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Journal of Integrative Agriculture 2016, 15(11): 2488–2496 RESEARCH ARTICLE Available online at www.sciencedirect.com ScienceDirect Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut DU Pei 1, 2* , LI Li-na 2, 3* , ZHANG Zhong-xin 2 , LIU Hua 2 , QIN Li 2 , HUANG Bing-yan 2 , DONG Wen-zhao 2 , TANG Feng-shou 2 , QI Zeng-jun 1 , ZHANG Xin-you 2 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, P.R.China 2 Industrial Crops Research Institute, Henan Academy of Agricultural Sciences/Key Laboratory of Oil Crops in Huang-Huai-Hai Plains, Ministry of Agriculture/Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou 450002, P.R.China 3 College of Agriculture, Henan University of Science and Technology, Luoyang 471023, P.R.China Abstract Interspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are (TTTAGGG) n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats (ITRs) are also commonly identified. In this study, the telomeric repeat was chromo- somally localized in 10 Arachis species through sequential GISH (genomic in situ hybridization) and FISH (fluorescence in situ hybridization) combined with 4’,6-diamidino-2-phenylindole (DAPI) staining. Six ITRs were identified such as in the centromeric region of chromosome B i 5 in Arachis ipaënsis, pericentromeric regions of chromosomes A s 5 in A. steno- sperma, B ho 7 in A. hoehnei and A v 5 in A. villosa, nucleolar organizer regions of chromosomes A s 3 in A. stenosperma and A di 3 in A. diogoi, subtelomeric regions of chromosomes B ho 9 in A. hoehnei and A du 7 in A. duranensis, and telomeric region of chromosome E s 7 in A. stenophylla. The distributions of the telomeric repeat, 5S rDNA, 45S rDNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S rDNA and 45S rDNA sites in this study, along with inversions detected on the long arms of chromosomes K b 10 and B ho 10, indicated frequent chromosomal rearrangements during evolution of Arachis species. Keywords: Arachis species, inversion, interstitial telomeric repeats, karyotype 1. Introduction The telomere is a specific DNA-protein structure with func- tions of regulating cell-senescence and carcinogenesis (Au- bert and Lansdorp 2008). It also protects chromosomes from exonuclease digestion (Blackburn 1991). In both animals and plants, telomere repeats generally comprise tandemly repeated DNA sequences of (TTAGGG) n and (TTTAGGG) n (Burr et al. 1992; Fajkus et al. 2005). Received 1 December, 2015 Accepted 24 May, 2016 Correspondence QI Zeng-jun, Tel: +86-25-84399029, Fax: +86- 25-84395344, E-mail: [email protected]; ZHANG Xin-you, Tel: +86-371-65729560, E-mail: [email protected] * These authors contributed equally to this study. © 2016, CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(16)61423-5
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Chromosome painting of telomeric repeats reveals new ... · Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut DU Pei1, 2*, LI Li-na2, 3*,

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Page 1: Chromosome painting of telomeric repeats reveals new ... · Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut DU Pei1, 2*, LI Li-na2, 3*,

Journal of Integrative Agriculture 2016, 15(11): 2488–2496

RESEARCH ARTICLE

Available online at www.sciencedirect.com

ScienceDirect

Chromosome painting of telomeric repeats reveals new evidence for genome evolution in peanut

DU Pei1, 2*, LI Li-na2, 3*, ZHANG Zhong-xin2, LIU Hua2, QIN Li2, HUANG Bing-yan2, DONG Wen-zhao2, TANG Feng-shou2, QI Zeng-jun1, ZHANG Xin-you2

1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, P.R.China2Industrial Crops Research Institute, Henan Academy of Agricultural Sciences/Key Laboratory of Oil Crops in Huang-Huai-Hai Plains, Ministry of Agriculture/Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou 450002, P.R.China

3College of Agriculture, Henan University of Science and Technology, Luoyang 471023, P.R.China

