Research Article |
Corresponding author: Honggang Wang ( hgwang@sdau.edu.cn ) Academic editor: Andrzej Joachimiak
© 2017 Fang He, Yuhai Wang, Yinguang Bao, Yingxue Ma, Xin Wang, Xingfeng Li, Honggang Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
He F, Wang Y, Bao Y, Ma Y, Wang X, Li X, Wang H (2017) Chromosomal constitutions of five wheat – Elytrigia elongata partial amphiploids as revealed by GISH, multicolor GISH and FISH. Comparative Cytogenetics 11(3): 525-540. https://doi.org/10.3897/CompCytogen.v11i3.11883
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A combination of meiotic pairing analysis and in situ hybridization (genomic in situ hybridization [GISH], multicolor GISH [mcGISH] and fluorescence in situ hybridization [FISH]) of five Triticum aestivum (Linnaeus, 1753) - Elytrigia elongata (Podpěra, 1902) (2n = 10x = 70) amphiploids was employed to investigate the genomic constitution and relationships between wheat and alien chromosomes. GISH, multicolor GISH and FISH patterns of mitotic chromosomes indicate that the genomic constitution of the five partial amphiploids (XY693, XY7430, SN19, SN20 and SN122) are 14A + 12B + 14D + 8Js + 8J, 12A + 16B + 14D + 2St + 8Js + 2J + 2 W-E, 14A + 14B + 14D + 4St + 8Js, 14A + 14B + 14D + 2St + 10Js + 2J, and 14A + 14B + 14D + 2St + 8Js + 4J, respectively. Analysis of meiotic chromosome pairing in the F1 hybrids between these five partial amphiploids suggests that SN20 and SN122 are the most closely related amphiploids and are somewhat related with XY693 and XY7430. However, the alien chromosome constitutions of SN19 differed from the other four amphiploids. In addition, a new pairing between wheat and E. elongata chromosomes was distinguished in some cells of the hybrids SN19 × XY7430, SN20 × XY7430 and SN122 × XY7430.
Elytrigia elongata , partial amphiploid, cytogenetic, in situ hybridization
Elytrigia (Á. Löve, 1980) species, such as those in the Triticeae tribe of the family Poaceae, contain excellent perennial forages with resistance to cold, drought, salinity, and various diseases (
Previous research indicated that E. elongata is an autodecaploid with the JJJJJ genomes proposed by
Wheat - alien amphiploids, which are stable and highly fertile, represent a crucial intermediate step in the transfer of agronomically useful genes from wheatgrass species to wheat (
Developed approximately 30 years ago in China, XY693 and XY7430 are two wheat - E. elongata partial amphiploids with 56 chromosomes. They possess many useful genes. XY693 exhibits excellent resistance to leaf rust, powdery mildew and Wheat streak mosaic virus (
Genomic in situ hybridization (GISH) is a widely used and effective method for detecting alien chromatin in wheat-alien species amphiploids. Multicolor GISH (mcGISH), which employs several different genomic probes, can be used to simultaneously visualize two or more genomes in a polyploid species.
In the present study, we used a combination of GISH, mcGISH and FISH to examine the cytogenetic composition of five partial amphiploids. In addition, the relationships between wheat and the alien chromosomes of the wheat - E. elongata amphiploids were assessed via the combination of the meiotic behavior of the F1 hybrids.
Plant materials used in this study included E. elongata, Pseudoroegneria spicata (Pursh, 1814) (StSt, 2n = 14), Triticum urartu (Gandilyan, 1972) (AA, 2n = 14), Aegilops speltoides (Gerlach & Dyer, 1980) (SS, 2n = 14), Aegilops tauschii (Cosson, 1850) (DD, 2n = 14), the common wheat Yannong15 and five partial amphiploids (XY693, XY7430, SN19, SN20 and SN122). E. elongata, T. urartu, A. speltoides and A. tauschii were provided by Prof. Zhensheng Li (formerly of the Northwest Institute of Botany at the Chinese Academy of Sciences, Yangling, China). Ps. spicata was provided by Prof. Lihui Li (Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, China). XY693 was derived from the cross T. aestivum ‘Xiao Yan 2’// T. aestivum ‘Lin 7’/ E. elongata. Similarly, XY7430 was derived from the cross T. aestivum ‘Xiang Yang 4’// T. aestivum ‘mi sui zao’/ E. elongata. The partial amphiploids SN19, SN20 and SN122 were novel germplasms developed from the hybridization of E. elongata, the common wheat Yannong15 and Shannongfu63 at the Agronomy College of Shandong Agricultural University, Tai’an, China. All plant materials are maintained in our laboratory through self-crosses.
