Research. Laining Zhang 1 *, Xiaoyu Yang 1 *, Li Tian 2, Lei Chen 3 and Weichang Yu 3,4. Summary. Introduction

Transcription

1 Research Identification of peanut (Arachis hypogaea) chromosomes using a fluorescence in situ hybridization system reveals multiple hybridization events during tetraploid peanut formation Laining Zhang 1 *, Xiaoyu Yang 1 *, Li Tian 2, Lei Chen 3 and Weichang Yu 3,4 1 School of Life Sciences, Institute of Plant Molecular Biology and Agricultural Biotechnology, State (China) Key Laboratory for Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin, Hong Kong; 2 Institute of Biological Chemistry, Washington State University, Pullman, WA , USA; 3 Shenzhen Research Institute, the Chinese University of Hong Kong, Shenzhen , China; 4 Present address: College of Life Sciences, Shenzhen University, Shenzhen, China Author for correspondence: Weichang Yu Tel: s: wyu@cuhkri.org.cn; wyu@szu.edu.cn Received: 1 October 2015 Accepted: 31 March 2016 doi: /nph Key words: chromosome evolution, chromosome marker, cultivated peanut (Arachis hypogaea), fluorescence in situ hybridization (FISH), interstitial telomere repeat (ITR), karyotyping, repetitive DNA. Summary The cultivated peanut Arachis hypogaea (AABB) is thought to have originated from the hybridization of Arachis duranensis (AA) and Arachis ipa ensis (BB) followed by spontaneous chromosome doubling. In this study, we cloned and analyzed chromosome markers from cultivated peanut and its wild relatives. A fluorescence in situ hybridization (FISH)-based karyotyping cocktail was developed with which to study the karyotypes and chromosome evolution of peanut and its wild relatives. Karyotypes were constructed in cultivated peanut and its two putative progenitors using our FISH-based karyotyping system. Comparative karyotyping analysis revealed that chromosome organization was highly conserved in cultivated peanut and its two putative progenitors, especially in the B genome chromosomes. However, variations existed between A. duranensis and the A genome chromosomes in cultivated peanut, especially for the distribution of the interstitial telomere repeats (ITRs). A search of additional A. duranensis varieties from different geographic regions revealed both numeric and positional variations of ITRs, which were similar to the variations in tetraploid peanut varieties. The results provide evidence for the origin of cultivated peanut from the two diploid ancestors, and also suggest that multiple hybridization events of A. ipa ensis with different varieties of A. duranensis may have occurred during the origination of peanut. Introduction Cultivated peanut (Arachis hypogaea; AABB; 2n = 4x = 40) is an allotetraploid in the Arachis section. This section contains 31 species, including diploids (2n = 2x = 20), tetraploids (2n = 4x = 40) and aneuploids (2n = 2x = 18) (Bertioli et al., 2009; Pandey et al., 2012). Cytogenetic evidence shows that three types of genome exist in this section: the A genome, B genome and D genome. The A genome is distinguished by the presence of a small pair of chromosomes (chromosome 9) (Nielen et al., 2012) and the appearance of a heterochromatic band near the centromeres after 4 0,6-diamidino-2- phenylindole (DAPI) staining (Lavia, 2000). By contrast, the aforementioned features are absent in the B and D genomes (Seijo et al., 2004). The D genome bears an asymmetrical karyotype with several submetacentric and subtelocentric chromosomes as compared with the A and B genomes (Robledo & Seijo, 2008). Cytogenetic and molecular data suggested *These authors contributed equally to this work. that the A and B genomes contributed to cultivated peanut (Smartt & Gregory, 1967; Kochert et al., 1991). Physical mapping with 5S and 45S rdna genes, genomic in situ hybridization (GISH) and molecular evolution studies of chloroplast DNA and 5S rdna provided independent evidence that Arachis duranensis (AA) and Arachis ipa ensis (BB) were the parental genome donors of A. hypogaea. Hybridization of these two diploid species followed by a spontaneous chromosome doubling was proposed as the mechanism for the formation of cultivated peanut (Seijo et al., 2004, 2007; Grabiele et al., 2012). As one of the only two tetraploid species in the Arachis section, and being reproductively isolated from other diploid relatives during evolution, cultivated peanut lacks genetic diversity (Milla et al., 2005). Therefore, high-density genetic mapping based on molecular markers is almost impossible and maps of A. hypogaea were mainly constructed using its diploid relatives (Pandey et al., 2012). In addition, morphologically similar chromosomes and the lack of chromosome markers make cytogenetic research in peanut difficult compared with other plant systems. Previous 1424

2 New Phytologist Research 1425 karyotyping studies in Arachis based on conventional technology and fluorescence in situ hybridization (FISH) with rdna genes showed high dependence on chromosome morphology and had a low degree of precision because of inadequate chromosome markers (Seijo et al., 2004; Lavia et al., 2009). These problems impede our understanding of peanut genome organization and evolution. To study the organization and evolution of the peanut genome, a karyotyping system with sufficient molecular markers is needed. FISH-based karyotyping relies on chromosome markers, which are independent of genetic diversity, and may complement genetic mapping in studies of chromosome organization and evolution. Chromosome identification systems employing FISH have been used to construct karyotypes or cytogenetic maps in rice (Oryza sativa), Sorghum bicolor, maize (Zea mays), Antirrhinum majus, poplar (Populus alba), common bean (Phaseolus vulgaris), soybean (Glycine max), melon (Cucumis melo) and Brassica napus (Cheng et al., 2001; Islam-Faridi et al., 2002; Kato et al., 2004; Zhang et al., 2005; Ribeiro et al., 2008; Pedrosa-Harand et al., 2009; Findley et al., 2010; Fonseca et al., 2010; Liu et al., 2010; Pires & Xiong, 2011). Large genomic clones, such as bacterial artificial chromosomes (BACs), provide reliable landmarks with which to discriminate individual chromosomes in many crops such as rice (Cheng et al., 2001) and Brassica (Pires & Xiong, 2011). In rice, 24 chromosome armspecific BACs identified all 12 chromosomes simultaneously to construct a karyotype (Cheng et al., 2001). In B. napus, karyotyping was constructed by genetically mapped BACs (Pires & Xiong, 2011). Integrated cytogenetic maps can be used to make comparisons among species or different generations within species to study chromosome evolution and phylogenetic relationships (Iovene et al., 2011; Mendes