AbstractInterspecific hybridization is an important approach to improve cultivated peanut varieties. Cytological markers such as tandem repeats will facilitate alien gene introgression in peanut. Telomeric repeats have also been frequently used in chromosome research. Most plant telomeric repeats are (TTTAGGG)n that are mainly distributed at the chromosome ends, although interstitial telomeric repeats (ITRs) are also commonly identified. In this study, the telomeric repeat was chromo-somally localized in 10 Arachis species through sequential GISH (genomic in situ hybridization) and FISH (fluorescence in situ hybridization) combined with 4’,6-diamidino-2-phenylindole (DAPI) staining. Six ITRs were identified such as in the centromeric region of chromosome Bi5 in Arachis ipaënsis, pericentromeric regions of chromosomes As5 in A. steno-sperma, Bho7 in A. hoehnei and Av5 in A. villosa, nucleolar organizer regions of chromosomes As3 in A. stenosperma and Adi3 in A. diogoi, subtelomeric regions of chromosomes Bho9 in A. hoehnei and Adu7 in A. duranensis, and telomeric region of chromosome Es7 in A. stenophylla. The distributions of the telomeric repeat, 5S rDNA, 45S rDNA and DAPI staining pattern provided not only ways of distinguishing different chromosomes, but also karyotypes with a higher resolution that could be used in evolutionary genome research. The distribution of telomeric repeats, 5S rDNA and 45S rDNA sites in this study, along with inversions detected on the long arms of chromosomes Kb10 and Bho10, indicated frequent chromosomal rearrangements during evolution of Arachis species.

Keywords: Arachis species, inversion, interstitial telomeric repeats, karyotype

1. Introduction

The telomere is a specific DNA-protein structure with func-tions of regulating cell-senescence and carcinogenesis (Au-bert and Lansdorp 2008). It also protects chromosomes from exonuclease digestion (Blackburn 1991). In both animals and plants, telomere repeats generally comprise tandemly repeated DNA sequences of (TTAGGG)n and (TTTAGGG)n (Burr et al. 1992; Fajkus et al. 2005).

Received 1 December, 2015 Accepted 24 May, 2016Correspondence QI Zeng-jun, Tel: +86-25-84399029, Fax: +86-25-84395344, E-mail: [email protected]; ZHANG Xin-you, Tel: +86-371-65729560, E-mail: [email protected]*These authors contributed equally to this study.

© 2016, CAAS. Published by Elsevier Ltd. This is an open access art ic le under the CC BY-NC-ND l icense (http:/ /creativecommons.org/licenses/by-nc-nd/4.0/)doi: 10.1016/S2095-3119(16)61423-5

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2489DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

Telomeric repeats are generally presented at chromo-some ends. Nevertheless, interstitial telomeric repeats (ITRs) occur in plants (Regad et al. 1994) and animals (Nergadze et al. 2004; Mattos et al. 2014; Scacchetti et al. 2015), including human (Park et al. 1992). In plants, ITRs are found at different chromosomes locations, including centromeric, rDNA, subtelomeric and telomeric regions (Gortner et al. 1998; Tek and Jiang 2004; Presting et al. 1996). Further analysis revealed that ITRs involved chro-mosome breakage, recombination and amplification (Lin and Yan 2008; Bolzan 2012).

Peanut (Arachis hypogaea, 2n=4x=40, genome AABB) is one of the most important food and forage crops in the world. Previous studies identified 80 annual and perennial species of Arachis in nine taxonomic sections, including Arachis (A, B and D), Caulorrhizae (C), Erectoides (E), Extranervosae (Ex), Heteranthae (H), Procumbentes (P), Trierectoides (Trie), Triseminatae (Tris) and Rhizomatosae (R) (Fernández and Krapovickas 1994; Krapovickas and Gregory 1994; Valls and Simpson 2005). However, the classification and phylogeny of many Arachis species are still in a state of flux. For example, FISH (fluorescence in situ hybridization) analysis using 45S and 5S rDNA and heterochromatin as probes led to re-designation of the genomes of A. benensis and A. trinitensis as F, those of A. batizocoi, A. cruziana and A. krapovickasii as K, and those of A. decora, A. palustris and A. praecox as G (Robledo and Seijo 2010; Silvestri et al. 2014). However, the P genome of A. chiquitana (Mallikarjuna 2005) was re-grouped into the A-genome due to its smallest “chromosome A” and similar patterns of 45S and 5S rDNA and heterochromatin as shown in other A-genome species (Robledo et al. 2009).

