Characterization of chromosome constitution in three wheat - Thinopyrum intermedium amphiploids revealed frequent rearrangement of alien and wheat chromosomes

Thinopyrum intermedium (2n = 6x = 42) is an important wild perennial Triticeae species exhibiting many potentially favorable traits for wheat improvement. Wheat-Th. intermedium partial amphiploids serve as a bridge to transfer desirable genes from Th. intermedium into common wheat. Three octoploid Trititrigia accessions (TE261–1, TE266–1, and TE346–1) with good resistances to stripe rust, powdery mildew and aphids were selected from hybrid progenies between Th. intermedium and the common wheat variety ‘Yannong 15’ (YN15). Genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH) and multicolor GISH (McGISH) analyses demonstrated that the three octoploid Trititrigia possess 42 wheat chromosomes and 14 Th. intermedium chromosomes. The 14 alien (Th. intermedium) chromosomes belong to a mixed genome consisting of J-, JS- and St-genome chromosomes rather than a single J, JS or St genome. Different types of chromosomal structural variation were also detected in the 1A, 6A, 6B, 2D and 7D chromosomes via FISH, McGISH and molecular marker analysis. The identity of the alien chromosomes and the variationes in the wheat chromosomes in the three Trititrigia octoploids were also different. The wheat-Th. intermedium partial amphiploids possess 14 alien chromosomes which belong to a mixed genome consisting of J-, JS- and St- chromosomes, and 42 wheat chromosomes with different structural variations. These accessions could be used as genetic resources in wheat breeding for the transfer of disease and pest resistance genes from Th. intermedium to common wheat.


Background
Thinopyrum intermedium (Host) Barkworth & D.R. Dewey [syn. Agropyron intermedium (Host) Beauvor and Elytrigia intermedia (Host) Nevski] (2n = 6x = 42; genome formula E e E e E b E b StSt, JJJ S J S StSt, EEVVStSt or J r J r J vs J vs StSt) is considered as a segmental autoallohexaploid, and its genome constitution hanging in doubt is a research hot subject in Triticeae crop research. The presence of S genome (later changed to St by Wang et al. [1]) in Th. intermedium was first reported by Liu and Wang [2] and confirmed by Zhang et al. [3], leading to the genome formula JJJJStSt or EEEEStSt, where J genome is from Th. bessarabicum (2n = 14, JJ or E b E b ), St genome is from Pseudoroegneria strigosa (2n = 14, StSt) and E genome is from Th. elongatum (2n = 14, EE or E e E e ). Also by using genomic in situ hybridization, Chen et al. considered that the genome constitution of Th. intermedium is JJJ S J S StSt where J S might be a modified J or E [4]. Ji et al. concluded that the genome constitution of Th. intermedium is E e E e E b E b StSt by using multicolor GISH (McGISH) [5]. Analyzing the chloroplast trnL-F sequence, granule-bound starch synthase I (GBSSI) and GISH, Mahelka et al. (2011) inferred that genomes of Th. intermedium are related to Ps. strigosa (StSt), Dasypyrum villosum (2n = 14, VV), and a complex origin from Th. elongatum (EE) and Aegilops tauschii (2n = 14, DD) [6]. J r J r J vs J vs StSt was proposed by Wang et al., who made use of EST-SSR markers to analyze the genome evolution of Th. intermedium, and J r and J vs refer to ancestral genomes of J e (E) and J b (J), respectively [7].
Trititrigia octoploids were developed from hybrid progenies between Th. intermedium and common wheat and inherited numerous excellent traits from Th. intermedium, such as resistance to powdery mildew, leaf rust, stem rust and stripe rust [19,20]. Furthermore, it is easier to successfully cross common wheat with Trititrigia octoploids than with Th. intermedium. Thus, Trititrigia octoploids are valuable germplasm to hybridize with common wheat, continually producing wheat-alien addition, substitution and translocation lines that can transfer beneficial traits from alien species into wheat [9,21,22].
Genomic in situ hybridization (GISH) is generally used to distinguish alien chromosomes in the common wheat background [30,31]. When Pseudoroegneria strigosa (2n = 2x = 14, StSt) genomic DNA was used as a probe in GISH analysis of Th. intermedium, the karyotype of Th. intermedium chromosomes could be classified into three groups. The first group is the St genome, completely labeled by probe signals, the second group is the J S genome labeled only in the centromere areas, and the third group is the J genome labeled only in the subtelomeric position [4,32]. As GISH with the St-genome DNA probe could effectively distinguish the three subgenome of Th. intermedium, it was widely used in the identification of Th. intermedium-derived wheat germplasms [19,21,28,33].
