Doubled haploid (DH) technology is an essential component in producing inbred lines for a competitive maize (Zea mays L.) breeding program. The R1-navajo (R1-nj) gene provides phenotypic marker that insures only variable reliability for seed selection of haploid embryos. Therefore, in the present study we outline a complex protocol for early stage genome size determination that integrates the phenotypic screening with the flow cytometry of nuclei from root tips and with the use of DNA isolated from seedlings for molecular marker-based genotyping. In a representative experiment with three genotypes, only 59% of the color marker pre-selected seeds were confirmed to be haploid by cytometric analysis of nuclei isolated from root tips. As a novel tool we have identified the UMC1152 SSR marker being polymorphic between the haploid inducer line (K405) and the K4390 hybrid as parents to screen seedlings pre-selected with the R1-navajo marker. Using this molecular marker, alleles characteristic for the inducer K405 line could not be detected in 83% of seedlings previously selected as haploid candidate. Seedlings identified as haploids were exposed to 0.06% colchicine solution for rediploidization. This procedure resulted in doubled haploids with 3% frequency relative to the initial population as it was quantified by the number of mature maize plants with fertile tassel. The described complex approach can support safer identification of haploids at early seedling stage in a hybrid population derived from crossing with a haploid inducer line.

中文翻译：

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通过将 R1-navajo 标记与流式细胞术或微卫星基因分型相结合，提高了玉米自交系生产的可靠性

双单倍体 (DH) 技术是为竞争性玉米 (Zea mays L.) 育种计划生产自交系的重要组成部分。R1-navajo (R1-nj) 基因提供表型标记，确保单倍体胚胎种子选择的可变可靠性。因此，在本研究中，我们概述了一个复杂的早期基因组大小测定方案，该方案将表型筛选与根尖细胞核的流式细胞术相结合，并使用从幼苗中分离的 DNA 进行基于分子标记的基因分型。在具有三种基因型的代表性实验中，通过对从根尖分离的细胞核进行细胞计量分析，仅 59% 的颜色标记预选种子被确认为单倍体。作为一种新工具，我们已确定 UMC1152 SSR 标记在单倍体诱导系 (K405) 和 K4390 杂种之间具有多态性，作为亲本筛选用 R1-navajo 标记预选的幼苗。使用该分子标记，在先前被选为单倍体候选者的 83% 的幼苗中无法检测到诱导剂 K405 系的等位基因特征。将鉴定为单倍体的幼苗暴露于 0.06% 秋水仙碱溶液中进行再二倍体化。该程序导致双倍单倍体，相对于初始种群的频率为 3%，因为它是通过具有可育雄穗的成熟玉米植物的数量来量化的。所描述的复杂方法可以支持在幼苗早期在与单倍体诱导系杂交的杂交种群中更安全地鉴定单倍体。