Telomeric repeats were identified through FISH in many species (Okazaki et al. 1993; Meyne et al. 1995). To our knowledge, the only ITR analysis using FISH analysis in peanut was conducted by Zhang (2013) in the species A. hypogaea, A. ipaënsis, and A. duranensis, with six ITRs

being identified in the species A. hypogaea and A. ipaënsis, and none in A. duranensis. In the current study, ITRs from 10 wild Arachis species were analyzed by FISH using telo-meric repeat probes combining with GISH (genomic in situ hybridization), 5S and 45S rDNA FISH and DAPI staining to: 1) characterize telomeric repeat distributions in Arachis species; 2) develop karyotypes with a higher resolution in these species; and 3) reveal new genomic evidence for evolution of Arachis species.

2. Materials and methods

2.1. Plant materials

Ten Arachis species, A. hypogaea, A. ipaënsis, A. duranen-sis, A. chiquitana, A. stenosperma, A. diogoi, A. villosa, A. batizocoi, A. hoehnei and A. stenophylla, were used in the study. The accession number, chromosome number and genome constitutions of the 10 Arachis species were provided in Table 1.

2.2. Chromosome preparation

Seeds of the 10 Arachis species were germinated on moist filter paper at 25°C for 7 d. Healthy lateral root tips were excised and pretreated with 2 mmol L-1 8-hydroxyquinoline for 3 h at 25°C, and fixed in absolute ethanol (3):glacial acetic acid (1) for 12 h at 4°C. Then, 0.3–0.5 mm of root tips meristem was excised and squashed in 45% glacial acetic acid and freezed at –80°C for 12 h. Thus, the spread chromosomes were dehydrated in 100% ethanol and dried in air after the cover slips were removed.

2.3. Cytogenetic analysis

Total genomic DNA was extracted from young fresh leaves of A. ipaënsis (2n=2x=20, BB) (Wang et al. 2002). The

Table 1 Plant materials used in this study and their chromosome constitution

Species Accession no.

Plant introduction (PI) no.

Chromosome no. Genome (references)

Arachis chiquitana 36 027 PI 476006 2n=2x=20 AA (Robledo et al. 2009)A. stenosperma 410 PI 338280 2n=2x=20 AA ( Robledo et al. 2009)A. diogoi 10 602 PI 276235 2n=2x=20 AA (Robledo et al. 2009)A. villosa 22 585 PI 298636 2n=2x=20 AA (Robledo et al. 2009)A. duranensis 7 988 PI 219823 2n=2x=20 AA (Robledo et al. 2009)A. hypogaea Z5 297 PI 319768 2n=4x=40 AABB (Seijo et al. 2004)A. ipaënsis 30 076 PI 468322 2n=2x=20 BB (Robledo and Seijo 2010)A. batizocoi 9 484 PI 298639 2n=2x=20 KK (Robledo and Seijo 2010)A. hoehnei 30 006 PI 468150 2n=2x=20 BB (Krapovickas and Gregory 1994; Holbrook and Stalker 2003)A. stenophylla 30 136 PI 468178 2n=2x=20 EE (Krapovickas and Gregory 1994; Holbrook and Stalker 2003)

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2490 DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

primer pair, F-5´-TTTAGGGTTTAGGGTTTAGGGTTTAG GGTTTAGGG-3´ and R-5´-AAATCCCAAATCCCAAATC CCAAATCCCAAATCCC-3´), modified from the primers using in amplifying human telomeric repeats (Ijdo et al. 1991), were used for specific amplification of plant telomeric repeats (TTTAGGG)n. Two clones of 5S and 45S rDNA of wheat (Triticum aestivum L.) used by Du et al. (2015), were provided by Dr. Bikram S Gill, Kansas State Univer-sity, USA. The 5S rDNA and (TTTAGGG)n were labeled with biotin-16-dUTP (Roche, Mannheim, Germany) by nick translation and detected with fluorescein anti-biotin (Roche, Mannheim, Germany). And 45S rDNA and total genomic DNA of A. ipaënsis were labeled with digoxigenin-11-dUTP (Roche, Mannheim, Germany) and detected with anti-digoxigenin-rhodamine (Roche, Mannheim, Germany).