et al., 2011; Xiong et al., 2011; Bonifacio et al., 2012; Ruiz-Herrera et al., 2012). In addition, FISH with BAC clone probes has been used in the assembly and validation of genome sequences (Chamala et al., 2013). Single-copy gene sequences have been used in karyotyping of mitotic chromosomes (Lamb et al., 2007) and pachytene chromosomes of maize (Wang et al., 2006). However, the detection of single-copy DNA sequences on plant chromosomes has been difficult because debris from the cell wall and cytoplasm reduces the accessibility of target DNA and increases background and consequently results in a relatively low signal-to-noise ratio (Jiang et al., 1995). In addition, a large number of repetitive sequences have frequently been used in FISH studies. For example, 5S and 45S rdnas have been used in FISH to study the relationships between peanut and its wild relatives (Seijo et al., 2004; Robledo et al., 2009; Seijo & Robledo, 2010). rdnas, telomeric and subtelomeric DNAs, centromere repeats and centromere retrotransposons have been used in the karyotyping of maize (Kato et al., 2004) and soybean (Findley et al., 2010). In a previous report, major repetitive DNA sequences from cultivated peanut Cot-1 DNA were cloned and analyzed (Zhang et al., 2012). Cot-1 DNA contains highly competitive DNA, which has a Cot value (the product of a sample s nucleotide concentration, the time of reassociation, and an appropriate buffer factor) of one (Zwick et al., 1997). Genome- and chromosome-specific markers were identified to distinguish A and B genomes in tetraploid peanut. A 115-bp tandem repetitive sequence was identified as a possible centromere repetitive DNA based on its localization in pericentromeric regions on most of the B-genome chromosomes. A partial centromeric retrotransposable element was also identified on B-genome chromosomes. These chromosome markers were reliable because they produced conserved patterns in peanut varieties from different origins, and thus could be used to perform karyotyping analysis of peanut. In this study, new FISH markers were cloned from A. duranensis, an A-genome diploid wild relative of cultivated peanut. Comparative analysis indicated that these markers were mostly conserved between A. duranensis and the cultivated peanut. The screen of peanut BAC clones (Yuksel & Paterson, 2005) also revealed chromosome-specific BAC clones that identified unique chromosomes based on chromosome localization patterns. By combining previously reported markers and our newly developed ones, we produced a peanut karyotyping cocktail that allowed the identification of almost all chromosomes in peanut and its two wild relatives, and the construction of the first FISH-based karyotypes in peanut and its two wild relatives. Comparative karyotyping analysis revealed the similarities and differences among cultivated peanut and the two wild relatives, A. duranensis and A. ipa ensis. This system will provide a molecular cytological tool for the identification of peanut chromosomes and the studies of chromosome evolution in Arachis. Materials and Methods Plant material Seeds of cultivated peanut (Arachis hypogaea L.) from China were the same as previously described (Zhang et al., 2012). All other tetraploid peanut varieties (PI , PI , PI and PI ) and diploid wild peanut Arachis duranensis Krapov. & W. C. Greg. (Grif 15035, Grif 15039, PI , PI , PI , PI , PI , PI , PI , PI , PI , PI , PI and PI ), Arachis ipa ensis Krapov. & W. C. Greg. (PI ), Arachis diogoi Hoehne (PI ), Arachis stenosperma Krapov. & W. C. Greg. (PI ) and Arachis villosa Benth. (Grif 7724) were obtained from the Plant Genetic Resources Conservation Unit of the Agricultural Research Service (ARS) of the United States Department of Agriculture (USDA). Cot-1 DNA preparation from Arachis duranensis and the cloning of repetitive DNA markers Cot-1 DNA was prepared as described previously with modifications (Zhang et al., 2012). Genomic DNA from a diploid species (A. duranensis) was isolated from young leaves. Isolated genomic DNA (70 lg) was fragmented to a size range from 100 to 1000 bp by heating at 95 C for 75 min in a PCR machine. The fragmented genomic DNA was diluted to a concentration of

3 1426 Research New Phytologist 373 ng ll 1 in 0.3 M NaCl, and subsequently subjected to reassociation at 65 C in a water bath for min. Single-stranded molecules were digested with S1 nuclease in a 37 C water bath for 8 min. The double-stranded DNA was purified with ethanol precipitation and checked on an agarose gel for correct size. The size of purified Cot-1 DNA ranged from 100 bp to 1 kb (data not shown). To check the quality of the Cot-1 DNA, 2 lg of purified Cot-1 DNA was labeled with Alexa Fluor dUTP (Thermo Fisher Scientific; and 100 ng of the probe was hybridized to peanut metaphase chromosomes by FISH. The cloning, sequencing, and screening of repetitive DNA markers from a Cot-1 library of A. duranensis were performed according to Zhang et al. (2012). All probes were labeled with Alexa Fluor dUTP (Thermo Fisher Scientific) and hybridized to metaphase chromosomes of peanut according to Zhang et al. (2012). Screening of peanut BAC clones for chromosome-specific markers Peanut BAC clones (324 clones; Supporting Information Table S1) were provided by Andrew Paterson from the University of Georgia (Yuksel & Paterson, 2005). To select BAC clones with low repetitive DNA content, colony hybridization was performed as follows. Cot-1 DNA (2 lg) from cultivated peanut (A. hypogaea) was labeled with digoxigenin (DIG)-11-dUTP (Roche; by nick translation according to the manufacturer s instructions. BAC clones were grown in liquid Luria broth (LB) medium in 96-well plates at 37 C overnight. Five microliters of liquid culture was dotted onto a Zeta-probe blotting membrane (Bio-Rad Laboratories; and the membrane was placed on an LB agar plate containing 12.5 lgml 1 of chloroamphenicol to allow the bacteria to grow overnight at 37 C. BAC clones on the membrane were treated as described by Ross et al. (1999) and subjected to in situ hybridization with the DIGlabeled Cot-1 DNA probe following the manufacturer s instructions (Roche). Plasmid DNA from BAC clones showing low hybridization to Cot-1 DNA (Table S1) was prepared using the Qiagen Plasmid Midi Kit (Qiagen; In a 50-ll probelabeling reaction, 5 lg of BAC DNA, 5 ll of109 nick translation buffer, 5 ll of nonlabeling dntp mix (2 mm datp, 2 mm dgtp and 2 mm dctp), 1.0 ll of 1 mm Alexa Fluor dutp (Thermo Fisher Scientific), 6.25 ll of DNA polymerase I (10 U ll 1 ; Thermo Fisher Scientific) and 1.0 ll ofdnasei (0.1 U ll 1 ; Roche) were mixed gently by pipetting and incubated at 15 C for 