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. 2017 May 18:154:13.
doi: 10.1186/s41065-017-0035-3. eCollection 2017.

Microdissection of the Ah01 chromosome in upland cotton and microcloning of resistance gene anologs from the single chromosome

Xinchuan Cao #  1   2 , Yuling Liu #  2   3 , Zhen Liu  3 , Fang Liu  2 , Yalei Wu  4 , Zhongli Zhou  2 , Xiaoyan Cai  2 , Xingxing Wang  2 , Zhenmei Zhang  2 , Yuhong Wang  2 , Zhimin Luo  3 , Renhai Peng  2   3 , Kunbo Wang  2
Affiliations

Microdissection of the Ah01 chromosome in upland cotton and microcloning of resistance gene anologs from the single chromosome

Xinchuan Cao et al. Hereditas. .

Abstract

Background: Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding.

Results: Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton.

Conclusions: Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.

Keywords: Chromosome microdissection; Microcloning; RGA; Upland cotton.

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Figures

Fig. 1
Fig. 1
Microdissection and collection of single mono-chromosomes by CellCut Plus laser manipulation. a Film-slide preparations of meiotic metaphase I chromosomes with one monomer chromosome (Ah01). b Film-slide preparations of meiotic metaphase I chromosomes with one microdissected chromosome. c The microdissected chromosome on the cap of a collection tube. Arrow indicates the Ah01 chromosome. Bar: 5 μm
Fig. 2
Fig. 2
Agarose gel electrophoresis of LA-PCR products. 1, 2: Negative controls 3 Product from the first round LA-PCR. 4, 7: Positive controls. 5, 6: Products from the second round LA-PCR. M: DNA marker
Fig. 3
Fig. 3
Southern blotting of products from the second round LA-PCR. 1: The negative control. 2, 3: Positive controls. 4, 5, 6: The second round LA-PCR products. 7: EcoRI digested genomic DNA. M: DNA marker
Fig. 4
Fig. 4
PAGE of SSR primer amplification product from single chromosome pool. 1–9: SSR primer amplification products from partial other chromosomes pool with Ah01 chromosome specific primer. 10: SSR primer amplification products from single chromosome pool with chromosome Ah01 special primer (arrow indicated). 11: The negative control. 12: The positive control. M: DNA marker
Fig. 5
Fig. 5
FISH signals of products from the second round LA-PCR. a Chromosomes stained with DAPI. b Signals fromproducts of LA-PCR II (red). c Signals fromchromosome Ah01 specific BAC (green, arrow indicated). d Signals from dual-FISH
Fig. 6
Fig. 6
Agarose gel electrophoresis (a) and Southern blotting (b) of P1/P2 primer PCR products. A-1, B-1: Negative controls. A-2, B-2: Single Ah01 chromosome as DNA template. A-3, B-3: Positive controls using 10 pg G. hirsutum genomic DNA as template. B-4: EcoRI digested genomic DNA of G. hirsutum. M: DNA marker
Fig. 7
Fig. 7
Cluster analysis of single chromosome RGA nucleotide sequenceswith those from other species
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