Sequential GISH/FISH was performed as described by Sepsi et al. (2008). Briefly, the Arachis chromosomes were identified by FISH and GISH. Where, FISH using 5S and 45S rDNA as probes combined with 4,6-diamidi-no-2-phenylindole (DAPI) staining; GISH using total genomic DNA of A. ipaënsis as probe, was used to distinguish A- and B-genome chromosomes. And FISH with the telomeric re-peats as probes were used to characterize the distribution of (TTTAGGG)n in Arachis chromosomes.

2.4. Imaging analysis

Chromosomes were photographed with a DMRX fluores-cence microscope (Leica, Germany). The merged images were further processed using Photoshop 6.0 for brightness, contrast, and intensity. To distinguish the signals produced by different probes in each chromosome after sequential FISH/GISH, the original green signal of 5S rDNA was con-verted to pseudo-color white, and the red signal of GISH converted to fuchsia, while the red signal of 45S rDNA and the green of telomeric repeat (TTTAGGG)n remained.

2.5. Karyotypic analysis

Chromosome size was measured in at least five metaphase cells of each species using the Image-Pro Plus 6.0 system. The centromeric index (i=Short arm length×100/Chromo-some length) was calculated and used to classify the chro-mosomes as described by Levan et al. (1964). Then chro-mosomes were grouped into metacentric (m, i=50–37.51), submetacentric (sm, i=37.50–25.01) and subtelocentric (st, i=25–12.51) based on the index. Mean karyotype value of each species was represented in ideograms. Chromosomes of the 10 Arachis species were distinguished according to Seijo et al. (2004) combined with the characterised distri-butions of telomeric repeats.

3. Results

3.1. Preliminary karyotype analysis in 10 Arachis species

Preliminary karyotypes of the tested Arachis species in this study were based on the distributions of 5S rDNA and 45S rDNA and the staining patterns of GISH and DAPI through sequential GISH/FISH (Fig. 1 and Appendixes A, B, C and D). The karyotypes were then used for chromosome mapping of telomeric repeats. Six Arachis karyotypes in-cluding A. batizocoi, A. diogoi, A. duranensis, A. hoehnei, A. hypogaea and A. ipaënsis agreed well with previous reports (Seijo et al. 2004; Robledo et al. 2009, 2010; Custó-dio et al. 2013). The karyotypes of A. batizocoi, A. diogoi, A. duranensis and A. hoehnei were same as previous reports

A B C

D E F

G H I

J K L

10 μm

10 μm

10 μm10 μm

10 μm10 μm

10 μm10 μm 10 μm

10 μm10 μm10 μm

Fig. 1 Sequential fluorescence in situ hybridization (FISH) of Arachis stenophylla (A-F) and A. batizocoi (G-L). A and G, 4’,6-diamidino-2-phenylindole (DAPI) staining (gray white). B and H, DAPI staining (blue). C and I, 5S rDNA labeled with biotin-16-dUTP and detected with fluorescein anti-biotin (green). D and J, 45S rDNA labeled with digoxigenin-11-dUTP and detected with anti-digoxigenin-rhodamine (red). E and K, (TTTAGGG)n labeled with biotin-16-dUTP and detected with fluorescein anti-biotin (green). F and L, merging of B to E and G to K. White, original green signal of C and I converted to pseudo white color shows 5S rDNA; red shows 45S rDNA; green shows the telomeric repeats (TTTAGGG)n.

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2491DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

(Fig. 2 and Appendix E-a, b, c and d), whereas karyotypes of A. hypogaea cultivar Z5297 and A. ipaënsis were similar to previous reports, and the only difference was shown by the DAPI staining patterns. Although DAPI bands that were previously observed only in A-genome chromosomes (Seijo et al. 2004; Robledo and Seijo 2010), clear DAPI bands were also observed on seven B-genome chromosomes (Fig. 2 and Appendix E-e and f).

