图1 种子钻孔和样品采集过程 (A) 种子组织的钻取; (B) 移液枪头收集种子组织; (C) 钻取种子组织并转移至384深孔板中; (D) 一列取样完成后贴胶带保护; (E) 钻头的清洁; (F) 移液枪头和手套的清洁; (G) 钻孔后的大豆种子; (H) 钻孔大豆种子播种后14天的幼苗。中间部分为整体流程简图, 种子取样经钻取、转移和清洁3个步骤, 再进行下一个种子的钻取。

Figure 1 Procedure for seed drilling and sample collection (A) Drilling of seed tissue; (B) Dilled tissue was collected by a pipette tip; (C) Transferring into a 384 deep-well plate; (D) When all wells in a row of the 384 deep-well plate were filled, the row will be sealed with a sticky tape; (E) Cleaning of the driller; (F) Cleaning of the tip and gloves; (G) Appearance of drilled seed; (H) Seedling of drilled seed (14 days after being sown). In the middle panel, the whole procedure is shown, in which seed sampling is performed by drilling, transferring and cleaning before proceeding to the next one.

图2 大豆遗传群体经种子钻取及DNA制备后的基因型鉴定 利用Satt557和S8两对引物进行单管PCR能准确鉴定两分子标记间的重组个体(箭头)。M: 分子质量标准品ΦX174 HaeIII; 1-19: Harosoy-E1与Harosoy (e1)杂交的F2群体(在每个泳道底部标注有每个个体2个分子标记的基因型)。

Figure 2 Genotyping of a soybean genetic population through seed-drilling and thereafter DNA extraction Accurate identification of recombinant occuring between two markers, Satt557 and S8, using one tube PCR (arrow). M: Molecular weight marker ΦX174 HaeIII; 1-19: F2 population that were derived from Harosoy-E1 × Harosoy (e1) (the genotypes of each individual were indicated at the bottom of each lane).

图3 种子钻孔法鉴定结果的验证 钻孔的大豆种子出苗后, 提取其叶片DNA进行相关引物的基因型鉴定, 以验证种子基因型鉴定结果的准确性。上部和下部凝胶图分别为Satt557和S8的验证结果(底部标注了2个分子标记的基因型)。

Figure 3 Verification of genotyping results obtained through seed-drilling DNA were extracted from leaves of the plants that were developed from the drilled seeds, thereafter genotyping of Satt557 and S8 were performed to verify the accuracy of genotyping data of seed. The upper and the bottom gels are shown the genotyping result of Satt557 and S8, respectively (the genotypes of each individual were indicated at the bottom of each lane).

图4 水稻、大豆和玉米种子大小及对应钻头型号 (A) 水稻种子(平均钻孔深度为(1.52±0.12) mm, 直径0.9 mm的钻头获取的平均组织重量为(1.54±0.12) mg, 直径1.1 mm的钻头获取的平均组织重量为(2.30±0.18) mg); (B) 小粒大豆种子(平均钻孔深度为(3.88±0.37) mm, 直径1.1 mm的钻头获取的平均组织重量为(5.85±0.99) mg, 直径1.2 mm的钻头获取的平均组织重量为(6.96±0.66) mg); (C) 大粒大豆种子(平均钻孔深度为(4.63±0.43) mm, 直径1.2 mm的钻头获取的平均组织重量为(8.31±0.79) mg, 直径1.3 mm的钻头获取的平均组织重量为(9.75±0.92) mg)。(D) 玉米种子(平均钻孔深度为(3.78±0.46) mm, 直径1.2 mm的钻头获取的平均组织重量为(6.79±0.83) mg, 直径1.3 mm的钻头获取的平均组织重量为(7.97±0.98) mg)。

Figure 4 The seed dimensions of rice, soybean and maize and the selection of their appropriate drillers (A) Rice seed (average drilling depth is (1.52±0.12) mm; average acquired tissue weight is (1.54±0.12) mg for the 0.9 mm diameter driller; average acquired tissue weight is (2.30±0.18) mg for the 1.1 mm diameter driller). (B) Soybean seed (small size) (average drilling depth is (3.88±0.37) mm; average acquired tissue weight is (5.85±0.99) mg for the 1.1 mm diameter driller; average acquired tissue weight is (6.96±0.66) mg for the 1.2 mm diameter driller). (C) Soybean seed (large size) (average drilling depth is (4.63±0.43) mm; average acquired tissue weight is (8.31±0.79) mg for the 1.2 mm diameter driller; average acquired tissue weight is (9.75±0.92) mg for the 1.3 mm diameter driller). (D) Maize seed (average drilling depth is (3.78±0.46) mm; average acquired tissue weight is (6.79±0.83) mg for the 1.2 mm diameter driller; average acquired tissue weight is (7.97±0.98) mg for the 1.3 mm diameter driller.