Characterization of genomic regions responsible for the outstanding microspore embryo-genic potential of the doubled haploid oilseed rape line DH4079 (Brassica napus L.)

在过去的几十年里，未成熟花粉粒(称为小孢子)的培养在油菜育种中变得越来越重要，因为它可以快速高效地产生完全纯合的双单倍体品系。尽管在优化组织培养方案方面取得了进展，但甘蓝型油菜在培养小孢子的胚胎发生反应方面仍然存在巨大差异。小孢子培养反应的遗传差异的遗传基础很大程度上是未知的，十字花科祖先拟南芥小孢子的非胚性反应仍然是一个谜。因此，甘蓝型油菜是探索小孢子胚胎发生的遗传基础的模式物种。春油菜品种托帕斯多年来一直以其优良的小孢子胚胎发生潜力而闻名。在此基础上育成了小孢子胚胎发生潜力进一步提高的双单倍体品系DH4079。DH4079目前被用作许多与小孢子胚胎发生相关的研究中的标准基因，包括那些研究诱导的胚性和非诱导的小孢子的基因表达谱。小孢子胚胎发生和胚芽直接转化的一些候选基因已经被确定。甘蓝型油菜是甘蓝型油菜的祖先种之一，其基因组序列已经公布，甘蓝型油菜和甘蓝型油菜的全基因组序列有望在短期内公布。利用这些基因组序列，可以确定候选基因在染色体上的物理位置。显然，目前还缺乏关于小孢子胚胎发生的表型数据，无法确定QTL和候选基因座的重叠位置，也缺乏迄今尚未被考虑的新基因座。在进行QTL定位时，对于感兴趣的性状(S)，DH群体的亲本应该尽可能不同。在胚胎发生能力方面，DH4079可能是已知的最好的甘蓝型油菜品系之一，每次实验可产生数千个胚胎。在本项目前期工作中，该自交系已与冬油菜自交系617杂交。这种品种已经在不同条件下的长期实验中被证明，在可比的条件下，每个实验产生的胚胎产量很低，最高可达50个胚胎。利用该杂交组合的一个无性系繁殖的F1植株，形成了207个单株的杂交群体。

During the past decades, the culture of immature pollen grains, called microspores has gained much importance in breeding of oilseed rape for the rapid and efficient generation of completely homozygous doubled haploid lines. Despite the progress achieved in optimizing tissue culture protocols, tremendous differences remain among Brassica napus genotypes in their embryogenic response of cultured microspores. The genetic basis of the genotypic differences in microspore culture response is largely unknown and the non-embryogenic response of microspores of the cruciferous ancestor species Arabidopsis thaliana remains a mystery. Hence, Brassica napus serves as a model species to explore the genetic basis of microspore embryogenesis. The spring oilseed rape cultivar Topas is known since years for its excellent microspore embryogenic potential. From this cultivar, the doubled haploid line DH4079 with an even further improved microspore embryogenic potential has been developed. DH4079 is currently used as standard genotype in many investigations related to microspore embryogenesis, including those investigating gene expression profiles in induced embryogenic and non-induced microspores. A number of candidate genes for microspore embryogenesis and for direct embryo to shoot conversion have been identified. The genome sequence of Brassica rapa, one of the ancestor species of Brassica napus has been published and the publication of the complete genome sequence of Brassica oleracea and Brassica napus is anticipated within short time. With those genome sequences the physical positions of the candidate genes can be identified on the chromosomes. Obviously, there is a lack of phenotypic data for microspore embryogenesis of DH populations to identify overlapping positions of QTL and candidate gene loci as well as to identify new loci which to date have not been considered. For QTL mapping the parents of the DH population should be as different as possible for the trait(s) of interest. Regarding the embryogenic capacity, DH4079 is probably one of the best Brassica napus lines known, giving thousands of embryos per experiment. In previous work to this project, this line has been crossed to inbred line 617 of winter oilseed rape cultivar Express. This cultivar has been shown in long term experiments under varying conditions to give a low embryo yield in the range of few up to 50 embryos per experiment, under comparable conditions. One clonally propagated F1-plant of this cross has been used to develop a DH population of 207 lines. The main aims of the present project is to develop a molecular linkage map of this population using SNP markers (Illumina Infinium Brassica 60k Chip), to map QTL for microspore embryogenesis and direct embryo to shoot conversion and to compare positions of QTL with those of candidate genes using the available Brassica genome sequences.