Almost all chromosomes of the E-genome of A. steno-phylla, showed clear DAPI bands; Es4, Es10 and Es9 had submetacentric and Es8 had subtelomeric bands. Chromo-some Es4 showed signals of 45S rDNA and Es10 had signals of 5S and 45S rDNA (Fig. 2 and Appendix E-g).

Compared to findings in Robledo et al. (2009), differenc-es were seen on chromosomes 4 and 7 in A. chiquitana, A. villosa and A. stenosperma. In A. chiquitana and A. villosa, heterogeneous distributions of 45S rDNA sites were presented on chromosome 7. For example, the 45S rDNA site on one chromosome Ac7 was much larger than the site on its homologous partner in A. chiquitana, while in A. villosa, only one homolog chromosome Av7 carried a 45S rDNA site (Fig. 2 and Appendix E-h and i). In A. stenosperma, a new but weak 45S rDNA site was ob-served on chromosome As4 in addition to sites on chromo-somes As2, As7 and As10 reported by Robledo et al. (2009)

(Fig. 2 and Appendix E-j).

3.2. Chromosome painting of telomeric repeats in the 10 Arachis species

Based on the above karyotypes, the chromosome positions of the telomeric repeat (TTTAGGG)n were determined for the 10 species through sequential GISH/FISH. This repeat was located in many chromosomal regions including the chromo-some ends. Among the chromosomal regions with telomeric repeats in the 10 species, six kinds of ITRs were observed in centromeric region (centromeric ITR), pericentromeric regions (pericentromeric ITR), intercalary region of long or short arms (long or short arm ITR), nucleolar organizer region (rDNA ITR), subtelomeric regions (subtelomeric ITR), and telomeric regions (telomeric ITR), respectively (Fig. 2).

In A. hypogaea cultivar Z5297, ITRs were observed on three chromosomes, with one centromeric ITR on chro-mosome Bhy5, one subtelomeric ITR on chromosome Ahy7 and one small ITR on the long arm of chromosome Ahy5. A. ipaënsis and A. hypogaea had the same ITR distribu-tions on B-genome chromosomes. The ITR distributions in A. duranensis and A. hypogaea showed differences on chro-mosomes 4 and 5 of the A-genome. Chromosome Adu4 car-ried an ITR on the short arm that was not obvious in the chro-

Ac

As

Adi

Av

Adu

Ahy

Bhy

Bi

Kb

Bho

Es

Arachis chiquitana

A. stenosperma

A. diogoi

A. villosa

A. duranensis

A. ipaёnsis

A. batizocoi

A. hoehnei

A. stenophylla

A. hypogaeaA

B

1DAPI FISH

2DAPI FISH

3DAPI FISH

4DAPI FISH

5DAPI FISH

6DAPI FISH

7DAPI FISH

8DAPI FISH

9DAPI FISH

10DAPI FISH

10 μm

Fig. 2 New karyotypes of ten Arachis species. The first column indicates chromosomes captured in white and black after DAPI counterstaining to show the characteristic bands of DAPI (gray white). Other color signals in the chromosomes were produced by different probes. White color, the original green signal of 5S rDNA converted to pseudo color white; red color, 45S rDNA; green color, telomeric repeats (TTTAGGG)n; fuchsia, original red signal of total genomic DNA converted to pseudo fuchsia color, shows GISH (genomic in situ hybridization) signal with total genomic DNA of Arachis ipaënsis labeled with digoxigenin-11-dUTP and detected with anti-digoxigenin-rhodamine; blue, chromosomes counterstained with DAPI. The same as below.

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2492 DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

Among them, six A-genome karyotypes (Ahy, Adu, Av, Adi, As and Ac) had similar karyotypic formulae and distributions of 5S rDNA, 45S rDNA and DAPI bands including the smallest “chromosome A” (Table 2). However, chromosomes 2, 3, 4, 5 and 7 showed different ITRs, which indicated genetic diversity among the A-genome chromosomes in these species (Fig. 3).

