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发挥了重要作用，可以快速有效地产生完全纯合的双单倍体株系。尽管在优化组织培养方案方面取得了进展，但不同基因型的甘蓝型油菜在培养小孢子的胚性反应方面仍然存在巨大差异。小孢子培养反应基因型差异的遗传基础在很大程度上是未知的，十字花科祖先种拟南芥的小孢子的非胚性反应仍然是一个谜。因此，甘蓝型油菜可以作为研究小孢子胚胎发生的遗传基础的模式种。春油菜品种Topas以其优良的小孢子胚性而闻名于世。在此基础上，获得了小孢子胚性进一步提高的双单倍体系DH4079。DH4079目前在许多与小孢子胚胎发生相关的研究中被用作标准基因型，包括研究诱导胚胎发生和非诱导小孢子的基因表达谱。小孢子胚胎发生和直接胚向茎转化的一些候选基因已经被确定。甘蓝型油菜的祖先种之一——油菜的基因组序列已经公布，预计短时间内将公布甘蓝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被识别出来。显然，缺乏DH群体小孢子胚胎发生的表型数据，无法确定QTL和候选基因位点的重叠位置，也无法确定迄今尚未考虑的新位点。对于QTL定位，DH群体的亲本应尽可能不同于感兴趣的性状。就胚胎发生能力而言，DH4079可能是已知的最好的甘蓝型油菜系之一，每次实验可产生数千个胚胎。在本项目前期工作中，已将该品系与冬季油菜品种速递的自交系617杂交。该品种在不同条件下的长期试验表明，在可比条件下，每次试验的胚胎产量较低，在少数到50个胚胎的范围内。该杂交的1个无性系繁殖的f1植株已培育出207个DH群体。本项目的主要目的是利用SNP标记（Illumina Infinium Brassica 60k Chip）建立该群体的分子连锁图谱，定位小孢子胚胎发生和直接胚到茎转化的QTL，并利用现有的芸苔基因组序列与候选基因的QTL位置进行比较。

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.