Seeds were germinated at 25°C on moist filter paper in Petri dishes, maintained at 4°C for approximately 24 h, and then transferred to 25°C. Roots 1- to 2-cm in length were cut and treated in ice water for approximately 24 h before fixation in Carnoy’s solution. After fixation, the root tips were stained and squashed in carbol fuchsin, and their mitotic chromosomes were observed under a microscope. When the plants reached the flag leaf stage, spikes were sampled and anthers at metaphase I (MI) of meiosis were fixed in Carnoy’s solution, dissociated in 1 M HCl at 60°C for 6 to 8 min, and homogenized in 1% acetocarmine. E. elongata and Ps. spicata DNA were labeled with fluorescein-12-dUTP by the nick translation method and used as probes. Sheared genomic DNA from YN15 (AABBDD, 2n = 42) was used as blocking DNA. The slides were counterstained with propidium iodide (PI, 0.25 mg/mL) in the Vectashield mounting medium (Vector Laboratories, USA).
The total genomic DNA was isolated from young leaves of E. elongata, T. urartu, A. speltoides and A. tauschii. The total genomic DNA of diploid E. elongata and T. urartu was labeled with fluorescein-12-dUTP, and the total genomic DNA of A. tauschii was labeled with Texas-red-5-dUCP via the nick translation method. The total genomic DNA of A. speltoides was used for blocking (at a ratio of 1:160). After hybridization, the slides were washed in 2 × saline sodium citrate (SSC) and mounted in Vectashield mounting medium (containing 1.5 mg/mL DAPI; Vector Laboratories, USA).
FISH was performed after mcGISH analysis using two probes, pTa535 labeled with fluorescein-12-dUTP, and pSc119.2 labeled with Texas-red-5-dUCP. The two probes were mixed at a ratio of 1:1 before hybridization. The slides were washed in 2 × SSC and mounted in Vectashield mounting medium (containing 1.5 mg/mL DAPI; Vector Laboratories, USA). The detailed procedures for the chromosome preparation and hybridization were previously described by
To further determine the five partial amphiploids by mcGISH, the mitotic chromosomes were probed using D-genomic DNA (A. tauschii) with Texas-red-5-dCTP (red), and the total genomic DNA of E. elongata and T. urartu was probed with fluorescein-12-dUTP (green) and blocked by the S-genome (A. speltoides) DNA. Using this technique, the A-, B-, and D-genomes from common wheat and the genomes from E. elongata could be simultaneously distinguished. The A-, B-, and D-genome chromosomes were labeled with yellow, brown/ gray and red/ pink fluorescence, respectively, whereas the alien chromosomes of E. elongata were labeled with green fluorescence.
Chromosome constitutions of these five wheat - E. elongata amphiploids (XY693, XY7430, SN19, SN20 and SN122) were analyzed using mcGISH (Table
Line | Chromosome no. | Origin of alien chromosomes | Genomic chromosome constitution* |
---|---|---|---|
XY693 | 2n=56 | 16 of E. elongata | 14A + 12B + 14D + 8Js + 8J |
XY7430 | 2n=56 | 12 of E. elongata | 12A + 16B + 14D + 2St + 8Js + 2J + 2 W-E |
SN19 | 2n=54 | 12 of E. elongata | 14A + 14B + 14D + 4St + 8Js |
SN20 | 2n=56 | 14 of E. elongata | 14A + 14B + 14D + 2St + 10Js + 2J |
SN122 | 2n=56 | 14 of E. elongata | 14A + 14B + 14D + 2St + 8Js + 4J |
McGISH patterns and FISH analysis of chromosomes inSN19 (A), SN20 (C) and SN122 (E). Yellow denotes the A-genome chromosomes, gray indicates the B-genome chromosomes, red represents the D-genome chromosomes and green denotes the E. elongata chromosomes or chromosomal fragments. The asterisks indicate the wheat - E. elongata translocation chromosomes. FISH on the same metaphase chromosome spreads are simultaneously presented in lines SN19 (B), SN20 (D) and SN122 (F) by pTa535 (green) and pSc119.2 (red).