The two B-genomes had the same karyotype formulas (Bhy=Bi=10m), which inclued the similar distribution of 5S and 45S rDNA on chromosomes 3, 7, and 10, DAPI bands on seven chromosomes, and a large centromeric ITR on chromosome 5 (Fig. 3).

The karyotype formulas of the remaining genomes Kb, Bho and Es were Kb=7m+3sm, Bho=9m+1sm and Es=6m+3sm+1st, respectively, they were quite different. Among them, the whole chromosome 9 had a large subtelomeric ITRs, the whole chromosome 4 had a 45S rDNA site, and the whole chromosome 10 had both 5S and 45S rDNA sites. Howev-er, chromosomes Kb3 and Kb8 both had a 5S rDNA site but Bho3, Bho8, Es3 and Es8 did not have that; Es7 had a large telomeric ITR in the short arm but Kb7 and Bho7 each had a small pericentromeric ITR in the long arm (Fig. 3). All of these indicated the variation among these chromosomes.

4. Discussion

45S and 5S rDNA as probes combined with DAPI staining method had been used by previous chromosome painting studies, and the karyotypes of most Arachis species were

Ac

As

Adi

Av

Adu

Ahy Bhy

Bi

Kb

Bho

EsA. chiquitana

A. stenosperma

A. diogoi

A. villosa

A. duranensisA. ipaёnsis

A. batizocoi

A. hoehnei

A. stenophylla

A. hypogaea

A genome

A B

E genome

B genome

K genome

B genome

DAPI bands45S rDNA5S rDNA(TTTAGGG)n

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

3 μm

Fig. 3 Idiogram karyotypes of 10 Arachis species. sm, submetacentric; st, subtelocentric.

mosome Ahy4, and chromosome Adu5 had an ITR on the long arm which was larger than the one on the chromosome Ahy5 (Fig. 2). The different ITR distributions (number and size) on the three A-genome chromosomes were also observed in the other four species (A. villosa, A. diogoi, A. stenosperma and A. chiquitana). A small rDNA ITR was presented in chro-mosomes Adi2 and As2, but was not seen in chromosomes Av2 and Ac2. Another small rDNA ITR observed in chromo-somes Av3, As3 and Ac3 was not detected on chromosome Adi3. In addition, one ITR observed only on the long arms of chromosomes Av5 and Adi5 and a small pericentromeric ITR was detected only on the short arm of chromosomes As5 and Ac5 (Figs. 3 and 4).

ITRs in A. batizocoi, A. hoehnei and A. stenophylla were observed on chromosomes 5, 7, 8 and 9. The ITR distribu-tions on chromosomes 5 and 9 were similar across the three species with a small pericentromeric ITR in chromosome 5 and the largest subtelomereic ITR covering almost entire short arm of chromosomes 9. As for chromosomes 7 and 8, a small pericentric ITR was presented in chromosomes Kb7 and Bho7, a large telomeric ITR presented in chromosome Es7, and an rDNA ITR was presented only in chromosome Kb8 (Fig. 2; Table 2).

3.3. Genomic analysis of the ten Arachis species

According to the chromosomal locations of telomeric repeats above and 5S rDNA, 45S rDNA and DAPI bands, karyotypes with higher resolution were developed for the 10 species.

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2493DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

established, which provided an efficient method for chro-mosome identification and genome evolutionary research on Arachis (Seijo et al. 2004; Robledo et al. 2009, 2010). This method was also adopted in the present study aiming to develop preliminary karyotypes of 10 Arachis species. All A- and B-genome chromosomes of cultivated peanut were distinguished with higher accuracy of multicolor GISH using total genomic DNAs from A- and B-genome donor species as probes (Du et al. 2015), instead of using DAPI staining (Seijo et al. 2004), because most B-genome chromosomes were stained by DAPI (She et al. 2012; Du et al. 2015). More importantly, chromosome painting using the telomeric repeat revealed six ITRs in Arachis distributed in telomeric (such as chromosome Es7), subtelomeric (Bho9 and Adu7), intercalary (Av5), nucleolar organizer (As3 and Adi3), pericentromeric (As5 and Bho7), and centromeric regions (Bi5). Moreover, some ITRs showed variation in size (Fig. 4-A); for exam-ple, chromosomes Kb9, Bho9 and Es9 had the largest ITRs which almost covering the entire short arms. Our results thus provide new cytogenetic markers for chromosome identification in peanut. After chromosome painting using the telomeric repeat combined with sequential GISH/FISH and DAPI staining, almost all chromosomes of the 10 spe-cies were clearly distinguished and karyotypes with higher resolution were developed for chromosome identification Ta

ble

2 N

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Spe

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(μm

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No.