2.5 h in a PCR machine. The reaction was stopped by the addition of 350 ll of stopping buffer (140 ng ll 1 autoclaved salmon sperm DNA in 5 9 Tris-acetate-EDTA (TAE) buffer; ph 5.2). The DNA probes were then precipitated by the addition of 0.1 volumes of 3 M sodium acetate (ph 5.2) and 2.5 volumes of 100% ethanol with incubation at 20 C overnight. The precipitated probes were dissolved in 2 9 saline sodium citrate (SSC) buffer to a final concentration of 100 ng ll 1. The BAC DNA probes were hybridized to peanut metaphase chromosomes according to the peanut FISH procedure (Zhang et al., 2012). Metaphase chromosome preparation, FISH marker DNA preparation and FISH procedure Chromosome preparation, probe labeling, and peanut FISH hybridization were performed according to a previously reported procedure (Zhang et al., 2012). The following FISH markers were prepared. The rdna genes (5S, 18S and 26S) were amplified using previously reported primers (Robledo & Seijo, 2008) from cultivated peanut genomic DNA. The amplified fragments were inserted into the PCR 2.1 vector (Thermo Fisher Scientific) and checked by sequencing (BGI, Shenzhen, China). A mixture of 18S and 26S rdna with a 1: 1 stoichiometric ratio was used in probe labeling to produce the 45S rdna probe. The Arabidopsis-type telomere repeat clone WY4T3X (Xu et al., 2012) containing three copies of the cloned Arabidopsis telomere DNA sequence from pat4 (Richards & Ausubel, 1988) was used to prepare the telomere DNA probe. A synthetic telomere oligo probe labeled with Texas Red was used in the analysis of the interstitial telomere repeats (ITRs) in A. duranensis varieties. The B-genome centromere repeat (JQ673497) was amplified using previously reported primers (Zhang et al., 2012). For karyotyping FISH analysis, a FISH cocktail containing eight probes was prepared according to the following recipe (Table S2): Alexa Fluor dUTP (green; Thermo Fisher Scientific)-labeled B-genome centromere satellite repeat (B-c) (0.25 ll of 100 ng ll 1 ), 45S (18S+26S) rdna (1.5 ll of 100 ng ll 1 ), and BAC clone 02D10 (1.5 ll of 100 ng ll 1 ); Alexa Fluor dUTP (far red; Thermo Fisher Scientific)-labeled heterochromatin band repeat (H-b) (0.25 ll of 100 ng ll 1 ) and telomere DNA (0.5 ll of 100 ng ll 1 ); Alexa Fluor dUTP (orange; Thermo Fisher Scientific)-labeled 5S rdna (1.0 ll of 100 ng ll 1 ), BAC clone 01B12 (1.5 ll of 100 ng ll 1 ), and BAC clone 02G01 (1.5 ll of 100 ng ll 1 ). FISH signals were examined with a Leica DM5500 fluorescence microscope and images were captured using a Leica DFC340 FX Digital charge-coupled device (CCD) camera (Leica Microsystems; The FISH images obtained from different channels were merged using the Leica CW4000 FISH software and analyzed using the Leica CW4000 KARYO software. ADOBE PHOTOSHOP 6.0 (Adobe Systems; was used to make the weak signal recognizable through background subtraction and imagefeature intensification. Sequence comparisons The cloning of H-b and B-c repetitive sequences from A. hypogaea, A. duranensis, and A. ipa ensis was performed as previously described (Zhang et al., 2012). Sequence alignment was performed at Biology Workbench ( with the CLUSTALW program.

4 New Phytologist Research 1427 Results Cloning of repetitive DNA markers from Arachis duranensis To find peanut chromosome markers, we previously developed a method to clone repetitive markers from Cot-1 DNA (Zhang et al., 2012). To produce more markers, especially A-genome chromosome markers, a Cot-1 library of 1500 clones from A. duranensis was constructed, of which 291 clones were sequenced to generate 202 insert sequences. Using the insert sequence as a query, a BLAST search against the National Center for Biotechnology Information (NCBI) database was performed. The 202 sequences were classified into five groups according to the BLAST results (Table S3, Fig. S1): 10 clones matched previously reported microsatellite DNA sequences in peanut, 56 clones had no homologs in the database, three clones showed high homology to rdna sequences from other species, 117 clones matched the FIDEL retrotransposon in peanut, and 16 clones were from the chloroplast genome. These results suggested that repetitive sequences account for a high percentage of this Cot-1 DNA library. An Alexa Fluor dUTP-labeled Cot-1 DNA probe was hybridized to peanut metaphase chromosomes by FISH. The Cot-1 DNA mainly localized to the pericentromeric regions of A-genome chromosomes, but also produced weak signals on B-genome chromosomes (Fig. S2), which demonstrated that this Cot-1 DNA contained A-genome repetitive DNA sequence. Chromosomal distribution of selected clones To select chromosome-specific DNA sequences, inserts from 46 unique clones were analyzed by FISH on metaphase chromosomes of peanut. Their chromosomal distributions in peanut are shown in Table 1. Among these clones, clone AD-9 had a distribution pattern similar to that of the heterochromatin band, which was a centromeric characteristic of A-genome chromosomes after staining with DAPI as a result of the binding of DAPI to the high-at region (Kapuscinski, 1995). Clone AD-9 hybridized to 18 chromosomes in A. duranensis (Fig. 1a c) and 18 A-genome chromosomes of A. hypogaea in the pericentromeric regions (Fig. 1g i), but produced no signals in A. ipa ensis (Fig. 1d f). Clone AD-45 hybridized to the pericentromeric regions of six A-genome chromosomes and 16 B-genome chromosomes in metaphase chromosomes of A. hypogaea (Fig. 1p r). It also hybridized to six chromosomes of A. duranensis (Fig. 1j l) and 16 chromosomes of A. ipa ensis (Fig. 1m o). Clone AD-47, AD-65, AD-88 and AD-112 showed A- genome-specific dispersed distributions on peanut metaphase Table 1 Distribution of selected clones from the Arachis duranensis Cot-1 library on peanut chromosomes Clone no. Size (bp) Classification a Chromosomal distribution b Clone no. Size (bp) Classification Chromosomal distribution S/M D N DA N C N D N D N D N D S/M D FIDEL D N D R No signal N D S/M C N D N D S/M D N DA N D N D N D N D N D N D N D N DA N D N D S/M D N DA S/M D N D N D N D S/M D N D N D N DA N D N D N D N D N D S/M D N D S/M D N D N D N D a Classification: C, chloroplast DNA; FIDEL, fairly long interdispersed euchromatic Long terminal repeat (LTR) retrotransposon (Nielen et al., 2010); N, unknown sequence in peanut; R, rdna; S/M, satellite or microsatellite DNA. b Chromosomal distribution: D, no clear signal, but the signal was recognizable on the entire chromosome; DA, dispersed signals on A-genome chromosomes; C, fluorescence in situ hybridization (FISH) signals were observed in the pericentromeric regions; no signal, no obvious signals were observed.

