Ji Lei, Jiwen Zhou, Hojie Sun, Wento Wn, Jin Xio, Chunxi Yun,Miroslv Krfiátová, Jroslv Dole?el, Hiyn Wng,, Xiue Wng

aState Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, Nanjing Agricultural University/JCIC-MCP,Nanjing 210095, Jiangsu, China

bInstitute of Experimental Botany, Czech Academy of Sciences, Centre of the Region Hana for Biotechnological and Agricultural Research,?lechtitel? 31, 77900 Olomouc, Czech Republic

Keywords:

ABSTRACT Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement. Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor, hampering accurate characterization of small segmental alien introgressions. We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6V(6VS).FISH with seven of them resulted in clear signals on H. villosa chromosomes. Using these, we constructed FISH karyotypes for H.villosa using oligo-6VS-1 and oligo-6VS-35 oligonucleotides and characterized the distribution of the two probes in five different H. villosa accessions. The new FISH probes can efficiently characterize H. villosa introgressions into wheat.

1. Introduction

Haynaldia villosa(L.)(Candargy,Dv;2n = 14,VV genome)Schur(syn.Dasypyrum villosum(L.)Schur)is a wild relative of wheat.Several genes of H.villosa have been transferred into common wheat as alien chromosome translocations, including powdery mildew resistance gene Pm21 and Pm55[1],wheat yellow mosaic(WYM)resistance gene Wss1[2,3],stem rust resistance gene Sr52[4],kernel softness genes[5,6],seed storage protein genes[7-9],yield-related genes[10]and cereal cyst nematode resistance gene CreV [11]. To facilitate more extensive use of H.villosa genes in wheat improvement,methods for rapid and precise identification of H. villosa chromosomes or chromosome segments in wheat genetic background are urgently needed.

The first attempt to characterize H.villosa chromosomes by fluorescence in situ hybridization(FISH)was by Uslu et al.

[12]who used repetitive DNA sequences as probes.However,only four of the seven chromosomes could be unambiguously distinguished. Yuan and Tomita [13] identified each of the seven H. villosa chromosomes using as probes a combination of the repetitive sequences pDvTU383, pTa71, and pTa794.Zhang et al. [14] constructed a molecular karyotype of H.villosa chromosomes using pSc119.2, pAs1, 45S rDNA, and 5S rDNA as FISH probes. Using a GAA microsatellite as a probe,Grosso et al. [15] identified all H. villosa chromosomes but 4V.Recently, Sun et al. [16] established a FISH karyotype of five different accessions of H. villosa using a set of cloned and synthesized oligonucleotide (oligo) probes. Thus, at present FISH with repetitive DNA probes can unambiguously identify individual chromosomes of H. villosa. However, the procedures are time-consuming,and some of the probes used,such as oligo-pSc119.2, oligo-pAs1, oligo-pDvAfa, (GAA)10, and(AAC)10are not H. villosa chromosome- or chromosome segment-specific. Thus, the development of H. villosa chromosome-specific or chromosome segment-specific oligo probes remains desirable.

The objective of the present study was to develop oligo probes specific for H. villosa chromosomes and investigate their potential application in chromosome engineering.

2. Materials and methods

2.1. Plant material

Six accessions of Haynaldia villosa were used. The accession 91C43 was kindly provided by the Cambridge University Botanic Garden. The five remaining accessions 2011I-84(PI368886), 2011I-87 (PI598390), 2011I-96 (PI598399), 2011I-113(W67286), and 2011I-133 (W621717) were obtained from the American Germplasm Resources Information Network.

Triticum aestivum cv. Chinese Spring and a Triticum durum-Haynaldia villosa amphiploid are maintained at the Cytogenetics Institute, Nanjing Agricultural University.

2.2. DNA sequence of H. villosa chromosome 6VS and oligonucleotide probe design

Sequence reads of chromosome arm 6VS were retrieved from the NCBI Sequence Read Archive (SRA), accession number PRJNA590539. Sets of 2,500,000 paired-end reads of 6VS were selected.Nucleotide sequences of 190 nt from 11 bp to 200 bp were selected from each read.Tandem repeats were identified using Repeatexplorer2 [17] (http://www.repeatexplorer.org)and clustered. Based on the tandem repeat sequences, oligo probes (55 ± 4) bp in length were developed using Oligo7 software [18] and synthesized by TsingKe Biological Technology(Beijing,China).The 5′ends of oligonucleotide sequences were labeled with 6-carboxyfluorescein (6-FAM, green) or 6-carboxytetramethylrhodamine (TAMRA, red). Probes oligopAs1-1,pAs1-4,and pAs1-6[16]were also used.

2.3. Cell cycle synchronization and preparation of mitotic chromosomes

Cell cycle synchronization and slide preparation followed Vrána et al. [19] with minor modifications. Seeds of H. villosa were soaked in water for 24 h and germinated on moist filter paper for 2 days at 25 °C.When the roots grew to about 2.5 cm long, the roots were treated with 2 μmol L?1amiprophosmethyl (APM) for 2.5 h to accumulate the cycling cells in metaphase, after which the root tips were cut off and treated in a nitrous oxide gas chamber for 1 h [16]. They were then fixed in ice-cold 90% acetic acid for 7 min,washed with sterile distilled water and stored in 70% ethanol at ?20 °C until use.