Although the mcGISH patterns of the mitotic chromosomes indicated that the wheat - E. elongata amphiploid SN19 has 54 chromosomes and the remaining four partial amphiploids have 56 chromosomes, the number of alien chromosomes ranged from 12 to 16, suggesting that chromosome deletion and substitution occurred in the wheat genome. Three-color FISH, with the simultaneous hybridization of the repetitive DNA probes pTa535 and pSc119.2, has been successfully employed on mitotic metaphase cells of these five partial amphiploids. Chromosome elimination and addition has been detected with these probes (Figs
GISH patterns of the mitotic chromosomes probed with St-genomic DNA from Ps. spicata and blocked with ABD-genomic DNA from Yannong15 wheat. A XY693 B XY7430 C SN19 D SN20 E SN122 F Karyotype of chromosomes from Fig.
Average metaphase I configuration per meiocyte in the F1 hybrids between partial amphiploids.
Hybrid | No. of plants | No. of cells | Meiotic configuration of amphiploids hybrids chromosomes | No. of alien chromosomes | Meiotic configuration of E. elongata chromosomes | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
I | IIring | IIrod | III | IV | I | IIring | IIIrod | ||||
SN20 × SN19 | 6 | 83 | 11.81 (7-21) |
17.54 (16-20) |
3.88 (1-6) |
0.04 (0-1) |
0.06 (0-1) |
13 | 9.1 (7-13) |
1.03 (0-2) |
0.92 (0-1) |
SN19 × SN122 | 5 | 64 | 12.08 (9-17) |
16.59 (16-18) |
4.75 (2-6) |
0 |
0.06 (0-1) |
13 | 9.45 (5-13) |
0.99 (0-3) |
0.78 (0-1) |
SN122 × SN20 | 5 | 79 | 3.44 (2-6) |
21.49 (17-24) |
4.78 (3-8) |
0.01 (0-1) |
0 |
14 | 2.5 (2-4) |
3.96 (3-4) |
1.79 (1-2) |
SN19 × XY7430 | 5 | 66 | 10.93 (7-18) |
16.37 (14-19) |
5.51 (2-9) |
0.05 (0-1) |
0.04 (0-1) |
12 | 9.32 (7-15) |
0.77 (0-5) |
0.57 (0-1) |
SN20 × XY7430 | 6 | 76 | 5.98 (4-12) |
17.2 (12-19) |
6.37 (4-9) |
0.11 (0-1) |
0.64 (0-3) |
13 | 4.18 (1-11) |
3.06 (1-5) |
1.35 (0-3) |
SN122 × XY7430 | 7 | 88 | 8.38 (4-14) |
15.7 (13-20) |
7.29 (3-9) |
0.27 (0-1) |
0.21 (0-1) |
13 | 6.1 (1-11) |
2.76 (1-4) |
0.69 (0-2) |
XY693 × SN122 | 6 | 94 | 5.98 (2-12) |
17.14 (16-19) |
7.68 (7-9) |
0.13 (0-1) |
0 |
15 | 5.26 (3-11) |
2.94 (1-4) |
1.93 (0-3) |
XY693 × SN19 | 7 | 69 | 11.61 (5-13) |
15.96 (15-17) |
5.05 (5-8) |
0.14 (0-1) |
0.24 (0-2) |
14 | 10.24 (2-12) |
0.87 (0-3) |
1.01 (0-3) |
XY693 × SN20 | 6 | 85 | 6.11 (2-10) |
17.34 (15-19) |
6.92 (4-9) |
0.14 (0-1) |
0.24 (0-2) |
15 | 5.68 (3-9) |
2.84 (2-4) |
1.82 (1-3) |
GISH patterns of meiotic chromosomes probed with E. elongata genomic DNA and blocked with Yannong15 wheat genomic DNA. A SN122 × XY7430 B SN20 × XY7430 C XY693 × SN20 D XY693 × SN122 E SN20 × SN19 F SN19 × SN122. Arrows indicate pairing between the wheat and E. elongata chromosomes.
