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Anchoring alien chromosome segment substitutions bearing gene(s) for resistance to mustard aphid in Brassica juncea-B. fruticulosa introgression lines and their possible disruption through gamma irradiation

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Abstract

Key message

Heavy doses of gamma irradiation can reduce linkage drag by disrupting large sized alien translocations and promoting exchanges between crop and wild genomes.

Abstract

Resistance to mustard aphid (Lipaphis erysimi) infestation was significantly improved in Brassica juncea through B. juncea-B. fruticulosa introgression. However, linkage drag caused by introgressed chromatin fragments has so far prevented the deployment of this resistance source in commercial cultivars. We investigated the patterns of donor chromatin segment substitutions in the introgression lines (ILs) through genomic in situ hybridization (GISH) coupled with B. juncea chromosome-specific oligonucleotide probes. These allowed identification of large chromosome translocations from B. fruticulosa in the terminal regions of chromosomes A05, B02, B03 and B04 in three founder ILs (AD-64, 101 and 104). Only AD-101 carried an additional translocation at the sub-terminal to intercalary position in both homologues of chromosome A01. We validated these translocations with a reciprocal blast hit analysis using shotgun sequencing of three ILs and species-specific contigs/scaffolds (kb sized) from a de novo assembly of B. fruticulosa. Alien segment substitution on chromosome A05 could not be validated. Current studies also endeavoured to break linkage drag by exposing seeds to a heavy dose (200kR) of gamma radiation. Reduction in the size of introgressed chromatin fragments was observed in many M3 plants. There was a complete loss of the alien chromosome fragment in one instance. A few M3 plants with novel patterns of chromosome segment substitutions displayed improved agronomic performance coupled with resistance to mustard aphid. SNPs in such genomic spaces should aid the development of markers to track introgressed DNA and allow application in plant breeding.

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Availability of data and material

Raw reads from sequencing data of the founder ILs are available at National Centre for Biotechnology Information as Bio-Project PRJNA662836 with bio sample IDs as SAMN18236321-23. Supply of plant materials will require prior approval from Biodiversity Authority of India.

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Acknowledgements

The research was carried with financial assistance from four different projects. Development of basic germplasm and sequencing of introgression lines were carried out with financial support from the ICAR National Professor Chair Project “Broadening the genetic base of Indian mustard (Brassica juncea) through alien introgressions and germplasm enhancement” funded by Indian Council of Agricultural Research (ICAR). Molecular cytogenetic experiments were conducted with financial assistance from Department of Biotechnology, Government of India, in the form of Centre of Excellence and Innovation in Biotechnology “Germplasm enhancement for crop architecture and defensive traits in Brassica”. Whole genome sequencing of Brassica fruticulosa was carried out with financial assistance from National Agricultural Science Fund aided project “Creating a fully characterized genetic resource pipeline for mustard improvement programme in India”. Part of the work was supported under the Newton-Bhabha Fund UK-India Pulses and Oilseeds Research Initiative, with funding from UK's Official Development Assistance Newton Fund awarded by UK Biotechnology and Biological Sciences Research Council (BB/R019819/1). Neha Agrawal acknowledges financial support from Jawaharlal Nehru Memorial Fund for the award of JNMF fellowship during one year of the studies.

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  1. Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, 141004, India

    Neha Agrawal, Mehak Gupta, Chhaya Atri, Javed Akhatar, Sarwan Kumar & Surinder S. Banga

  2. Department of Genetics and Genome Biology, University of Leicester, Leicester, UK

    Pat (J. S.) Heslop-Harrison

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  1. Neha Agrawal
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Contributions

S.S.B., N.A. and P.H.H. designed the research. S.S.B. and C.A. developed and maintained the introgression lines. N.A. and M.G. conducted cytogenetic studies. J.A. and N.A. carried out bioinformatics analysis. N.A and M.G interpreted the data and wrote the paper. S.S.B. and P.H.H. edited the manuscript and supervised the studies. All authors have read and approved this version of manuscript.

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Correspondence to Surinder S. Banga.

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Supplementary Information

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Supplementary file1 (DOCX 13 kb)

122_2021_3886_MOESM2_ESM.jpg

Multicolour in situ hybridization on mitotic chromosome spreads of (a-c) AD-101 and (d-f) AD-64/AD-104 using (a, d) B. fruticulosa genomic DNA (green) and Cent Br2 (Red), (b, e) B. nigra genomic DNA and (c, f) three oligo libraries.

Supplementary file2 (JPG 1971 kb)

122_2021_3886_MOESM3_ESM.jpg

Multicolour in situ hybridization on mitotic chromosome spreads of control non-introgressed euploid B. juncea B. juncea using (a) B. nigra genomic DNA (green) (b) B. fruticulosa genomic DNA (green) and Cent Br2 (Red) showing no hybridization signals from B. fruticulosa.)

Supplementary file3 (JPG 1232 kb)

122_2021_3886_MOESM4_ESM.tif

SNP density at the predicted sites introgressed fragments on chromosomes A01, B02, B03 and B04 of identified introgression lines compared to B. juncea recipient parent. SNP density (within 1 Mb window) was estimated and plotted by r-package “CMplot” (https://github.com/YinLiLin/CMplot).Chromosome cartoons reflecting SNP density were drawn manually.(TIFF 8507 kb)

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Agrawal, N., Gupta, M., Atri, C. et al. Anchoring alien chromosome segment substitutions bearing gene(s) for resistance to mustard aphid in Brassica juncea-B. fruticulosa introgression lines and their possible disruption through gamma irradiation. Theor Appl Genet 134, 3209–3224 (2021). https://doi.org/10.1007/s00122-021-03886-z

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