Identification of salt stress-tolerant candidate genes in the BC2F2 population at the seedling stages of G. hirsutum and G. darwinii using NGS-based bulked segregant analysis

Muhammad Shehzad; Allah Ditta; Xiaoyan Cai; Shafeeq Ur Rahman; Yanchao Xu; Kunbo Wang; Zhongli Zhou; Liu Fang

doi:10.3389/fpls.2023.1125805

Identification of salt stress-tolerant candidate genes in the BC₂F₂ population at the seedling stages of G. hirsutum and G. darwinii using NGS-based bulked segregant analysis

Front Plant Sci. 2023 Jul 3:14:1125805. doi: 10.3389/fpls.2023.1125805. eCollection 2023.

Authors

Muhammad Shehzad¹, Allah Ditta^{1

2}, Xiaoyan Cai^{1

3}, Shafeeq Ur Rahman⁴, Yanchao Xu^{1

3}, Kunbo Wang¹, Zhongli Zhou¹, Liu Fang^{1

3

5}

Affiliations

¹ State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, Henan, China.
² Plant Breeding and Genetics Division, Cotton Group, Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Punjab, Pakistan.
³ National Nanfan Research Institute of Chinese Academy of Agriculture Sciences, Sanya, China.
⁴ MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing, China.
⁵ School of Agricultural Sciences, Zhengzhou University, Zhengzhou, Henan, China.

Abstract

Salinity is a major threat to the yield and productivity of cotton seedlings. In the present study, we developed a BC₂F₂ population of cotton plants from Gossypium darwinii (5-7) and Gossypium hirsutum (CCRI 12-4) salt-susceptible parents to identify salt-resistant candidate genes. The Illumina HiSeq™ strategy was used with bulked segregant analysis. Salt-resistant and salt-susceptible DNA bulks were pooled by using 30 plants from a BC₂F₂ population. Next-generation sequencing (NGS) technology was used for the sequencing of parents and both bulks. Four significant genomic regions were identified: the first genomic region was located on chromosome 18 (1.86 Mb), the second and third genomic regions were on chromosome 25 (1.06 Mb and 1.94 Mb, respectively), and the fourth was on chromosome 8 (1.41 Mb). The reads of bulk1 and bulk2 were aligned to the G. darwinii and G. hirsutum genomes, respectively, leading to the identification of 20,664,007 single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels). After the screening, 6,573 polymorphic markers were obtained after filtration of the candidate regions. The SNP indices in resistant and susceptible bulks and Δ(SNP-index) values of resistant and susceptible bulks were measured. Based on the higher Δ(SNP-index) value, six effective polymorphic SNPs were selected in a different chromosome. Six effective SNPs were linked to five candidate genes in four genomic regions. Further validation of these five candidate genes was carried out using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), resulting in an expression profile that showed two highly upregulated genes in the salt-tolerant species G. darwinii, i.e., Gohir.D05G367800 and Gohir.D12G239100; however, the opposite was shown in G. hirsutum, for which all genes, except one, showed partial expression. The results indicated that Gohir.D05G367800 and Gohir.D12G239100 may be salt-tolerant genes. We are confident that this study could be helpful for the cloning, transformation, and development of salt-resistant cotton varieties.

Keywords: bulked segregant analysis; candidate gene; cotton; polymorphic markers; salt stress.

Grants and funding

The National Key R&D Program of China (2021YFE0101200), National Natural Science Foundation of China (32171994, 32272090, 32072023), the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-88) and Nanfan Special Project of National Nanfan Research Institute of Chinese Academy of Agriculture Sciences (YBXM2309) were funded to this research.