5 1428 Research New Phytologist chromosomes (data not shown). The remaining clones showed dispersed localization on both A- and B-genome chromosomes, and thus were not used in FISH karyotyping analysis. Cloning and characterization of heterochromatin band sequences in peanut and wild species To clone the full length of the clone AD-9 repeat unit, two primers, AD-9F (5-ATTACCGGATAATGCTATTTTTTCAT AAAT-3 0 ) and AD-9R (5-ATTATAAAGATTTAAAAAATATC CAATAAA-3 0 ), were designed based on the clone AD-9 sequence, and used in PCR amplifications with A. hypogaea genomic DNA as a template. PCR products were sequenced and analyzed with a TANDEM REPEATS FINDER program (Benson, 1999; A 317-bp fragment in A. hypogaea were found to be the repeat unit and named heterochromatin-band (H-b-Ah) (GenBank no. KF957858). FISH hybridization with the Alexa Fluor dUTP-labeled H-b-Ah repeat produced 22 signals on chromosomes of A. hypogaea (Fig. 2g i): 18 on the A-genome chromosomes and four on the B-genome chromosomes. Similar to AD-9, H-b hybridized to 18 chromosomes of A. duranensis in the pericentromeric regions (Fig. 2a c). The H-b probe also hybridized to four chromosomes in A. ipa ensis (Fig. 2d f). This probe was thus used to replace the AD-9 clone in subsequent FISH and karyotyping analysis. Homologs of the H-b-Ah sequences were PCR amplified and cloned from the two wild relatives A. duranensis and A. ipa ensis, respectively, using two PCR primers: H-b-Ah F (5 0 -TTTA AAAAATATCCAATAAAC-3 0 ) and H-b-Ah R (5 0 -ATCTTT ATTTTTCATCTTTTT-3 0 ). A 318-bp repetitive unit from A. duranensis and a 317-bp repetitive unit from A. ipa ensis were amplified and named H-b-Ad (GenBank no. KF957859) and H- b-ai (GenBank no. KF957860), respectively. Sequence analysis showed that all three cloned heterochromatin band DNA repeats had a high AT content, with 85.80% in H-b-Ah, 85.22% in H- b-ad and 84.86% in H-b-Ai (Fig. 2j). Alignment of the three heterochromatin band sequences showed high conservation of AT-rich regions, although variations including deletion, addition and substitution exist among them. Cloning and chromosomal distribution of the B-genome centromere repeat in peanut and the wild species Arachis duranensis and Arachis ipa ensis The B-genome centromere repeat of 115 bp has previously been reported to be a chromosome marker in tetraploid peanut (Zhang et al., 2012). To test its utility as a chromosome marker in the species A. ipa ensis and A. duranensis, FISH analyses were performed on metaphase chromosomes of A. ipa ensis and A. duranensis (Fig. 3). It labeled 16 chromosomes of A. ipa ensis in the pericentromeric regions (Fig. 3a c). This chromosome localization matched the pattern on the B-genome chromosomes of A. hypogaea. In A. duranensis, it labeled the pericentromeric regions of 10 chromosomes (Fig. 3d f). In comparison, it only labeled six to eight A-genome chromosomes in A. hypogaea (Fig. 3g i; Zhang et al., 2012). The localization on extra chromosomes in A. duranensis may be attributable to loss of the repeat in A-genome chromosomes of A. hypogaea after the formation of the cultivated peanut species, or the complicated genetic background in A. hypogaea may have made it difficult to detect the signals. Homologs of the B-genome centromere repeat (B-c) sequences were PCR amplified from A. duranensis and A. ipa ensis, using the primers B-c-Ah F (5 0 -AAAAAAAACAGAAAAAAAAT-3 0 ) and B-c-Ah R (5 0 -AACGTTGTGCTGACCTCGTT-3 0 ) designed based on B-c-Ah. A 164-bp repetitive unit from A. duranensis and a 103-bp repetitive unit from A. ipa ensis were identified and named B-c-Ad (GenBank no. KF957861) and B-c-Ai (GenBank no. KF957862), respectively. Although variations in both repeat length and composition existed, they all shared a 95-bp highly conserved region (Fig. 3j, boxed region). Distribution of telomere repeat in peanut The Arabidopsis-type telomere DNA repeat (TTTAGGG) is conserved in most higher plants (Richards & Ausubel, 1988). To study the telomere structure of peanut and its wild relatives, the Arabidopsis telomere DNA was labeled and hybridized to chromosomes from A. hypogaea, A. duranensis and A. ipa ensis. Telomere DNA hybridized to all chromosomal ends (Fig. 4), indicating that there might be high homology and conservation of telomere DNA between Arabidopsis and peanut. In addition, two strong signals were observed in the pericentromeric regions of a pair of B-genome chromosomes in A. hypogaea (Fig. 4g i) and homologs in A. ipa ensis (Fig. 4d f). Strong signals were obtained in the intercalary regions of a pair of A-genome chromosomes (A5) and the subtelomeric regions of another pair of A-genome chromosomes (A7) in A. hypogaea (Fig. 4g i), but were not observed in homologous chromosomes in A. duranensis (Fig. 4a c). The unique nonterminal chromosomal localizations of telomere DNA made it a good candidate marker for peanut chromosome identifications. The strong signals in the pericentric regions distinguished one of the largest chromosomes (B1) in A. hypogaea and A. ipa ensis, which had no other chromosome markers on them. Screening of peanut BAC clones for chromosome-specific FISH markers To screen peanut BAC clones for FISH markers, 324 BAC clones (Yuksel & Paterson, 2005) were first analyzed by dot hybridization with Cot-1 DNA probes to remove clones with highly repetitive sequences. Sixty-eight BAC clones showed weak hybridization to Cot-1 DNA, and probably contained a low amount of repetitive DNA (Fig. S3). FISH probes were made from 56 individual BAC clones (Table S1), and hybridized to metaphase peanut chromosomes to observe their chromosome localizations. Twenty-six clones produced unique chromosomal localization patterns. Three clones (01B12, 02D10 and 02G01) were chosen because of their high signal intensity, low interference with other repetitive DNA markers, and complementation with other chromosome markers. BAC clone 02D10 hybridized

