A Rapid, Non-destructive and Continuous Sampling Technique and DNA Extraction for Soybean Seed

doi:10.11983/CBB20095

Abstract

Abstract: Establishment of a simple, quick, non-destructive, and continuous sampling procedure, can save planting cost and accelerate gene functional analysis and breeding procedures. In this study, we created a rapid, non-destructive, and continuous sampling technique using micro electric driller and air pump. We also optimized the high throughput DNA extraction method for the 384 deep-well plate and genotyping method. Furthermore, this technique could be applied in rice, maize and other crops for seed sampling and high throughput genotyping.

Key words: soybean (Glycine max), seed, drilling, continuing sampling, 384 deep-well plate, genotyping

Zhengjun Xia, Yuzhuo Li, Jinlong Zhu, Hongyan Wu, Kun Xu, Hong Zhai. A Rapid, Non-destructive and Continuous Sampling Technique and DNA Extraction for Soybean Seed[J]. Chinese Bulletin of Botany, 2021, 56(1): 56-61.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20095

https://www.chinbullbotany.com/EN/Y2021/V56/I1/56

Figures/Tables 4

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.

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).

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).

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.

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