Fluorescence in situ hybridization (FISH) and multicolor GISH (McGISH) are generally used to distinguish different chromosomes, analyze the chromosomal constitution and detect chromosomal variations in cytological studies [19,20,34,35]. The available FISH probes that reveal fine banding patterns in wheat chromosomes include the Aegilops tauschii clone pAs1 [36,37], the rye clone pSc119.2 [38], the GAA-satellite sequence [39], T. aestivum clone pTa535 [40] and many other oligonucleotides [41,42]. Wheat chromosomes can be clearly distinguished using a combination of FISH probes including the pAs1 clone and the GAA-satellite sequence. Chromosome painting using this combination of FISH probes has been achieved in the common wheat cultivar 'Chinese Spring' (CS) [43,44] in which all chromosomes of A, B and D subgenomes were labeled with specific signals, with the signals on the B-and D-genome chromosomes being particularly abundant. Through combined McGISH and FISH analysis, the positions and characteristics of chromosomal structural variations in such materials can be revealed.
In this study, the chromosomal constitution and chromosomal variations in three Trititrigia octoploids (TE261-1, TE266-1 and TE346-1) exhibiting good resistance to stripe rust, powdery mildew and aphids were investigated. The genetic characteristics, types of chromosome variation of the three Trititrigia octoploids were revealed using GISH, FISH, McGISH and molecular markers.

Results
Chromosomal constitution of the three Trititrigia octoploids The root tip chromosome analysis showed that chromosome number of TE261-1, TE266-1 and TE346-1 was 2n = 56. Observation of meiotic chromosomes in PMCs revealed that most chromosomes in the observed cells formed 28 bivalents at meiotic metaphase I, 14 alien chromosomes formed 7 bivalents were observed in GISH analysis, indicating high cytological stability (Supplemental Fig. 1, Supplemental Table 1).
GISH, FISH and McGISH were used to identify the chromosomal constitution of TE261-1, TE266-1 and TE346-1. The GISH ( Fig. 1-A1) and FISH ( Fig. 1-A2) results indicated that TE261-1 contained 42 wheat chromosomes and 14 Th. intermedium chromosomes (Fig. 2), including one pair of St-genome chromosomes that were completely labeled with probe signals, one pair of J-genome chromosomes labeled only in the telomeres, three pairs of J S -genome chromosomes with obvious labeling in centromere areas, one pair of acrocentric chromosomes from the J S genome, and one pair of J-St translocated chromosomes. The J S acrocentric chromosomes (Fig. 2, Supplemental Fig. 1-A2), were first discovered in the TE series of Trititrigia octoploids.
The results revealed that TE266-1 also contained 42 wheat chromosomes and 14 Th. intermedium chromosomes ( Fig. 1-B1, B2, B3 and Fig. 2), including two pairs Variation in the wheat chromosomes in the three Trititrigia octoploids FISH analysis were used to identify different wheat chromosomes of TE261-1, TE266-1, TE346-1 and YN15 while using labled (GAA) 8 (red signals) and pAs1 (green signals) as probes. McGISH analysis were also conducted while using labeled A (green signals) and D (red signals) genomes as probes, B-genome DNA (gray) is used for blocking. The wheat chromosome variations in the three Trititrigia octoploids compared with their common wheat parent YN15 based on FISH and McGISH patterns. For the A-genome chromosomes, the hybridization signals of pAs1 are not visible on 1A in TE261-1, TE266-1 and TE346-1 (Fig. 3D). An absence of (GAA) 8 signals on 6AL and additional pAs1 signals at the 6AS subtelomeric position were observed in TE261-1 and TE346-1, while only the absence of (GAA) 8 signals on 6AL was observed in TE266-1 (Fig. 3D).
Compared with YN15, the signals on the 6B chromosome in TE261-1, TE266-1 and TE346-1 also changed ( Fig. 3A-D). Interspersed green signals for the pAs1 probe were detected on 6BS of TE261-1 and TE266-1, and the length of the 6BS chromosomes appeared to be extended in TE266-1 and TE346-1. Faint red (GAA) 8 signals were also observed on the 6BL chromosomes in the three Trititrigia octoploids.
