Developing Yellow-seeded Brassica Napus Through Interspecific Hybridization In Brassica And Studies On The Hybrids And Their Progenies

Yellow-seeded Brassica napus possesses a thiner seed coat,which is associated with higher oil,higher protein and lower fibre contents when compared to black-seeded ones in the same background.However,no naturally yellow-seeded forms of B.napus exist. Though many investigations to breed yellow-seeded B.napus have been carried out in the last several years,only few stable yellow-seeded forms have been developed.Several reasons have been attributed for the complicated inheritance of this trait and the failures to obtain truly yellow-seeded B.oleracea genotypes.Recent years,it was reported that yellow-seeded B.oleracea var.acephala mutants were found and breed true in Southwest University,China.The present investigation is to combine the genes for yellow seed color in A/C genome of other species into B.napus via interspecific hybridization of yellow-seeded species viz.B.oleracea var.acephala×B.rapa,B.rapa×B.oleracea var. acephala,B.juncea×B.oIeracea var.acephala,and B.carinata×B.rapa.By taking advantage of cytogenetic,genomic in situ hybridization(GISH),amplified fragment length polymorphisms(AFLP) and simple sequence repeat(SSR) technology,the morphological,cytological and molecular characterizations of the hybrids and their progenies were conducted.This paper provides new evidence for the genomic changes in synthetic B.napus,and also provides clues for the rules of B-chromosomes elimination in progenies of B.juncea×B.oleracea.The main results were as follows:1.Reciprocal crosses were conducted between three yellow-seeded varieties of B. rapa and five yellow- or brown-seeded accessions of B.oleracea var.acephala.Hybrids were obtained from five crosses of B.rapa×B.oleracea,three crosses;of B.oleracea×B. rapa and two reciprocal crosses.To eatablish an efficient reproducible culture system for different Brassica interspecific crosses,ovary culture and embryo culture were conducted in crosses B.rapa×B.oleracea and oleracea×B.rapa,respectively.A higher rate of hybrid production was recorded when ovaries were cultured at 4-7 days after pollination (DAP).Of different culture media,medium E(MS with half strength macronutrients) showed good response for ovaries from all the crosses,the highest rate of hybrid production reaching 45%in B.rapa(1151)×B.oleracea(T₂).In embryo culture,the hybrid rate was significantly enhanced at 16-18 DAP,up to 48.1%in B.oleracea(T₃)×B.rapa(JB₂).2.For ten crosses,meiotic behavior of resynthesized B.napus was observed.PMCs of newly allotetraploids from four crosses presented normal meiotic:progression.The pollen stainability of these plants was above 92.7%.Meiosis in the amphidiploids of cross 1151×T₂ was characterized by high frequencies of univalents and multivalents per PMC at diakinesis/metaphase I and notably unbalanced chromosome segregations at anaphase I (AI).In all the plants observed in 1151×T₂,18.95-44.3%of PMCs exhibited a segregation of 18:20(n-1:n+l) at AI,which was caused by nondisjunction of one bivalent or the distribution of two homologous univalents to the same pole.Meiosis proceeded normally after AI then,which led to the formation of viable n-1 and n+1 gametes and high pollen fertility of these plants.Amphidiploids from other crosses presented abnormal chromosome behavior at different meiotic phases,such as precocious division of centromere,chromosome laggards and elimination which led to the poor pollen stainability and seed set.3.Micro@ore culture was carried out using amphidiploids of cross 1151×T₂ in an attempt to isolate Brassica nullisomics for the first time.Four nullisomics(2n=36),two nullihaploids(2n=18) and one tetrasomic haploid(2n=20) were identified cytologically and characterized for their morphology and by using AFLP molecular markers.Five nullihaploids/nullisomics were supposed to be absent of one or one pair of C-genome chromosome(s),and one nullihaploid was supposed to be absent of one A- genome chromosome.Moreover,we presumed that MD-9 lost one pair of C-genome