For slide preparation, root tips were washed in ddH2O for 5 min.One or two 2 mm-long meristems were placed in 20 μL of 4% cellulase Onozuka R-10 (Yakult, Tokyo, Japan), 1% pectolyase Y23 (Karlan, Osaka, Japan) in KCl buffer(75 mmol L?1KCl, 7.5 mmol L?1EDTA, pH 4.0) and incubated for 50 min at 37 °C. Digested meristems were washed for 5 min in Tris-EDTA buffer and twice in 70% ethanol. Meristems were separated with a needle in 20 μL of acetic acidmethanol mix (9:1) and immediately dropped onto precleaned glass slides and placed in a humid chamber. Finally,dried preparations were UV cross-linked.

2.4.FISH and fluorescence microscopy and imaging

Total genomic DNA of H.villosa was labeled with fluorescein-12-dUTP by nick translation and used as a probe for genomic in situ hybridization (GISH). Oligo-FISH was performed on metaphase spreads following Cuadrado et al. [20] with minor modifications. The slides were examined under fluorescence microscope (Olympus, BX51, Olympus, Tokyo, Japan) and separate images for each optical filter set were captured using a CCD camera(Olympus,DP72,Olympus,Tokyo,Japan).The images were optimized for contrast and brightness using Adobe Photoshop CC 14.2.

3. Results and discussion

The seven designed oligo probes produced strong and distinct fluorescent bands on H. villosa chromosomes (Tables S1, S2). Six probes (oligo-6VS-1, oligo-6VS-35, oligo-6VS-57,oligo-6VS-44, oligo-6VS-56, and oligo-6VS-73) produced hybridization signals on every chromosome of H. villosa (Fig. 1-A-F). Oligo-6VS-18 probe produced signals on five chromosome pairs (Fig. 1-G). To identify the chromosomes of H. villosa to which oligo-6VS-18 hybridized, oligo-pAs1 was also used.The oligo-6VSCL18 probe produced no signals on chromosomes 5V and 7V (Fig. 1-G).

To identify oligo probes specific for H. villosa, probes were used simultaneously with total genomic DNA of H. villosa for oligo-FISH/GISH in the T. durum-H. villosa amphiploid(AABBVV) and the common wheat Chinese Spring. These experiments showed that oligo-6VS-1, oligo-6VS-18, oligo-6VS-44, oligo-6VS-56, oligo-6VS-57, and oligo-6VS-73 probes produced hybridization signals exclusively on chromosomes of H.villosa.Hybridization signals with oligo-6VS-1 occurred in telomeric, subtelomeric, centromeric and pericentromeric chromosome regions (Fig. 2-A). Whereas three probes (oligo-6VS-57, oligo-6VS-44, and oligo-6VS-18) produced distinct hybridization signals in telomeric regions (Fig. 2-B-D), two probes (oligo-6VS-56 and oligo-6VS-73) generated signals in both telomeric and subtelomeric regions of H.villosa chromosomes(Fig.2-E,F).No hybridization signals were observed on T. durum A- and B-genome chromosomes or common wheat A-, B-, and D-genome chromosome with any of these probes(Figs. 2-A-F and S1-A, C-G). The oligo-6VS-35 probe produced hybridization signals on chromosomes of H. villosa and some chromosomes of the A and B subgenomes in T. durum (Fig. 2-G)and the A, B, and D genomes in common wheat.

Fig.1- FISH with oligonucleotide probes on mitotic metaphase spreads of H.villosa 91C43.(A1-A3 to E1-E3) Hybridization patterns of oligo-6VS-1,oligo-6VS-35,oligo-6VS-57, oligo-6VS-44,and oligo-6VS-56(red).(F1-F3) Hybridization pattern of oligo-6VS-73(green).(G1-G3) Hybridization of oligo-6VS-18(green)and oligo-pAs1(red).No signals were observed on chromosomes 5V and 7V.All the chromosomes were blue.Scale bars,10 μm.

Fig.2-FISH with oligonucleotide probes on mitotic metaphase spreads of T.durum-H.villosum amphiploid(AABBVV genome).(A1-A4 to E1-E4 and G1-G4) Simultaneous FISH and GISH with oligo-6VS-1,oligo-6VS-57,oligo-6VS-44, oligo-6VS-18,oligo-6VS-56,oligo-6VS-35(red)and H.villosa genomic DNA(green).(D1-D4)Simultaneous FISH and GISH with oligo-6VS-18(green)and H.villosa genomic DNA(green).No hybridization signals of oligo-6VS-18 were found on chromosomes 5V and 7V.(F1-F4)Simultaneous FISH and GISH with oligo-6VS-73(green)and H. villosa genomic DNA(green).All the chromosomes were blue.Scale bars,10 μm.