McGISH patterns and FISH analysis of chromosomes in XY693 (A), XY7430 (B). Yellow denotes the A-genome chromosomes, gray indicates the B-genome chromosomes, red represents the D-genome chromosomes and green denotes the E. elongata chromosomes or chromosomal fragments. The asterisks indicate the wheat - E. elongata translocation chromosomes. FISH on the same metaphase chromosome spreads are simultaneously presented in lines XY693 (C), XY7430 (D), by pTa535 (green) and pSc119.2 (red).
The alien chromosome constitutions of five wheat - E. elongata amphiploids were analyzed using GISH (Table
Probing the partial amphiploid XY693 with St genomic DNA and blocking with wheat DNA revealed that 16 chromosomes emitted greenish-yellow hybridization signals: a Js type of signal was detected on 4 pairs of chromosomes, a J type of signal on 8 chromosomes, and four translocation signals on the telomere of four wheat chromosomes (Fig.
Five partial wheat - E. elongata amphiploids were crossed with each other, and their meiotic chromosome behavior was studied in all nine F1 hybrids (except XY693 × XY7430). E. elongata chromatin was distinguished in the pollen mother cells (PMCs) at MI of the F1 hybrids using GISH, and the values for each meiotic parameter are presented in Table
As presented in Table
In contrast to the regular meiotic behavior of the parents, the values for the F1 hybrids were highly variable. Based on the lowest frequencies of unpaired chromosomes, the most closely related strains were SN20 and SN122. Only an average of 2.5 E. elongata chromosome univalents per cell was observed in the hybrid SN122 × SN20. Another grouping involved SN20 × XY7430 (Fig.
To date, numerous studies have reported that many of the chromosomes contained in alien genomes are products of translocations either between Elytrigia chromosomes or with wheat chromosomes. Using GISH and mcGISH,
The genomic composition of the decaploid species E. elongata has been a subject of interest for a considerable time (
Chromosome counting on the metaphase spreads after GISH revealed 16 alien chromosomes in the partial amphiploid XY693 and 12 alien chromosomes in the partial amphiploid XY7430. However, the chromosome number of these two amphiploids was consistently 56. This observation suggests that the chromosome substitutions occurred between the E. elongata genome and wheat genome. Similar results were obtained in previous studies.
The data from the GISH and meiotic chromosome pairing analysis in the F1 hybrids between different partial amphiploids were useful in identifying similar alien genomes. Based on the lowest frequency of E. elongata unpaired chromosomes, the two strains SN20 and SN122 would appear to be the most closely related of all the partial amphiploids examined. The highest frequency of the univalent E. elongata chromosome in the hybrids SN20 × SN19, SN19 × SN122, XY693 × SN19 and SN19 × XY7430 suggested that the alien chromosome constitutions of SN19 differed from the other four amphiploids. High E. elongata chromosome univalent and bivalent frequencies were observed in the other hybrids, including SN20 × XY7430, SN122 × XY7430, XY693 × SN122 and XY693 × SN20. These results indicate that the partial amphiploids SN20 and SN122 were related to XY693 and XY7430. Previous reports demonstrated that the alien chromosome constitutions of XY693 and XY7430 differed (
Partial wheat – E. elongata amphiploids, with a high cross-compatibility with wheat, are desirable ‘‘bridge’’ materials for transferring disease resistance genes from E. elongata to wheat. In previous studies, these five partial amphiploids demonstrated regular meiotic behavior and are highly fertile. Based on the chromosome constitution of these amphiploids, we manipulated Elytrigia chromosomes in these amphiploids to eliminate unwanted Elytrigia chromatin and to introduce useful agronomic traits into wheat. Due to the unpredictable and unstable meiotic behavior of the F1 hybrids between the lines, the combining of traits by intercrossing partial amphiploids is not a promising alternative.
This research was supported by the National Key Research and Development Plan of China (2016YFD0102004) and National Natural Science Foundation of China (Grant No. 31501298).