|dc.description.abstracteng||Microspore culture is a very powerful technique in breeding of oilseed rape for the rapid and efficient generation of completely homozygous doubled haploid (DH) lines. Despite the progress achieved in optimizing tissue culture protocols, tremendous differences remain among Brassica napus genotypes in their embryogenic response and direct embryo to plant conversion, representing a hindrance in the development of double haploid populations for plant breeding purposes.
To understand the genetic factors underlying microspore culture response, the following objectives were addressed: (a) to develop a doubled haploid population from the cross of the highly embryogenic line DH4079 and the low embryogenic inbred line Express 617; (2) to characterize the doubled haploid population for its microspore embryogenic potential, its direct embryo to plant conversion as well as for microspore density, embryo survival, root regeneration and secondary embryogenesis; (3) to develop an Illumina SNP-chip based molecular marker map, to identify QTL for those traits and to localize candidate genes within QTL confidence intervals for the traits of interest; and (4) to study the gene expression pattern in low-temperature treated microspore derived embryos with the aim to identify differentially expressed genes which may be involved in the enhanced direct embryo to plant conversion after cold induction.
In vitro propagated F1-plants of the cross DH4079 x Express 617 were used to generate a DH population of 207 lines that were seed propagated and used as the source of microspores, which were cultured following a standard protocol. The number of microspores and microspore embryogenic potential, defined as the percentage of microspores developing embryos, were recorded. Embryos at the late cotyledonary stage were transferred to solid medium to induce plantlet regeneration and following a cold treatment, the survival, secondary embryogenesis, direct embryo to plant conversion and root regeneration were scored. Experiments were repeated five times and mean values obtained from 81 to 98 lines were used for QTL mapping was performed based on an Illumina Infinium Brassica 60K SNP molecular linkage. Large and significant genetic differences were observed between the genotypes for all traits. Microspore number varied from 58,900 to 148,700 /ml and microspore embryogenic potential ranged from 0 to 3.6 %. Embryo survival varied from 26 % to 99 %, and direct embryo to plant conversion was found to range from 13 % to 85 %. Root regeneration varied from 14 % to 95 %, and secondary embryogenesis occurred in 4 % up to 91 % of the embryos. Analysis of variance revealed a predominant effect of the genotype influencing microspore culture response and high broad-sense heritabilities ranging from 66 % for the microspore number to 86 % for microspore embryogenic potential were detected. A linkage map based on 1,414 SNP markers was developed of which 49 % exhibited distorted segregation clustered over all linkage groups, except for chromosome A04. Together, ten QTL were mapped on linkage groups A01, A02, A05, A10, C04 and C06. A positive correlation between survival of embryos, direct embryo to plant conversion and root regeneration, as well as overlapping QTL confidence intervals indicated the presence of either a ‘hotspot’ for loci controlling the regeneration of plants resulting from microspore derived embryos or a pleiotropic gene influencing several traits. BLAST-analysis revealed the presence of nineteen candidate genes within the 95 % confidence interval of QTL. Among those, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED PEPTIDE 1, CLAVATA3/EMBRYO SURROUNDING REGION-RELATED PEPTIDE 25, WUSCHEL RELATED HOMEOBOX 1, WUSCHEL RELATED HOMEOBOX 2, WUSCHEL RELATED HOMEOBOX 5, CUP-SHAPED COTYLEDON 3, SCARECROW and NO-APICAL-MERISTEM stand out, because they control cell fate and are responsible for root and shoot meristem initiation and maintenance, which is of major factor influencing microspore embryogenic potential and further direct embryo to plant conversion.
In a further step, expression profiles of cold-treated and not cold-treated embryos were assessed through MACE (Massive Analysis of cDNA Ends). Microspore culture of genotypes Express 617 and DH4079 was performed following a standard protocol and microspore derived embryos at the late cotyledonary stage were cold treated for 10 days at 2 °C, while control embryos were incubated for 10 days at 20 °C. After this, cold-treated and control embryos were cultured together for 10 days at 20 °C before they were frozen in liquid nitrogen. MACE was performed by GenXPro GmbH, Frankfurt am Main, Germany. MACE revealed 86,557 different transcripts, 2,606 exhibited a differential expression (log2FoldChange>2.3) of which 207 transcripts were up- or down-regulated in both genotypes when embryos were cold treated. Of the 207 transcripts, 187 transcripts were annotated to known gene sequences and the top ten up- and down-regulated cDNA sequences were used for BLAST analysis to identify orthologous genes in the B. napus, B. oleracea, B. rapa and A. thaliana databases. Genes involved in plant defense, stress tolerance and cell detoxification were up-regulated (MATE efflux family protein, AP2/ERF transcription factors, Betv1/MLP like protein), as well as genes encoding for carbohydrate and lipid transporters (SWEET12 and SRPBCC ligand-binding domain-containing protein). Conversely, genes encoding proteins related to stress tolerance with an increasing accumulation during embryo development (M17, LEA49 and glycine-rich protein), were down-regulated in cold-treated embryos.
Results of this study revealed a predominant effect of genotypic factors influencing embryogenic potential and direct embryo to plant conversion in the DH4079 x Express 617 population. QTL analysis and MACE are complementary tools that allow the identification of genes controlling microspore culture response. An increased knowledge of the genetic factors involved in microspore culture would facilitate the allele transfer from high-responsive genotypes to less-responsive lines of agronomic interest and its employment in the development of double haploid populations during plant breeding programs.||de