Fig.3- FISH with oligonucleotide probes oligo-6VS-35 and oligo-6VS-1 on mitotic metaphase spreads of six H.villosa accessions. All the chromosomes were blue.Scale bar,10 μm.

Probes oligo-6VS-1, oligo-6VS-18, oligo-6VS-44, oligo-6VS-56, oligo-6VS-57, and oligo-6VS-73 produced hybridization signals on chromosomes of H. villosa, but not in the tested durum wheat and common wheat Chinese Spring (Fig. S1-A,C-G). B2DSC [21] was used to predict the copy number and physical distribution of these probes in the Chinese Spring genome,using default parameters(pident = 85,qcovhsp = 80)for the blast and filter steps. The sequences of oligo-6VS-18,oligo-6VS-56, and oligo-6VS-57 probes did not match Chinese Spring reference sequences. These findings suggested that signal absence of these three probes was due to no copy numbers for detection in Chinese Spring (Table S3). The sequences of probe oligo-6VS-44 matched on chromosome 4A, 2D and 4D, with the predicted copy number 3, 59 and 48,respectively (Fig. S2). The sequence of probe oligo-6VS-1 matched on chromosome 1B, 3B and 4B with the largest predicted copy numbers of 3, 1, and 2, respectively (Fig. S3).The sequence of probe oligo-6VS-73 matched on every chromosome of Chinese Spring (Fig. S4). These findings suggested that signal absence was due to copy numbers too low for detection in Chinese Spring(Table S3).

The consensus sequence of oligo-6VS-35 probe showed 92% identity with the published tandem repeat pSc119.2 isolated from Secale cereale (Fig. S5). The hybridization signals of oligo-6VS-35 probe were almost identical to, but better defined and more intense than, those of the oligo-pSc119.2 probe (Fig. S6).Hybridization signals with the oligo-6VS-1 probe were located almost exclusively in telomeric regions of the short arms and subtelomeric regions of long arms of every chromosome of H.villosa(Fig.S7).Thus,an oligo-based FISH karyotype of H.villosa could be constructed using FISH with the oligo-6VS-1 probe in combination with the oligo-6VS-35 probe(Fig.3).

After the oligo-6VS-35 and oligo-6VS-1 probes were tested in H.villosa line 91C43,they were used for FISH in five other H.villosa accessions: 2011I-84, 2011I-87, 2011I-96, 2011I-113, and 2011I-133. Except for a few dissimilarities, no marked differences in hybridization signal distribution were observed among the six accessions of H. villosa, confirming the utility of such repetitive sequences for the identification of individual chromosomes in this species (Fig.3). Minor differences in the distribution of hybridization signals of the two probes were observed between accession 91C43 and the other five accessions(Fig.3).

We observed differences in probe hybridization patterns between members of chromosome pairs, indicating heterozygosity. For example, one telomeric signal of oligo-6VS-35 was missing in one of the 3V chromosomes in accession 2011I-113, in one of the 2V chromosomes in accessions 2011I-84 and 2011I-96, and in chromosome 1V of accessions 2011I-84 and 2011I-113. This finding agrees with those of previous cytogenetic studies in H. villosa. For example, Xiao et al. [22] found different signal patterns for oligo-3B117.3 probes between members of chromosome pairs 3V and 7V in H. villosa accession W621717. Sun et al. [16] found that hybridization signals of oligo-pAs1 and oligo-pDvAfa probes in centromeric regions on chromosome 5V and telomeric signals of VP14 probe on chromosome 3V of H. villosa were heterozygous. This phenomenon is a consequence of the biology of H. villosa, an annual diploid cross-pollinating species. Such heterozygosity has been reported for other open-pollinated grass species, including Secale cereale [23] and the diploid Aegilops cristatum [24,25].

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.02.008.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

This research was supported by the National Key Research and Development Program of China (2016YFD0102001), the National Natural Science Foundation of China (31571653,31771782, 31201204, 31501305), International Cooperation and Exchange Programme of the National Natural Science Foundation of China (31661143005), Introducing the Technique to Exploring the Genetic Germplasm Based on the Chromosome Sorting and Sequencing (2015-Z41), the Special Fund of Jiangsu Province for the Transformation of Scientific and Technological Achievements (BA2017138). JD and MK were supported by European Regional Development FundProject “Plants as a Tool for Sustainable Global Development” (CZ.02.1.01/0.0/0.0/16_019/0000827).

Author contributions

Haiyan Wang, Xiue Wang and Jin Xiao designed the experimental plan.Jia Lei,Jiawen Zhou,Haojie Sun and Chunxia Yuan performed all experiments.Wentao Wan and Jin Xiao analyzed the sequence of 6VS.Miroslava Karafiátová and Jaroslav Dole?el isolated chromosome 6VS. Haiyan Wang, Xiue Wang, Jia Lei,Jiawen Zhou, and Jaroslav Dole?el wrote the manuscript. All authors have read and approved the final manuscript.