Noble Research Institute, LLC, Ardmore, OK, 73401, USA.
Key Laboratory of National Forestry and Grassland Administration on Grassland Resources and Ecology in the Yellow River Delta, College of Grassland Science, Qingdao Agricultural University, Qingdao, Shandong, 266109, China.
Plant J. 2022 Jul;111(2):608-616. doi: 10.1111/tpj.15797. Epub 2022 Jun 1.
Though Medicago truncatula Tnt1 mutants are widely used by researchers in the legume community, they are mainly used for reverse genetics because of the availability of the BLAST-searchable large-scale flanking sequence tags database. However, these mutants should have also been used extensively for forward genetic screens, an effort that has been hindered due to the lack of a compatible genetic crossing partner for the M. truncatula genotype R108, from which Tnt1 mutants were generated. In this study, we selected three Medicago HapMap lines (HM017, HM018 and HM022) and performed reciprocal genetic crosses with R108. After phenotypic analyses in F1 and F2 progenies, HM017 was identified as a compatible crossing partner with R108. By comparing the assembled genomic sequences of HM017 and R108, we developed and confirmed 318 Indel markers evenly distributed across the eight chromosomes of the M. truncatula genome. To validate the effectiveness of these markers, by employing the map-based cloning approach, we cloned the causative gene in the dwarf mutant crs isolated from the Tnt1 mutant population, identifying it as gibberellin 3-β-dioxygenase 1, using some of the confirmed Indel markers. The primer sequences and the size difference of each marker were made available for users in the web-based database. The identification of the crossing partner for R108 and the generation of Indel markers will enhance the forward genetics and the overall usage of the Tnt1 mutants.
尽管蒺藜苜蓿 Tnt1 突变体被豆科植物研究人员广泛使用，但由于可利用 Blast 可搜索的大规模侧翼序列标签数据库，它们主要用于反向遗传学。然而，这些突变体也应该被广泛用于正向遗传学筛选，由于缺乏与 Tnt1 突变体产生的 R108 基因型相兼容的遗传杂交伙伴，这种努力受到了阻碍。在这项研究中，我们选择了三个 Medicago HapMap 系（HM017、HM018 和 HM022），并与 R108 进行了正反交。在 F1 和 F2 后代的表型分析后，HM017 被鉴定为与 R108 相兼容的杂交伙伴。通过比较 HM017 和 R108 的组装基因组序列，我们开发并验证了 318 个均匀分布在蒺藜苜蓿基因组 8 条染色体上的 Indel 标记。为了验证这些标记的有效性，我们采用基于图谱的克隆方法，从 Tnt1 突变体群体中分离出的矮化突变体 crs 克隆了其原因基因，确定其为赤霉素 3-β-双加氧酶 1，使用了一些确认的 Indel 标记。这些标记的引物序列和每个标记的大小差异已在基于网络的数据库中提供给用户。R108 杂交伙伴的鉴定和 Indel 标记的产生将增强 Tnt1 突变体的正向遗传学和整体应用。
