Research Article |
Corresponding author: Kunbo Wang ( wkbcri@163.com ) Corresponding author: Fang Liu ( liufcri@163.com ) Academic editor: Alexander Belyayev
© 2017 Yuling Liu, Zhen Liu, Renhai Peng, Yuhong Wang, Zhongli Zhou, Xiaoyan Cai, Xingxing Wang, Zhenmei Zhang, Kunbo Wang, Fang Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Liu Y, Liu Z, Peng R, Wang Y, Zhou Z, Cai X, Wang X, Zhang Z, Wang K, Liu F (2017) Cytogenetic maps of homoeologous chromosomes A h01 and D h01 and their integration with the genome assembly in Gossypium hirsutum. Comparative Cytogenetics 11(2): 405-420. https://doi.org/10.3897/CompCytogen.v11i2.12824
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Cytogenetic maps of Gossypium hirsutum (Linnaeus, 1753) homoeologous chromosomes Ah01 and Dh01 were constructed by fluorescence in situ hybridization (FISH), using eleven homoeologous-chromosomes-shared bacterial artificial chromosomes (BACs) clones and one chromosome-specific BAC clone respectively. We compared the cytogenetic maps with the genetic linkage and draft genome assembly maps based on a standardized map unit, relative map position (RMP), which allowed a global view of the relationship of genetic and physical distances along each chromosome, and assembly quality of the draft genome assembly map. By integration of cytogenetic maps with sequence maps of the two chromosomes (Ah01 and Dh01), we inferred the locations of two scaffolds and speculated that some homologous sequences belonging to homoeologous chromosomes were removed as repetitiveness during the sequence assembly. The result offers molecular tools for cotton genomics research and also provides valuable information for the improvement of the draft genome assembly.
cotton, BAC, FISH, physical map, draft genome assembly
The genus Gossypium (Linnaeus, 1753) includes approximately 47 diploid species (2n = 2x = 26) that are divided into eight genome groups, named as A-G and K genome (
The uneven distribution of recombination events on chromosomes results in divergence between genetic distance and physical distance, which limits the application of genetic map in guiding genome sequence assembly and map-based cloning (
Tetraploid cotton contains too many chromosomes (2n = 4x = 52) and it is difficult to prepare chromosomes due to large amounts of secondary metabolites in cells. So research on cotton cytogenetic maps has lagged behind other crops. Moreover, previous cotton FISH mapping was mainly limited to the use of repetitive DNA (
Structure analysis of homoeologous chromosomes in allotetraploid cotton plays an important guiding role in sequence assembly, map-based cloning, and so on.
G. hirsutum (Linnaeus, 1753) accession TM-1 was used for cytological studies. BACs used for FISH mapping were identified by screening two genomic BAC libraries derived from G. herbaceum (Linnaeus, 1753) var. africenum (
The screening was performed using bacteria liquid-PCR according to the protocol previously described (
Chromosome preparation and FISH were conducted according to the previous protocols (
Slides were examined under a Zeiss Imager M1 microscope. Images were captured and merged using MetaSystems isis software with a CCD camera (MetaSystems CoolCube 1) attached to a Zeiss Imager M1 microscope. To determine physical positions of signals, only chromosomes without apparent morphological distortion were introduced and their physical positions of signals were measured using MetaSystems isis. Final image adjustments were performed using Adobe Photoshop CS3 software.