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No.

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5S

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No.

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No.

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62±0

.13

46.2

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561

(Ac 3

)3

(Ac 2

, Ac 7

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(Ac 9

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Ac 8

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4.37

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943

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1 (A

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4 (A

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3 (A

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251

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72±0

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37.2

2±1.

101

(Adu

3)2

(Adu

2, A

du10

)3

(Adu

4, A

du5,

Adu

7)1

(Adu

9) (+

++),

9 (A

du1-

Adu

8, A

du10

) (++

)

A. h

ypog

aea

9m+1

sm3.

92±0

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39.2

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001

(Ahy

3)2

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2, A

hy10

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(Ahy

5, A

hy7)

1 (A

hy9)

(+++

+), 9

(Ahy

1-A

hy8,

Ahy

10) (

+++)

10m

4.40

±0.2

243

.95±

1.13

1 (B

hy3)

3 (B

hy3,

Bhy

7, B

hy10

)1

(Bhy

5)2

(Bhy

1, B

hy4)

(++)

, 5 (B

hy5-

Bhy

9) (+

)A

. ipa

ënsi

s10

m4.

21±0

.17

42.0

7±0.

581

(Bi 3

)3

(Bi 3

, Bi 7

, Bi 1

0)1(

Bi 5

)2

(Bi 1

, Bi 4

) (++

), 5

(Bi 5-

Bi 9

) (+)

A. b

atiz

ocoi

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78±0

.15

37.8

5±0.

783

(Kb 3

, Kb 8

, Kb 1

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(Kb 4

, Kb 1

0)4

(Kb 5

, Kb 7

, Kb 8

, Kb 9

)1

(Kb 9

) (++

+), 7

(Kb 1

, Kb 2

, Kb 4-

Kb 6

, Kb 8

, Kb 1

0) (+

+), 1

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) (+)

A. h

oehn

ei9m

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±0.1

742

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1 (B

ho10

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4, B

ho10

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(Bho

5, B

ho7,

Bho

9)1

(Bho

9) (+

++),

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Bho

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ho7,

Bho

8, B

ho10

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), 1

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ylla

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431

(Es 1

0)2

(Es 4

, Es 1

0)3

(Es 5

, Es 7

, Es 9

)1

(Es 9

) (++

+), 8

(Es 1

, Es 2

, Es 4-

Es 8

, Es 1

0) (+

+), 1

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) (+)

1) m

, met

acen

tric;

sm

, sub

met

acen

tric;

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c.2) IT

Rs,

inte

rstit

ial t

elom

eric

repe

ats.

3) th

e nu

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repr

esen

t the

inte

nsity

of D

AP

I ban

ds.

Val

ues

are

mea

ns±S

E.

DAPI bands45S rDNA

5S rDNA(TTTAGGG)n

Es7

Bho9

Adu7

As7

Ac7

Adi7

Adu7

Kb10

Bho10

As7

3 μm10 μm

As3

Adi2

As5

Bi5

Bho7

Av5

Ac8

A B

C

Fig. 4 Organizations of telomeric repeats (A): 45S rDNA (B), 45S (B) and 5S rDNAs (C) in individual chromosomes of different Arachis genomes.

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2494 DU Pei et al. Journal of Integrative Agriculture 2016, 15(11): 2488–2496

and evolutionary research in Arachis. To our knowledge, this is the first time to report the karyotype of A. stenophylla.