6 New Phytologist Research 1429 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) Fig. 1 Chromosomal localization of selected clones from an Arachis duranensis Cot-1 library. (a i) Clone AD-9 on metaphase chromosomes of (a c) A. duranensis,(d f) A. ipa ensis, and (g i) A. hypogaea;(j r) clone AD-45 on metaphase chromosomes of (j l) A. duranensis,(m o) A. ipa ensis, and (p r) A. hypogaea. Clones AD-9 and AD-45 were labeled with Alexa Fluor dUTP and hybridized to metaphase chromosomes of A. duranensis, A. ipa ensis, and A. hypogaea, respectively. 4 0,6-diamidino-2- phenylindole (DAPI) staining of the chromosomes (left panels), fluorescence in situ hybridization (FISH) signals (red; middle panels) and merged images (right panels) are shown. White arrows denoted signals for clones AD-9 and AD-45 on chromosomes of the A genome in A. duranensis and A. hypogaea, and white arrowheads denoted signals for clones AD-9 and AD-45 on chromosomes of the B genome in A. ipa ensis and A. hypogaea. Bars, 10 lm. (m) (n) (o) (p) (q) (r) to the distal regions of B6 chromosomes (Fig. 5a). 01B12 localized to the distal regions of chromosomes A5 and B5 (Fig. 5b). 02G01 localized to the pericentromeric regions, and produced signals in pericentromeric regions of 16 B-genome chromosomes and two A-genome chromosomes (Fig. 5c e). Karyotyping analysis of peanut and its two wild relatives Peanut metaphase chromosomes were arranged and numbered according to their relative sizes and the presence of rdna markers according to Seijo et al. (2004). FISH probes were hybridized

7 1430 Research New Phytologist (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Fig. 2 Chromosomal localizations of the heterochromatin band (H-b) sequence and the alignment of sequences from the three species. Alexa Fluor dutp (red)-labeled H-b-Ah from Arachis hypogaea was hybridized on metaphase chromosomes of (a c) A. duranensis,(d f) A. ipa ensis, and (g i) A. hypogaea.4 0,6-diamidino-2-phenylindole (DAPI) staining of the chromosomes (a, d, g), fluorescence in situ hybridization (FISH) signals (red; b, e, h) and merged images (c, f, i) are shown. White arrows denote signals for H-b on chromosomes of the A genome in A. duranensis and A. hypogaea, and white arrowheads denote signals for H-b on chromosomes of the B genome in A. ipa ensis and A. hypogaea. Bars, 10 lm. H-b sequences from the three species were amplified by PCR and alignment was performed with the CLUSTALW program (j). H-b sequences from A. hypogaea, A. duranensis and A. ipa ensis are represented by H-b-Ah, H-b-Ad and H-b-Ai, respectively. A consensus sequence is presented below the alignment to show conserved nucleotides shared by two or more sequences. N represents a nucleotide not conserved among the three sequences, and - represents nucleotide insertion or deletion. to metaphase chromosomes to localize FISH markers to each chromosome, and the hybridization patterns are shown in Table 2. A collection of eight chromosome markers were combined based on their chromosome localizations to make a karyotyping probe cocktail (Table S2). By applying this cocktail to A. hypogaea, almost all homologous chromosomes could be identified based on the distribution patterns and signal intensity of the probes (Fig. 6). Genome-A and -B chromosomes were distinguished by the DAPI staining of the heterochromatin band. Individual chromosomes in A. hypogaea were distinguished using FISH markers as follows: chromosome A1 had strong signals for

8 New Phytologist Research 1431 (a) (b) (c) (d) (e) (f) Fig. 3 Chromosomal localizations of a B- genome centromere repeat (B-c) sequence and the alignment of sequences from the three species. Alexa Fluor dUTP (red)- labeled B-c-Ah from Arachis hypogaea was hybridized on metaphase chromosomes of (a c) A. duranensis,(d f) A. ipa ensis, and (g i) A. hypogaea, respectively. 4 0,6- diamidino-2-phenylindole (DAPI) staining of the chromosomes (a, d, g), fluorescence in situ hybridization (FISH) signals (red; b, e, h) and merged images (c, f, i) are shown. White arrows denote signals for B-c-Ah on chromosomes of the A genome in A. duranensis and A. hypogaea, and white arrowheads denote signals for B-c-Ah on chromosomes of the B genome in A. ipa ensis and A. hypogaea. Bars, 10 lm. B-c sequences were PCR amplified from A. duranensis (B-c- Ad), A. ipa ensis (B-c-Ai), and A. hypogaea (B-c-Ah), respectively. Sequence alignment was performed with the CLUSTALW program (j). A consensus sequence is presented below the alignment to show conserved nucleotides shared by two or more sequences. N represents a nucleotide not conserved among the three sequences, and - represents nucleotide insertion or deletion. (g) (j) (h) (i) the BAC clone 02G01 (red) and H-b (white) in the pericentromeric regions; chromosome A2 had overlapping signals for 45S rdna (green) and B-c (green) in the pericentromeric regions; chromosome A3 had signals for 5S rdna (red), B-c (green) and H-b (white) in the pericentromeric regions; chromosomes A4, A6 and A8 were all labeled by the H-b probe, but chromosome A4 could be distinguished because it had a stronger signal intensity than both chromosomes A6 and A8. Chromosome A8 was smaller than A4 and A6, and had weaker H-b signals than A4 and A6. However, the differences between A6 and A8 were small, and sometimes it was difficult to distinguish them; chromosome A5 had signals for BAC clone 01B12 (red) in the subtelomere region, H-b (white) in the pericentromeric region and telomere repeat (white) in the intercalary region; chromosome A7 had signals for telomere DNA repeat (white) in the subtelemeric region and H-b in the pericentromeric region; chromosome A9 had signals for both B-c (green) and H-b (white), and was the smallest chromosome; chromosome A10 had strong signals for 45S rdna (green) in the pericentromeric region, H-b in the pericentromeric region, and a satellite; chromosome B1 had strong signals for telomere DNA repeat (white) in the pericentromeric region; chromosome B2 was not labeled by any probe; chromosome B3 had overlapping signals for 5S and BAC clone 02G01 (red), and overlapping signals for 45S rdnas and B-c (green); chromosomes B4 and B9 were labeled by B-c (green) and BAC clone 02G01 (red). The B-c signals on B4 were weaker than those on B9; chromosome B5 had signals for BAC clone 01B12 (red) at the chromosome end, and B-c (green) and BAC clone 02G01 (red) in the pericentromeric region; chromosome B6 had signals for BAC clone 02D10 (green) at the chromosome end, and B-c (green), H-b (white) and BAC clone 02G01 (red) in the pericentromeric region; chromosome B7 had signals for 45S rdna (green) in the subtelomeric region, and B-c (green) and BAC clone 02G01 (red) in the pericentromeric region; chromosome B8 was labeled by B-c (green), BAC clone 02G01 (red) and H-b (white) in the pericentromeric region; chromosome B10 had signals for 45S rdna (green), B-c

