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获得了非常重要的作用，以快速有效地产生完全纯合的双单倍体系。尽管在优化组织培养方案方面取得了进展，但甘蓝型油菜不同基因型在培养小孢子的胚性反应方面仍存在巨大差异。小孢子培养反应的基因型差异的遗传基础在很大程度上是未知的，十字花科祖先物种拟南芥的小孢子的非胚发生反应仍然是一个谜。因此，甘蓝型油菜可以作为探索小孢子胚胎发生遗传基础的模式植物。多年来，春油菜品种Topas以其优异的小孢子胚胎发生潜力而闻名。从该品种，双单倍体系DH4079具有更进一步提高的小孢子胚胎发生潜力已被开发。DH4079目前在许多与小孢子胚胎发生相关的研究中用作标准基因型，包括研究诱导的胚胎发生和非诱导的小孢子中的基因表达谱的研究。已经鉴定了许多小孢子胚胎发生和直接胚向芽转化的候选基因。甘蓝型油菜的祖先种之一芜菁的基因组序列已经公布，预计在短时间内将公布甘蓝型油菜和甘蓝型油菜的全基因组序列。有了这些基因组序列，候选基因的物理位置就可以在染色体上确定。很明显，DH群体的小孢子胚胎发生缺乏表型数据来鉴定QTL和候选基因位点的重叠位置，以及鉴定迄今尚未考虑的新位点。对于QTL作图，DH群体的亲本对于感兴趣的性状应尽可能不同。关于胚胎发生能力，DH4079可能是已知的最好的甘蓝型油菜品系之一，每次实验产生数千个胚胎。在本项目前期工作中，该品系与冬油菜品种Express的自交系617杂交。在不同条件下的长期实验中，该品种已显示出在可比条件下，每次实验的胚产量在几个至50个胚的范围内。该杂交的一个克隆繁殖的F1植物已用于开发207个系的DH群体。本项目的主要目的是利用SNP标记（Illumina Infinium Brassica 60k Chip）构建该群体的分子连锁图，定位小孢子胚胎发生和直接胚向芽转化的QTL，并利用可用的芸苔属基因组序列比较QTL与候选基因的位置。

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.