The signals of the D-genome chromosomes in TE261-1, TE266-1 and TE346-1 appeared to vary significantly. FISH and McGISH results indicated that in TE266-1, the 2D chromosomes were replaced with one pair of A-D translocated chromosomes; the main body of the A-D translocated chromosomes was derived from 2A chromosomes according to the pAs1 signals, while part of the long arm was replaced with a 2D chromosome segment ( Fig. 3-B, 3-D).
The FISH results showed that the original strong pAs1 signals on 7DS or 7DL in YN15 were replaced with faint (GAA) 8 and pAs1 signals in TE261-1, TE266-1 and TE346-1. The McGISH results indicated that the partial long arm of the 7D chromosome in TE261-1 was replaced with the chromosome segment of the A-genome chromosome ( Fig. 1-A3, Fig. 3-D). However, the translocated A-genome chromosome segment was located on the short arm in TE266-1 and TE346-1, not the long arm as in TE261-1. The source of the A-genome chromosome segment was uncertain due to the lack of a specific FISH signal in this A-genome chromosome segment.
Specific molecular markers of the wheat chromosomes were employed to verify the chromosomal variation and detect the variation type in the three Trititrigia octoploids. The specific band (~300 bp) of YN15 amplified with the specific marker Xmag3124, which was located on 1A, was not observed in the three Trititrigia octoploids ( Fig. 4 and Supplemental Fig. 4). The specific band (~150 bp) of YN15 detected by marker GPW4344, which was located on 6A (Supplemental Fig. 2 and Supplemental Fig. 4), were also absent in the three Trititrigia octoploids, and a 6A specific band (~300 bp) absent in TE346-1 were The sequences of the primers used for these 16 chromosome-specific markers were employed to run local BLAST searches against the database of the T. aestivum (Chinese Spring, CS) whole-genome sequence.  Eleven markers were screened according to screening criteria including the appropriate chromosomes, initial position, terminal position and degree of strict matching (Supplemental Table 2). For example, the chromosomes mapped by BLAST should be matched the specific chromosomes detected by PCR; the higher matching degree means the better option, and the 3′ of primer must be matched; the absolute value of margin between forward primer initial position and reverse primer terminal, should match the size of specific bands; and last, the appropriate position mapped by the markers were picked out. Compared BLAST with PCR results after screening, the size of specific bands amplified with majority markers were similar (Fig. 4), but the 7D specific band (~240 bp) detected by Barc053 was different from BLAST result (295 bp), and difference exist in Ppd-D1 (R1) result of PCR (250 bp) and BLAST (413 bp). The difference could be attributed to the sequence diversity between CS and YN15. The screened markers were then labeled at the specific location of the corresponding chromosomes using MapGene2Chromosomes v2, according to their physical position on the chromosomes (Supplemental Fig. 3). The positions of the variations detected with markers were consistent with the FISH signals on the 6A, 6BL, 2D and 7D chromosomes, while the variation at the 1AL telomere was detected with markers, rather than FISH signals.

Phenotypic evaluation of the three Trititrigia octoploids
The reactions to stripe rust, powdery mildew and aphids in TE261-1, TE266-1 and TE346-1 were evaluated at the seedling and adult plant stages using the wheat parents YN15 and Th. intermedium as controls (Table 1). At the seedling stage, TE261-1, TE266-1, and TE346-1 were immune to the stripe rust race CYR32 and resistant to the powdery mildew race E09. At the adult stage, all three Trititrigia octoploids showed good resistance to stripe rust and powdery mildew in the field. TE261-1 and TE346-1 also showed moderate resistance to aphids at the adult stage, while TE266-1 and YN15 were susceptible. As the common wheat parent YN15 is susceptible to stripe rust, powdery mildew and aphids, whereas Th. intermedium is immune to all three, we deduce that the resistance of Trititrigia is derived from Th. intermedium.

Discussion
Th. intermedium is a valuable perennial species of Triticeae for wheat improvement due to its resistance to a number of wheat diseases and pests as well as its stress tolerance and high crossability with various Triticum species [8][9][10][11]45]. Partial wheat-Th. intermedium amphiploids showing high cross-compatibility with wheat are desirable 'bridge' materials for transferring desirable genes from Th. intermedium to common wheat [19,20].
Chromosome counting in metaphase spreads revealed that the chromosome number of these three octoploids of Trititrigia was consistently 2n = 56. Molecular cytogenetic analysis then revealed that the three Trititrigia accessions all carry 42 wheat chromosomes and 14 Th. intermedium chromosomes and that the 14 alien (Th. intermedium) chromosomes are composed of a mixed genome consisting of J-, J S -and St-genome chromosomes rather than a single J, J S or St genome, similar to other reported of Trititrigia octoploids [21,46,47]. The mechanism underlying the formation of the mixed genome is uncertain, and with development of new techniques and methods, such as oligo-FISH painting system [48] further research will contribute to elucidating the homologous relationship and integrality between the J, J S and St genomes and the origin and evolution of Th. intermedium.