The RMP unit was used as standardized map unit for comparative analysis between different types of maps. The RMP values for the SSR linkage map were the percentage from the genetic location (cM) of each locus along the total length (cM) of the corresponding linkage group. The RMP values of the cytogenetic map were the percentage of the distance (μm) from the FISH signal site to the end of the short arm showed relative to the total length of the chromosome (μm) (
To construct the cytogenetic maps of chromosomes Ah01 and Dh01 of G. hirsutum, an initial set of 47 SSR markers shared by both chromosomes of Ah01 and Dh01 from a whole genome marker map (WGMM) (
SSR | BAC | Loc. in D-genome sequence | Chr.15 cM |
RMP (%)*2 | Loc. in tetraploid | |||
---|---|---|---|---|---|---|---|---|
Chr. | Start bp | End bp | ||||||
NAU2015 | 305A19 | Chr02 | 61962135 | 61962910 | 12.6 | 7.14 | Chr.01 | Chr.15 |
NAU3254 | 348I20 | Chr02 | 60694684 | 60699332 | 29.1 | 16.49 | Chr.01 | Chr.15 |
NAU2474 | 144E04 | Chr02 | 59155451 | 59156001 | 39.5 | 22.39 | Chr.01 | Chr.15 |
NAU3433 | 64M24 | Chr02 | 55462914 | 55463585 | 53.6 | 30.38 | Chr.01 | Chr.15 |
BNL2921 | 400N03 | Chr02 | 27353761 | 27353982 | 73.3 | 41.55 | Chr.01 | Chr.15 |
NAU4891 | 118G12 | Chr02 | 15429614 | 15428837 | 86.2 | 48.86 | Chr.01 | Chr.15 |
NAU3135 | 85P13 | Chr02 | 11717323 | 11717890 | 90.2 | 51.13 | Chr.01 | Chr.15 |
BNL3888b | 164I21 | Chr02 | 11188812 | 11189229 | 90.3 | 51.19 | Chr.01 | Chr.15 |
BNL3580 | 421E24 | Chr02 | 7879846 | 7880283 | 93.4 | 52.94 | Chr.01 | Chr.15 |
NAU4044 | 400L15 | Chr02 | 2312144 | 2313542 | 111.5 | 63.20 | Chr.01 | Chr.15 |
HAU076 | 378J07 | --*3 | -- | -- | -- | -- | Chr.01 | -- |
TMB0062 | 423C18 | --*3 | -- | -- | -- | -- | Chr.01 | -- |
SSR markers | BAC library | Screened BAC clones |
---|---|---|
HAU2861 | 1* | 22K17; 22L15; 22L18; 67J23; 75D24; 75E24; 108E08; 108E24; 130M09; 151C24; 151E18 |
NAU3433 | 1* | 41J08; 41K08; 46K02; 64M20; 64M24; 78G20; 78H20 |
NAU3053 | 1* | 22K18; 22L17; 67I12; 75C23; 75E24;107P10; 107P24 |
NAU4891 | 1* | 50H19; 51C14; 51H12; 56J17; 118G11; 118G12 |
Gh649 | 1* | 99L01; 136O19; 136P17 |
NAU2095 | 1* | 52B01 |
Gh216 | 1* | 50P23; 57I23; 79A06; 79A12; 79B07; 101K10; 101K12; 146P05 |
NAU5163 | 2* | 141H01; 158M07; 158N09; 158L08; 159L07; 159L08 |
BNL3888b | 2* | 164I21; 164I22 |
NAU3254 | 2* | 348I18; 348I20; 348I21; 348H17; 348J19 |
CIR049 | 2* | 256N07 |
BNL2921 | 2* | 400N03; 400L02 |
BNL3580 | 2* | 421E24 |
NAU2015 | 2* | 305A19 |
NAU4044 | 2* | 400L15 |
NAU2474 | 2* | 144E04; 165B11 |
NAU3135 | 2* | 85P13; 377G04; 377H05; 247P16; 247P17; 325M09; 325M10 |
TMB0062 | 2* | 298N21; 403A13; 423C18; 423C19; 424A12 |
HAU076 | 2* | 249G03; 249G04; 249I5; 325N10; 378J07; 398J05; 398H05; 249G05 |
By dual-color FISH on mitotic chromosomes, the order of the two BACs was determined along the chromosomes based on the genetic positions of their corresponding SSR markers. Results showed, among the 12 positive BAC clones, 11 BAC clones were homoeologous-specific BACs because they generated signals on both chromosomes of Ah01 and Dh01, indicating sequence homology between these BACs retained in Ah01 and Dh01 (Fig.
The order of two BACs on metaphase chromosome of G. hirsutum (AD1) TM-1 using Dual-color-FISH. a 305A19(green)/348I20(red) b 305A19 (green)/64M24(red) c 144E04 (red)/64M24(green) d 64M24 (red)/400N03(green) e 305A19 (red)/378J07(green) f 118G12(red)/164I24(green) g 423C18 (red)/400L15(green) h 85P13 (green)/400L15(red) i 85P13 (red)/421E24(green) j 85P13 (red)/164I24(green) k D1 (red)/118G12(green) l 378J07 (green)/ A1 (red). Bar = 5 µm.
The genetic distances of SSR markers associated with the corresponding BACs were also converted into the relative positions in the corresponding linkage map (Fig.
Physical locations of FISH-mapped BACs in G. hirsutum draft genome assembly and cytogenetic map.
