldbe genomewide and affected many genes including plant phenotype, the petal color, thesynthesis of fatty acids and glucosinolate.4. Inheritance pattern of oleic acid content. By using new line '291', 'Oro' andZhongyou 821 with 69%, 57% and 25% oleic acid, respectively, reciprocal crosses'291'×Zhongyou 821, 'Oro'×Zhongyou 821 and '291'×'Oro' were made. Oleic acid inseeds of parents, F₁, F₂ and BC₁ generations were analyzed by half-seed method. Thegenetic analyses on '291'×Zhongyou 821 cross were repeated for two years with F₃ seedsalso studied. In crosses '291'×Zhongyou 821 and 'Oro'×Zhongyou 821 no maternal orcytoplasmic for oleic acid content was observed and the F₁ mean was significantly lowerthan mid-parent value, indicating partial dominance for high content. On the contrary,there existed partial maternal effect for oleic acid content in cross '291'×'Oro' and the F₁mean was nearly equal to the mid-parent value, and the difference in F₂ means ofreciprocal crosses was unsignicant, showing no cytoplasmic effect. The analysis of theoleic acid content in the segregating populations from the cross of '291'×Zhongyou 821for two years indicated that the segregation pattern fitted the model of two alleles at twoindependent loci with partial dominance for low concentration, O_l and O₂. The correlationcoefficients between oleic and erucic acids in two years were r=-0.92 and r=-0.93. Theresults in two years revealed that the environmental effect on oleic acid content was notsignificant. The segregation pattern in the cross 'Oro'×Zhongyou 821 was explained onthe basis of two alleles at one loci, O_l. The correlation coefficient between oleic anderucic acids was r=-0.91. In cross '291'×'Oro', it was difficult to classify low,intermediate and high oleic acid types in the F₂, BC₁ populations, but more than 25% ofthe F₂ seeds and more than 50% of F₁×'291' seeds showed the values of '291', indicatingthe segregation for oleic acid was controlled by two alleles at one locus, O₂. Thegenotypes proposed for the three parents were o_lo_lo₂o₂ for '291', o_lo_lO₂O₂ for B. napus cv.'Oro' and O_lO_lO₂O₂ for Zhongyou 821. The increased oleic acid (～10%) in new B. napusline '291' compared to 'Oro' was attributed to the effect of recessive gene o₂.