Two types of structural variation in the alien chromosomes were detected: J-St translocated chromosomes, which were present in all three Trititrigia octoploids, and J S acrocentric chromosomes in TE261-1 (Fig. 2), which might have formed via the centromere breakage of one pair of J S -genome chromosomes. J-St translocated chromosomes also exist in many TE series of Trititrigia octoploids [21,26,28], but J S acrocentric chromosomes were first discovered in the TE series of Trititrigia octoploids. The formation of these two types of variations might occur through chromosome breakage and fusion when F 1 hybrids obtained through distant hybridization are backcrossed with hexaploid wheat [34].
Although the alien chromosomal constitution of three Trititrigia octoploids was different, some of the alien chromosomes were probably identical according to the GISH and FISH signals. For example, the J-genome chromosomes with the satellite sequence and the J-St  (Fig. 2). In addition to the structural variation in the alien chromosomes, structural variations, such as the variations detected in the 1A, 6A, 6B, 2D and 7D chromosomes, occurred in the wheat chromosomes as well. The results indicated that during the formation of partial amphiploids, the wheat chromosomes underwent various types of chromosomal recombination due to chromosome segments introgressed from Th. intermedium into the common wheat chromosomes, although the introgressed segments were too small to detect via GISH. The structural variations may have been generated via recombination between different wheat chromosomes, such as homoeologous chromosomal recombination between A-, B-, and D-genome chromosomes, influenced by the Th. intermedium chromosomes. The variation in chromosomal patterns of repetitive sequences (e.g. chromosome 6B) could be also caused by repetitive sequence reorganization (amplification/loss). After hybridization, genomes can undergo genetic and epigenetic changes (genomic shock) which can reorganize their structure.
Similar chromosomal variations were detected in the 1A and 6A chromosomes of the three Trititrigia and might have been derived from the same origin in the earlier generations of backcrosses. Additionally, there were distinct variations in the 6B, 2D and 7D chromosomes of these Trititrigia octoploids, such as pAs1 signals at different positions of 6BS in TE266-1 and TE346-1 and the 2A-2D translocation in TE266-1. The variations in the 7D chromosomes of the three Trititrigia octoploids were also different. McGISH indicated that the partial 7DS chromosomes of TE266-1 and TE346-1 were replaced with an A-genome chromosome segment, but the corresponding position in TE261-1 was on the 7DL chromosome. The original position replaced in 7DL or 7DS was difficult to discern via FISH because the signals on the two arms of the 7D chromosomes were similar. The results of molecular marker analyses also supported the results of FISH and McGISH in TE261-1. Additional work will be needed to obtain strong evidence indicating how these chromosomal recombinations occurred as well as the effect of these chromosomal variations.
The drastic and abundant chromosome variations were detected in these three Trititrigia octoploids, a kind of generic hybrid between wheat and Th. intermedium. However, we guess the more drastic variations might exist in early-generation of Trititrigia octoploids, and then backcrosses with YN15 resulted in reducing variability and increasing stability. We hypothesized that Th. intermedium, as a natural generic hybrid, possessed drastic chromosomal variation causing difficulty to confirm the progenitor diploid species.
Although FISH results using general probes consisting of (GAA) 8 and pAs1 were more abundant, colorful and characteristic to distinguish most common wheat chromosomes, it was still insufficient to identify the alien chromosomes in present amphiploids. Li et al. had reported an efficient Oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae [48], while using novel comparative genomebased oligo painting FISH procedure. These would be beneficial and helpful for determining the alien Thinopyrum chromosomes in these amphiploids in the future studies.
Phenotypic evaluation indicated the successful transfer of stripe rust and powdery mildew resistance from Th. intermedium to these three Trititrigia octoploids and that TE261-1 also exhibited moderate resistance to aphids. In addition, the chromosomal constitution of TE261-1, TE266-1 and TE346-1 is different from that of other TE series of Trititrigia octoploids reported previously [21,26,28]. Therefore, these accessions could be employed as bridge parents for crossing with wheat to develop addition, substitution or translocation lines to generate new rust-, powdery mildew-and aphid-resistant germplasms for wheat breeding.