BAC | SSR marker | Loc. in AD1*1 draft genome | Loc. in AD1 Cytogenetic map *3 | ||||
---|---|---|---|---|---|---|---|
No. of chromosome | Start (bp) | End (bp) | RMP(%)*2 | Dh01 RMP(%) | Ah01 RMP(%) | ||
305A19 | NAU2015 | Dh01 | 60681011 | 60681490 | 1.26 | 3.00±0 | 4.51±0.41 |
378J07 | HAU076 | Ah01 | 96488204 | 96488397 | 3.40 | / | 8.01±0.48 |
144E04 | NAU2474 | Dh01 | 57722851 | 57723034 | 6.07 | 4.33±0.47 | / |
scaffold183_A01 | 19925 | 20108 | / | / | 9.01±1.25 | ||
348I20 | NAU3254 | Dh01 | 59322542 | 59322834 | 3.47 | 8.33±0.47 | 10.02±0.51 |
64M24 | NAU3433 | Ah01 | 90268406 | 90268610 | 9.63 | / | 15.00±0.47 |
Dh01 | 53813626 | 53813830 | 12.44 | 11.33±1.24 | / | ||
400N03 | BNL2921 | Ah01 | 40133025 | 40133182 | 59.82 | 84.66±0.47 | 61.99±0.94 |
423C18 | TMB0062 | Ah01 | 17562250 | 17562499 | 82.42 | 70.66±5.24 | 74.11±0.36 |
118G12 | NAU4891 | Ah01 | 17991434 | 17991731 | 81.99 | 79.33±4.49 | 84.01±1.10 |
85P13 | NAU3135 | Ah01 | 11722545 | 11722728 | 88.26 | / | 88.07±0.19 |
Dh01 | 9387192 | 9387374 | 84.73 | 85.33±0.47 | / | ||
D1 | BNL3902*4 | Dh01 | 26803236 | 26803427 | 56.38 | 69.66±0.94 | / |
164I21 | BNL3888b | Ah01 | 11084705 | 11084886 | 88.90 | 88.66±1.69 | 90.98±0.27 |
421E24 | BNL3580 | Ah01 | 7078093 | 7078309 | 92.91 | 89.00±1.41 | 92.99±0.65 |
400L15 | NAU4044 | Ah01 | 2245730 | 2245951 | 97.75 | / | 96.01±1.19 |
scaffold3710_D01 | 109956 | 110177 | / | 90.33±1.24 | / |
To compare our cytogenetic maps directly to the draft genome assembly map (
Integrated cytogenetic /genome assembly maps of G. hirsutum Ah01/Dh01. a Relative map position of BACs mapping to Dh01 of the AD1-NBI draft genome b Cytogenetic map of G. hirsutum Chromosome Dh01 based on 12 BAC clones c Cytogenetic map of G. hirsutum Ah01 based on 12 BAC clones d Relative map position of BACs mapping to Ah01 of the AD1-NBI draft genome. Arrow-head in a and d represent the locations of scaffold3710_D01 and scaffold183_A01 in the draft genome (AD1-NBI) respectively.
In cotton, more than 30 genetic maps have been published, including several integrated maps with higher marker density (
In total, the integrated genetic and cytogenetic maps can serve as a template to facilitate sequence assembly, because the maps provided information on the distribution of genetic markers across chromosomes and the linkage gaps derived from recombination suppression.
As a typical allotetraploid, which contains two sub-genomes originating from related ancestor species with different genome sizes, G. hirsutum has been studied on its homoeologous chromosomes for a long time. Results revealed that fragment additivity (
The e-PCR can be used to search for sub-sequences that closely match the primers of SSRs, which can help to identify the genome positions of SSRs within the reference genome sequence (
Mis-assemblies are common when draft genome sequences have been generated by de novo assembly of sequences obtained with NGS technologies (
Here, we constructed the cytogenetic maps of homoeologous chromosomes Ah01 and Dh01 using shared-BACs. By integration of cytogenetic maps and the cytogenetic and genome assembly maps, we identified the positions of two scaffolds in chromosome (Fig.
We demonstrated concordant orders and RMP of markers between the sequence map and physical map based on FISH. By integration of cytogenetic maps with sequence maps of the two chromosomes, we inferred the locations of the two scaffolds, and speculated some homologous sequences belonging to homoeologous chromosomes were removed as repetitiveness during the process of sequence assembly. Our study not only offers molecular tools for cotton genomics research, but also provides valuable information for the improvement of the draft genome assembly.
We deeply thank Prof. Tianzhen Zhang (Nanjing Agricultural University, China) for providing the chromosome-specific BAC clones, Prof. Zhiying Ma (Heibei Agricultural University, China) for supplying the BAC library. The research was sponsored by a grant from the National Natural Science Foundation of China (No. 31471548), PhD Scientific Research Fund of Anyang Institute of Technology (No. BSJ2016005), State Key Laboratory of Cotton Biology Open Fund (No. CB2017A06), State Key Laboratory of Cotton Biology Open Fund (No. CB2015A21).