9 1432 Research New Phytologist (a) (b) (c) (d) (e) (f) Fig. 4 Chromosomal distribution of telomeric DNA repeats. Arabidopsis telomeric repeat DNA was labeled with Alexa Flour dutp (red) or Alexa Flour dUTP (green) and hybridized to metaphase chromosomes of (a c) Arachis duranensis,(d f) A. ipa ensis and (g i) A. hypogaea, respectively. 4 0,6-diamidino-2-phenylindole (DAPI) staining of the chromosomes (left panels), fluorescence in situ hybridization (FISH) signals (middle panels) and merged images (right panels) are shown. White arrows, arrowheads and stars denote interstitial telomere repeat (ITR) signals in pericentromeric regions of B1 chromosomes (e, f, h, i), intercalary regions of chromosome A5 (h, i) and subtelomeric regions of chromosome A7 (h, i) in tetraploid peanut and the two diploid wild species. Bars, 10 lm. (g) (h) (i) (a) (b) (c) (d) (e) (green) and BAC clone 02G01 (red) in the pericentromeric region. Similarly, the cocktail was applied to A. duranensis (Fig. 7a f) and A. ipa ensis (Fig. 7g l). An ideogram was drawn to illustrate Fig. 5 Chromosomal distribution of bacterial artificial chromosome (BAC) clones in Arachis hypogaea. (a) Clone 02D10; (b) clone 01B12; (c e) clone 02G01. BAC clone DNA was labeled with Alexa Fluor dUTP (red) and hybridized to metaphase chromosomes. Metaphase chromosomes were visualized with 4 0,6-diamidino-2- phenylindole (DAPI) staining (blue). For colocalization analysis (c e), H-b was labeled with Alexa Fluor dUTP (green), mixed with Alexa Fluor dUTP (red)-labeled BAC clone 02G01, and hybridized to metaphase chromosomes of A. hypogaea. Co-localization of H-b (green; c) and BAC clone 02G01 (red; d) on a pair of A genome chromosomes is shown in merged images (e; arrow heads). Arrows denote fluorescence in situ hybridization (FISH) signals for BAC clones. Bars, 10 lm. the FISH karyotypes of the three species (Fig. 8). The karyotype of A. duranensis was similar to the A genome of cultivated peanut except for the following differences: A. duranensis chromosomes 5 and 7 lacked the proximal telomere DNA repeat signals that

10 New Phytologist Research 1433 Table 2 Characteristics of Arachis hypogaea chromosomes by fluorescence in situ hybridization (FISH) markers Chromosome Marker no. Marker(s) a positions b Chromosome no. Marker(s) A1 02G01 C B1 Telo C H-b C A2 45S rdna C B2 no n/a A3 5S rdna C B3 02G01 C 5S rdna C H-b C 45S rdnas C A4 H-b C B4 02G01 C A5 01B12 T B5 01B12 T H-b C 02G01 C Telo c I A6 H-b C B6 02D10 C 02G01 C H-b C A7 H-b C B7 02G01 C Telo ST 45S rdna I A8 H-b C B8 02G01 C H-b C A9 B9 02G01 C H-b C A10 45S rdna C B10 02G01 C 45S rdna C H-b C Marker positions a The eight FISH markers used were B-genome centromere satellite repeat (B-c), heterochromatin band repeat (H-b), 5S rdna, 45S rdna, Arabidopsis telomeric repeat DNA (Telo), and bacterial artificial chromosome (BAC) clones 02D10, 02G01 and 01B12, as described in the Materials and Methods section. b Marker positions: C, pericentromeric region; T, telomeric region; I, intercalary region; ST, subtelomeric region; n/a, not applicable. c Only telomere repeat signals at the interstitial sites are listed. were present on the A5 and A7 chromosomes in A. hypogaea; A. duranensis chromosome 7 had B-c centromere repeat signals that were absent in A7 chromosomes in A. hypogaea. The karyotype of A. ipa ensis resembled the B genome of cultivated peanut with few variations (Table 3). Chromosome variation among cultivated peanut varieties These chromosome markers allow us to check for variations among cultivated peanut varieties. Four additional cultivated peanut varieties were obtained from the Plant Genetic Resources Conservation Unit of USDA (PI from Paraguay, PI from Argentina, PI from Uruguay, and PI from Turkey) and analyzed using different FISH markers (Figs S4 S9). The results are summarized in Table 4. Most of the markers were conserved among the tetraploid peanut varieties, except for small amounts of variation in the number of signals for the repetitive markers. This variation may reflect the abundances of the repetitive sequences in varieties cultivated in different regions because of the regional isolation and high inbreeding of cultivated peanut. However, variations in both the number and the position of the ITR signals were observed on A-genome chromosomes, while the ITR signals in the pericentromeric regions of a B-genome chromosome (B1) were conserved. Three chromosomal locations were observed for the ITR signals: subtelomeric regions of chromosome A7, intercalary regions of chromosome A5, and pericentromeric regions of chromosome A4. PI from Paraguay, PI from Turkey, and the karyotyped Chinese variety had signals in both the subtelomeric regions of chromosome A7 and the intercalary regions of chromosome A5; PI from Uruguay had signals in the intercalary regions of chromosome A5 and the pericentromeric regions of chromosome A4; PI from Argentina had signals only in the intercalary regions of chromosome A5. ITR variations among diploid A-genome Arachis species and A. duranensis varieties The origin of cultivated peanut was proposed to be from the hybridization of A. duranensis and A. ipa ensis followed by a chromosome doubling (Seijo et al., 2004, 2007; Grabiele et al., 2012). The karyotypic differences between the A genomes of tetraploid peanut and A. duranensis and the variations of ITRs on A-genome chromosomes among tetraploid peanut prompted us to search for other A. duranensis varieties from regions where peanut originated to see if other varieties had the same ITRs as the A-genome chromosomes of tetraploid peanut. FISH results for 13 additional A. duranensis varieties were analyzed after hybridization with the telomeric DNA repeat probe (Fig. S10). The numbers and chromosomal positions of ITRs in these varieties are summarized in Table 5. ITRs in these varieties ranged from four to 12 loci, and most of these ITRs were localized in the intercalary, centromeric and subtelomeric regions. Large variations were also observed among other diploid A-genome species, from zero in A. diogoi to eight ITRs in A. villosa (Fig. S6a i). Discussion Chromosome identification and karyotyping of peanut FISH allows the observation of targeted DNA directly in cytogenetic