The locations of ITRs generally indicate that chromosome fusion (Azzalin et al. 2001), and chromosome breakage or rearrangement (Lin and Yan 2008; Bolzan 2012). Our re-search indicated that various chromosome rearrangements had occurred and the telomeric repeat might be a useful tool for studying genomic evolution in Arachis species. Previous studies also indicated that ITRs or telo-box elements could be presented in the promoter regions of some genes in Ara-bidopsis thaliana (Manevski et al. 2000), and telo-box might have function in regulating eukaryotic elongation factor 1A (EFIA) or ribosomal protein gene expression (Tremousaygue et al. 1999). Further analysis of various ITRs in peanut might reveal their possible roles.

In addition to various ITRs found in Arachis, various rDNA distributions were also established in this study. For ex-ample, homologous chromosomes of Ac7 and Adi7 showed different rDNA sizes, and the 45S rDNA signal of one of the Adu7 homologs was missing (Fig. 4-B). Heterogeneous ITRs previously reported in Solanum (He et al. 2013) were attributed to unequal crossing over. Our results further indicated that chromosomal rearrangements might have occurred relatively frequently in Arachis. And the typical ex-ample was one obvious inversion detected in chromosomes Kb10 and Bho10 (Fig. 4-C). Further analysis of the detailed structural variation will be important for genetic research and chromosome engineering in Arachis.

Previous reports concluded that A. duranensis and A. ipaënsis were likely donors of the A- and B-genomes in cultivated peanut (Robledo et al. 2009, 2010; Koppolu et al. 2010; Nielen et al. 2010). In this study, the diversity between the A-genomes of A. duranensis (AduAdu) and cultivated peanut (AhyAhyBhyBhy) on chromosomes 4 and 5 was obvious. Also, both the diversity between Adu and Bi genomes in A. duranensis and A. ipaënsis on chromo-somes 2, 3, 4, 5, 7 and 10, and diversity between Ahy and Bhy genomes in cultivated peanut on chromosomes 2, 3, 5, 7 and 10 were more obvious. These results indicated that chromosomal rearrangements had occurred in these six chromosomes. Recent genome sequencing also indicated that six chromosomes of the Adu and Bi genome had frequent rearrangements, including inversions and translocations, whereas the other four chromosomes maintained high collinearity (Bertioli et al. 2016).

The genomes in A. hoehnei and A. batizocoi were previ-ously grouped in B-genome, and A. stenophylla was grouped into E-genome species in section Erectoides (Krapovickas and Gregory 1994). However, Robledo and Seijo (2010) suggested that A. batizocoi should be allocated to a new genome named “K” based on rDNA FISH. Our study showed different distributions of telomeric repeats on chromosomes

5, 7, 8 and 9 in A. batizocoi and A. ipaënsis, which provided evidence for the new K designation.

The genomic origin of A. hoehnei (Bho) was controversial (Tallury et al. 2005; Friend et al. 2010; Custódio et al. 2013; Moretzsohn et al. 2013). The present characteristic patterns of the telomeric repeat suggested that the genome Bho of A. hoehnei might be similar to E-genome and different from A- and B-genomes (Fig. 2). However, due to the genetic complexity, it is necessary to use additional landmarks or whole genome sequencing to determine the real origin and differentiation of genome Bho .

5. Conclusion

Chromosome painting reveals various distributions of the telomeric repeat and rDNA in Arachis. These markers com-bining with sequential GISH/FISH and DAPI staining allowed the development of higher resolution karyotypes for the 10 Arachis species. These analyses not only distinguished most chromosomes but also revealed many chromosomal rearrangements in Arachis species. The markers could be extended to other Arachis species and should facilitate both evolutionary genomic research and introgression of alien genes into cultivated peanut via interspecific hybridization.

Acknowledgements

We thank Dr. B S Gill (Plant Pathology Department, Kan-sas State University, Manhattan, KS, USA) for providing clones of 45S rDNA and 5S rDNA for this study and Dr. Cai Xiwen (Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA) and Dr. Chu Chenggen (Monsanto Company, 21120 Hwy 30, Filer, ID 83328, USA) for reviewing the manuscript. This research was supported by the China Agriculture Research System (CARS-14), the Henan Provincial Agriculture Research System, China (S2012-05) and the Major Technology Research and Devel-opment Program of Henan Province, China (141100110600).

Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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