Conclusions
Three octoploid Trititrigia accessions (TE261-1, TE266-1, and TE346-1) with good resistances to diseases and aphids were characterized by GISH, FISH, McGISH and molecular marker analyses. Molecular cytogenetic analysis revealed that these Trititrigia accessions all carry 42 wheat chromosomes and 14 Th. intermedium chromosomes, and the 14 alien (Th. intermedium) chromosomes are composed of a mixed genome consisting of J-, J S -and St-genome chromosomes rather than a single J, J S or St genome. Different structural variations occurred in the wheat chromosomes, such as the variations detected in the 1A, 6A, 6B, 2D and 7D chromosomes as well. Results indicated that the wheat-Th. intermedium partial amphiploids possess 14 alien chromosomes which belong to a mixed genome consisting of J-, J S -and St-chromosomes, and 42 wheat chromosomes with different structural variations.

Chromosome slide preparation, meiotic preparation, GISH, FISH and McGISH
Fresh root tips were collected from germinating seeds treated with nitrous oxide (N 2 O) for 2 h [53] and then immersed in 90% glacial acetic acid to fix cell division. The preparation of root tip cell chromosome slides and analysis was performed referring to the method of Han [46]. When the flag leaf of wheat was spread, young spikes were sampled and anthers at metaphase I (MI) of meiosis were fixed in Carnoy's solution. The meiotic chromosomes were prepared from pollen mother cells (PMCs) by knocking and pressing anthers in 45% acetic acid, after which the chromosome configuration in PMC metaphase I (MI) was observed.
The reaction volume for in situ hybridization was 10 μL per slide. GISH was conducted using a probe of 50 ng St-genome DNA, with a block of 6000 ng YN15 DNA; FISH was performed with 5 ng (GAA) 8  Evaluation of resistance to powdery mildew, stripe rust and aphids The powdery mildew and stripe rust resistance of plants in the seedling stage were tested in a plant incubator. The wheat variety Huixianhong was employed for inoculation of the Blumeria graminis f. sp. tritici (Bgt) pathotype E09 and the Puccinia striiformis f. sp. tritici (Pst) pathotype CYR32. Thirty seeds of the test materials were grown in seedling plates and placed in a plant incubator under 12 h light (25,000 lx) at 25°C and 12 h of darkness at 17°C, with 70% relative humidity. Huixianhong and YN15 were used as controls. Inoculation with E09 and CYR32 was achieved by smearing at the one-leaf stage, and the gradient of infection was investigated when Huixianhong had been completely infected; these experiments were repeated three times. The powdery mildew and stripe rust resistances of adult-stage plants were tested via natural infection in an open field. The resistance evaluation criteria for powdery mildew and stripe rust followed the methods of Sheng [57] and Li et al. [58]. In the scale of powdery mildew resistance, 0 is immune; 0; is nearly immune with necrotic flecks; 1 is highly resistant with small lesions (< 1 mm) and thin mycelium; 2 is moderately resistant with small lesions (< 1 mm) and a slightly thick mycelium; 3 is moderately susceptible with lesions (> 1 mm) and a thick mycelium; and 4 is highly susceptible with lesions (> 1 mm) and a consecutive thick mycelium. In the scale of stripe rust resistance, 0 is immune; 0; is nearly immune with tiny flecks; 1 is highly resistant with yellowish-white flecks; 2 is moderately resistant with necrotic plaques around small spore stripes; 3 is moderately susceptible with fading around spore stripes; and 4 is highly susceptible with large spore stripes.
For the identification of aphid resistance, natural aphids were employed, and the evaluation was performed during the wheat grain-filling stage. The test materials were sown on two 1.4-m-long rows with YN15 as CK, Th. intermedium planting in pot were put nearby to evaluate the aphid resistance. The investigation of aphid injury and the classification criteria for aphid resistance followed the method of Liu et al. [59]. The number of aphids on 10 seriously aphid-damaged stems was determined for all materials; then, using this number as the numerator and the average number of aphids on YN15 as the denominator, the ratio was computed as the evaluation criterion: 0 is immune with no aphids; 1 is highly resistant with a ratio between 0.01 and 0.30; 2 is moderately resistant with a ratio between 0.31 and 0.6; 3 is slightly resistant with a ratio between 0.61 and 0.90; 4 is slightly susceptible with a ratio between 0.91 and 1.2; 5 is moderately susceptible with a ratio between 1.21 and 1.5; and 6 is highly susceptible, with a ratio > 1.5.