preparations, thus providing reliable markers for chromosome identification and karyotyping studies (Jiang & Gill, 2006). FISH-based karyotyping can integrate genetic and physical maps in plants (Jiang & Gill, 2006; Walling et al., 2013) and accelerate genetic and genomic studies. However, the development of a karyotyping system in peanut was not easy because peanut has 40 morphologically similar metacentric chromosomes and there are insufficient numbers of reliable chromosome markers available. Previous karyotyping based on chromosome morphology and rdna markers could not unambiguously identify all chromosomes and thus has limited utility in peanut genome studies (Lavia, 2000; Robledo et al., 2009; Seijo & Robledo, 2010). To solve this problem, we developed a set of FISH markers through the cloning of de novo repetitive DNA from Cot-1 libraries of cultivated peanut (Zhang et al., 2012) and a wild relative (A. duranensis), the screening of peanut BAC clones (Yuksel & Paterson, 2005), and the use of previously reported markers such as

11 1434 Research New Phytologist (a) (b) (c) (d) (e) (f) Fig. 6 Fluorescence in situ hybridization (FISH) karyotyping of Arachis hypogaea. A FISH probe cocktail containing eight probes (Alexa Fluor dUTPlabeled bacterial artificial chromosome (BAC) clone 02D10, B-genome centromere satellite repeat B-c and 45S rdna, pseudocolored green in images; Alexa Fluor dUTP-labeled 5S rdna and BAC clones 02G01 and 01B12, pseudocolored red in images; Alexa Fluor dUTP-labeled Arabidopsis telomeric repeat DNA and heterochromatin band repeat H-b, pseudocolored white in images) was hybridized to metaphase chromosomes of A. hypogaea. (a) Captured karyotyping images of A. hypogaea; (b) chromosomes of A. hypogaea arranged using the Leica CW4000 karyotyping system; (c) 4 0,6- diamidino-2-phenylindole (DAPI)-stained chromosomes (changed to black/white form using the Leica CW4000 karyotyping system); (d) signals for BAC clone 02D10, B-genome centromere satellite repeat B-c and 45S rdna (green); (e) signals for 5S rdna, and BAC clones 02G01 and 01B12 probes (red); (f) signals for Arabidopsis telomeric repeat DNA and heterochromatin band repeat H-b (white). Bar, 10 lm. telomere DNA (Richards & Ausubel, 1988) and rdna (Seijo et al., 2004; Robledo et al., 2009; Seijo & Robledo, 2010). By applying the karyotyping probe cocktails in cultivated peanut, most chromosomes were labeled by one or more markers, with the exception of chromosome B2. This allowed us to identify almost all chromosomes, and to produce the FISH-based karyotype of cultivated peanut and its two diploid relatives. This karyotyping system should be applicable to peanut chromosome studies, genomics, physical mapping, phylogenetics and evolutionary studies. Repetitive sequences in the centromeres of peanut In a previous report, we cloned a centromere satellite repeat (B-c) and a retrotransposon fragment in peanut (Zhang et al., 2012). In this study, another centromere-specific satellite repeat H-b was cloned and localized mostly in the pericentromeric regions of A-genome chromosomes. The centromere is an essential component of the chromosome for sister chromatid adhesion, kinetochore formation and chromosome segregation during eukaryotic cell division. In a functional centromere, the centromere histone H3 variant (CENH3) binds to centromere DNA and recruits other centromere proteins to form the kinetochore to ensure faithful chromosome segregation during mitosis and meiosis (Lamb et al., 2007; Tek et al., 2010; Wang et al., 2011). Two classes of repetitive sequences (centromere satellite repeat and centromere-specific retrotransposons) are usually found in the centromeres of most plant species. For example, megabase arrays of the CentO satellite repeat and the centromere-specific retrotransposon CRR were found in the centromeres of all rice chromosomes (Cheng et al., 2002). Similarly, CentC and CRM are the major repetitive DNA sequences in maize centromeres (Zhong et al., 2002). In this study, we cloned an H-b repetitive sequence, whose distribution seems to be similar to that of the heterochromatin band in A-genome centromeres. However, the H-b probe only labeled 18 A-genome chromosomes in both cultivated peanut and A. duranensis, and it also labeled a few B-genome chromosomes (Fig. 2). In comparison, DAPI staining revealed the heterochromatin bands on all A-genome chromosomes of peanut and A. duranensis, but no heterochromatin bands were observed in the B-genome chromosomes of peanut and A. ipa ensis. Sequence analysis showed the existence of highly conserved H-b repeats in all three species, although no heterochromatin bands were observed on B-genome chromosomes (Fig. 2j). These results may suggest that H-b could be a centromere-specific sequence rather than a heterochromatin band sequence as we originally thought. The cloned repetitive sequences (B-c and H-b) in peanut resemble the centromere satellite DNA, although their functions in peanut centromeres were not studied because the peanut CenH3 gene was not cloned. Sequence comparison of the H-b and B-c satellite repeats revealed variations at the sequence level, although they were generally conserved among the three species (Figs 2j, 3j). The presence of the H-b and B-c

12 New Phytologist Research 1435 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) Fig. 7 Fluorescence in situ hybridization (FISH) karyotyping of Arachis duranensis and A. ipa ensis. A FISH probe cocktail containing eight probes (Alexa Fluor dUTP-labeled BAC clone 02D10, B-genome centromere satellite repeat B-c and 45S rdna, pseudocolored green in images; Alexa Fluor dUTP-labeled 5S rdna, and bacterial artificial chromosome (BAC) clones 02G01 and 01B12, pseudocolored red in images; Alexa Fluor dUTPlabeled Arabidopsis telomeric repeat DNA and heterochromatin band repeat H-b, pseudocolored white) was hybridized to metaphase chromosomes of (a f) A. duranensis and (g l) A. ipa ensis. (a, g) Captured karyotyping images; (b, h) chromosomes arranged using the Leica CW4000 karyotyping system; (c, i) 4 0,6-diamidino-2-phenylindole (DAPI)-stained chromosomes (changed to black/white form using the Leica CW4000 karyotyping system); (d, j) signals for BAC clone 02D10, B-genome centromere satellite repeat B-c and 45S rdna (green); (e, k) signals for 5S rdna, and BAC clone 02G01 and 01B12 probes (red); (f, l) signals for the Arabidopsis telomeric DNA repeat and heterochromatin band repeat H-b (white). Bars, 10 lm. homologs in A. duranensis and A. ipa ensis might suggest a common origin from which the AA- and BB-genome species have evolved. The chromosomal localization of the two satellite repeats (B-c and H-b) in tetraploid peanut is similar to the sum of signals in the two wild diploid relatives. The presence of two different satellite repeats in the centromeres has also been reported in other polyploid or paleopolyploid species such as soybean (Tek et al., 2010). Wild relatives of peanut and chromosome evolution Cultivated peanut, A. hypogaea, was thought to be formed by hybridization of two diploid ancestors, A. duranensis and A. ipa ensis, followed by chromosome doubling (Seijo et al., 2007; Pandey et al., 2012). To study the relationship between cultivated peanut and its wild relatives, the peanut FISH karyotyping cocktail was applied to the two wild species (Fig. 7) to produce their karyotypes. Comparing the A and B genomes of cultivated peanut, we found that every chromosome in the wild species had a counterpart in cultivated peanut. Chromosomes of the cultivated peanut B genome and A. ipa ensis had identical patterns as well as signal intensities of each probe (Figs 6, 7), suggesting that large chromosome rearrangements did not occur in the B-genome chromosomes during evolution. This result supports previous proposals that A. ipa ensis might be one of the ancestors of cultivated peanut and the origin of cultivated peanut was a recent event (3500 yr ago) (Seijo et al., 2004, 2007; Seijo & Robledo, 2010; Nielen et al., 2012). By contrast, variation was observed between A-genome chromosomes and the other putative ancestor, A. duranensis.

13 1436 Research New Phytologist (a) (b) (c) Fig. 8 Schematic diagram of fluorescence in situ hybridization (FISH) karyotypes of peanut and its two wild relatives. (a) Arachis duranensis (AA genome), (b) A. hypogaea (AABB genome) and (c) A. ipa ensis (BB genome). FISH signals are represented by different shapes (stars, triangles, bars and dots) as shown in the diagram. B-c, B-genome centromere satellite repeat; H-b, heterochromatin band repeat; Telomere, telomere DNA repeat; 45S, 45S rdna; 5S, 5S rdna; 02D10, 01B12 and 02G01 are the three bacterial artificial chromosome (BAC) clones used in karyotyping. The sizes of the shapes represent their relative signal intensities on each chromosome. The position of each signal is drawn approximately at its chromosomal localization except for overlapping pericentromeric signals for B-c and H-b, which are separated artificially to show them individually in some chromosomes. Chromosomes A5 and A7 had clear ITR signals on the arms that were absent in the karyotyped A. duranensis variety, although most of the A-genome chromosomes in peanut had identical patterns to those of A. duranensis (Figs 6, 7). Analyses of four more cultivated peanut varieties revealed more variation of the ITRs. In addition to the intercalary ITRs on A5 and the subtelomeric ITRs on A7, ITRs in pericentromeric regions of chromosome A4 were also observed. The number of ITRs on the A-genome chromosomes varied among varieties from different geographic regions (Table 4). Interestingly, the two varieties from China and Turkey, which were not the place of origin of cultivated peanut, had similar karyotypes and identical ITR patterns. By contrast, it seems that the three varieties from South America, which are thought to be the place of origin of cultivated peanut, had more variation in karyotypes and the ITR signals. More variation in the place of origin than at other sites where peanut was introduced and cultivated as a conventional inbred crop is expected because peanut is a self-pollinated plant and is the only cultivated tetraploid species in the genus, although more investigation is needed before drawing this conclusion. Non-end telomere repeats have also been reported in other higher plants and animals as intrachromosomal or interstitial Table 3 Characteristics of Arachis duranensis and A. ipa ensis chromosomes by fluorescence in situ hybridization (FISH) karotyping probes A. duranensis chromosome Marker no. Marker(s) a positions b A. ipa ensis chromosome no. Marker(s) A1 02G01 C B1 Telo c C H-b C A2 45S rdna C B2 no n/a A3 5S rdna C B3 02G01 C 5S rdna C H-b C 45S rdnas C A4 H-b C B4 02G01 C A5 01B12 T B5 01B12 T H-b C 02G01 C A6 H-b C B6 02D10 C 02G01 C A7 B7 02G01 C H-b C 45S rdna I A8 H-b C B8 02G01 C H-b C A9 B9 02G01 C H-b C A10 45S rdna C B10 02G01 C 45S rdna C H-b C Marker positions a The eight FISH markers used were B-genome centromere satellite repeat (B-c), heterochromatin band repeat (H-b), 5S rdna, 45S rdna, Arabidopsis telomeric repeat DNA (Telo), and bacterial artificial chromosome (BAC) clones 02D10, 02G01, and 01B12, as described in the Materials and Methods section. b Marker positions: C, pericentromeric region; T, telomeric region; I, intercalary region; n/a, not applicable. c Only telomere repeat signals at the interstitial sites are listed. telomeric repeats (Uchida et al., 2002; Ruiz-Herrera et al., 2008; He et al., 2013). The prominent FISH signals of telomere repeats on interstitial sites of peanut chromosomes B1, A5 and A7 suggested that they were large telomere arrays. The ITR on B1 chromosome seems to be in the pericentromeric region, while the arrays on A5 and A7 were localized to the chromosome arms. ITRs were proposed to be the relics of chromosome rearrangement events such as chromosome fusions (Uchida et al., 2002; Ruiz-Herrera et al., 2008) and paracentric inversions (Peters et al., 2012). In such scenarios, chromosome fusion or an inversion event would have happened in A5 and A7 of A. hypogaea, but no apparent morphological differences were observed between A. duranensis and the A-genome chromosomes of A. hypogaea. In addition, if paracentric inversion events occurred, recombinations would happen and result in the segregation of normal offspring and those with paracentric inversions. We examined four different peanut varieties from different geographic regions. Although ITR variations existed among them, they all had ITR signals on one or two pairs of A-genome chromosomes. This observation argues against the hypothesis that ITRs originated from paracentric inversions, although we could