Marker-Assisted Backcross Breeding of Drought-Tolerant Maize Lines Transformed by Vacuolar H+-Pyrophosphatase Gene (AnVP1) from Ammopiptanthus nanus

Maize is highly sensitive to water deficit but has high transpiration and biomass production, leading to a substantial water demand. Genetic engineering can overcome reproductive isolation and utilize drought-tolerant genes from distant species. <i>Ammopiptanthus nanus</i> is a relic of the Tertiary glaciation that can adapt to harsh environmental conditions. In our previous study, five maize homozygous T₈ lines overexpressing the <i>AnVP1</i> gene from <i>Ammopiptanthus nanus</i> were generated and showed the enhancement of drought tolerance. However, the recipient inbred line Zh-1 was poor in yield and agronomic performance. In the present study, the <i>AnVP1</i> gene was backcrossed from donor parent L10 (one of the five T₈ lines) into recurrent parent Chang 7-2 (one of the elite parents of the commercial hybrid Zhengdan 958). In total, 103 InDel markers were developed and used for assisted background selection. After two generations of foreground selection through glufosinate spraying, the detection of CP4 EPSP MAb1 strips, and the PCR amplification of the <i>AnVP1</i> gene, along with the similarity of agronomic traits to the recurrent parent, and background selection assisted by these InDel markers, the transgenic <i>AnVP1</i> gene became homozygous in the BC₂ lines. The average recovery rate of the genetic background of the recurrent parent reached 74.80% in the BC₁ population and 91.93% in the BC₂ population, respectively. The results of RT-PCR and RT-qPCR indicated the stable expression of the <i>AnVP1</i> gene in the two ultimately selected BC₂F₃ lines, BC₂-36-12 and BC₂-5-15. The drought tolerance of these two BC₂F₃ lines were significantly improved compared to the recurrent parent Chang 7-2, as revealed by their wilting phenotype and survival rate of seedlings. This improvement was related to the enhancement of water-retention ability, as indicated by higher RWC and the reduction in damage, as shown by the decrease in REL, MDA, and H₂O₂ under drought stress. The result of field evaluation in two arid and semi-arid environments indicated that the drought tolerance of Chang 7-2 was significantly improved. This study suggests that the improved Chang 7-2 can be crossed with Zheng 58 to develop the transgenic commercial hybrid Zhengdan 958.