chromosomes carrying the genes for plant height,the size and number of petals,branch habit,erucic acid and glucosinolates biosynthesis.4.To study the dynamics of genome change after allopolyploidization,we surveyed the amphidiploids(A₁ and A₂ plants) of 1151×T₂ with AFLP and SSR molecular markers. Also amphidiploids(A₁ plants) of other crosses were surveyed by AFLP molecular markers.Extensive genomic changes were detected in all the amphidiploids,involving loss of parental restriction or SSR fragments,gaining of novel fragments and reappearance of parental fragments in A₂ plants that lost in A₁ plant.The frequency of losing parental fragments was much higher than that of gaining novel fragments.For all the reciprocally synthetic B.napus,the data showed that the frequency of sequence losing in B.rapa was much higher than in the B.oleracea.The results suggested that the biased sequence elimination was not caused by cytoplasmic factors.5.Nine hexaploids were produced from cross B.juncea×B.rapa and a subsequent chromosome doubling.Ninety-three pentaploids(AABCC) and twenty-seven tetraploids (ABCC) were obtained after hybridize the hexaploids with resynthesized B.napus and B. oleracea,respectively.GISH analysis of pentaploids and tetraploids revealed that a maximum of two bivalents formed by autosyndesis within B genome at diakinesis,and a maximum of two and four B-chromosomes paired with A/C chromosomes in pentaploids and tetraploids,respectively.B-chromosomes could be eliminated through B-chromosome laggards and precocious division of chromatids at anaphase I.However, the frequency of the B-chromosome abnormity in pentaploids was much higher than in tetraploids,indicating that new-type B.napus can be obtained more easily from selfed progenies of pentaploids.6.Pentaploids(AABCC) and tetraploids(ABCC) were selfed to eliminate B-chromosomes and generate newly B.napus.Pentaploids had better seed set than tetraploids.However,siliques per flower and seeds per silique of tetraploids were greatly enhanced when pollinated with the pollen of resynthesized B.napus.More derivatives of the ABCC×AACC cross exhibited a chromosome number adjacent to 38.Thus, hybridization between tetraploids and resynthesized B.napus can also be conducted to accelerate the elimination of B-chromosome and to generate newly B.napus.7.Eight and seventeen trigenomic aneuploids with doubled chromosomes were obtained through microspore culture of hexaploids(AABBCC) and tetraploids(ABCC), respectively.Out of the eight aneuploids derived from hexaploids,six aneuploids with 2n＞54 were revealed by GISH to contain eight pairs of B-chromosomes in it.They exhibited normal meiosis procession in most cases.Higher pollen fertility(78.23-86.85%) of these plants indicates that they are promising materials to develop new subspecies in Brassica.GISH analysis found 1-6 pairs of B-chromosomes in aneuploids from tetraploids.Some of them had 82.43%stainable pollen,and seed set was achieved from self-pollinated.These aneuploids are important materials in rapeseed improvement and genetic analysis.8.For the first time,yellow-seeded B.napus were obtained through interspecific hybridization between yellow-seeded B.rapa and yellow-seeded B.oleracea var. acephala,and between yellow-seeded B.juncea and yellow-seeded B.oleracea var. acephala.Meanwhile,hybridizations of yellowed B.carinata and yellowed B.rapa also succeeded in developing yellow-seeded B.napus.In crosses between B.rapa and B. oleracea var.acephaIa,yellow and yellow-brown seeded B.napus plants segregated in the advanced generations or doubled haploid(DH) populations of six crosses.The fact that A₂ plants of JB_×T₃ produced yellow seeds,whereas in its reciprocal crosses no yellow seeds but brown or black seeds were obtained,indicated cytoplasmic effect on seed color.Additionally,the prevalence of various interactions between the two parental genomes gave rise to novel variations which may be of practical interest.For example, new genotypes of long-pod,earliness,big-seed and double low materials were connected with the newly resynthesized B.napus in this study.