Monitoring the introgression of E genome chromosomes into triticale using multicolor GISH

Abstract

Hybrids were made between triticale (AABBRR) line T182 and a Thinopyrum amphiploid 8801(AABBEE) and progenies advanced up to F₆. Multicolor genomic in situ hybridization (McGISH) revealed different ratios of R and E genome chromosomes in each of the lines examined. R and E genome chromosomes were eliminated by F₅ in two of the lines studied. The ratios of R:E genome chromosomes varied from 2:8 to 12:2 in other F₆ lines. Translocations between R and E genome chromosomes were detected as early as F₃. A single wheat/R translocation was detected in one line in F₆.

Keywords:

• multicolor GISH
• introgression
• Thinopyrum elongatum
• triticale

Introduction

Interspecific hybridization has, since the nineteenth century, been an important way to enrich the primary gene pool of wheat. Over the past 50 years, numerous crosses have been made between wheat and its relatives to transfer desirable genes into wheat. The Thinopyrum species, which carry important traits such as disease resistance (Martin et al. 1976; Wang and Liang 1977; Zhang et al. 2004) and stress tolerance (Foster et al. 1988; McDonald et al. 2001; Colmer et al. 2006), have been utilized frequently. Jauhar and Peterson (1998, 2001) identified Fusarium head blight (FHB) resistance in hybrid derivatives from crosses between durum wheat cultivars and Th. elongatum, as well as an accession of Thinopyrum junceiforme. Colmer et al. (2006) screened salinity tolerance of wheat relatives and found that the Thinopyrum species showed very high performance. In fact, many reports proved that wheat-elongatum amphiploids and chromosomes addition lines can enhance the salt tolerance of wheat (Dvořák and Ross 1986; Colmer et al. 1995). Resistance genes for Claviceps purpurea fungus were also present in Thinopyrum species (Cox et al. 2005). To date, at least 18 different genes have been introgressed into wheat from various Thinopyrum species (Fedak 2000; Ceoloni et al. 2000). In many cases, producing wheat/Thinopyrum amphiploids is only the first step in the process of transferring the desired genes from Thinopyrum to wheat (Xu et al. 1994; Zhang et al. 1996). Subsequently, additional manipulations need to be carried out between these amphiploids and wheat, barley, and triticale, in order to transfer the traits.

Triticale, the first synthetic hybrid cereal crop produced between the female parent wheat and the male parent rye, is being grown worldwide. Hexaploid triticale (2n = 42, AABBRR) has proven to be the most commercially successful to date, and most breeding and research continues with this type because of its superior vigor and reproductive stability compared to the octaploid type. However, it lacks the DD chromosomes from Triticum tauschii, which donated the baking quality genes to bread wheat. Moreover, it always requires substantial breeding work to remove the inherent problems which it expresses. These problems generally include partial floret sterility, shriveled seed, low yield potential, poor adaptation to local production conditions, and poor agronomic characteristics. Hence it has been treated as a minor crop in many countries. However, recent reports indicate that it has a potential to be an industrial crop, providing raw materials for biofuel, etc., for its production in marginal lands (Lam et al. 2001; Mergoum et al. 2009). Thus, it is necessary to broaden the genetic base of triticales by using multiple approaches. One approach is to synthesize new combinations of primary triticales using a diversity of wheat and rye parents. Another is to integrate the extensive variability of Thinopyrum into triticale.

Genomic in situ hybridization (GISH) is a powerful technique to identify alien chromatin in wheat–alien hybrids (Le et al. 1989; Fedak and Han 2005). The multicolor GISH technique enables three or more genomes of a polyploid species to be detected simultaneously, thus providing a more detailed genome analysis.

In this study, we report the introgression of E genome chromosomes into hexaploid triticale, utilizing multicolor GISH to monitor the process.

Materials and methods

The amphiploid 8801(AABBEE) with the E genome from Thinopyrum elongatum [Host] D. Dewey, derived from the hybrid Triticum turgidum (L.) (AABB)/Thinopyrum elongatum (EE) (Han, F. P, pc) is an excellent source of FHB resistance. We produced a hybrid between the amphiploid 8801 and the Canadian triticale × Triticasecale witt) line T182 (AABBRR) (Salmon, D. pc). All generations of progeny plants were grown in controlled-environment chambers at day/night temperatures of 20/15°C and a 16-h photoperiod. The F2 to f6 progenies were obtained by selfing of the previous generation.

Somatic chromosomes counts were determined on sections of root apical meristems that were pretreated in ice water at 4°°C for 24 h and fixed in 3:1 ethanol:acetic acid fixative solution. Chromosome staining was carried out by standard methods using 2% aceto carmine or Feulgen solution after hydrolysis in 1 N HCl for six minutes.

For studies of chromosome behavior at meiosis, young inflorescences were collected and fixed in Carnoy’s solution for 48 hours then transferred to 70% ethanol and stored in refrigerated conditions until used. Anthers were then squashed in 2% aceto carmine. Chromosome configurations were scored in a minimum of 20 cells per line.

For multicolor GISH, genomic DNAs of rye and Th. elongatum were extracted using the standard CTAB method (Kidwell and Osborn 1992), then purified with NaAc. Purified DNAs were labeled with biotinylated-dUTP and digoxigenin-dUTP respectively using nick translation kits (Roche, Germany). Total genomic DNA of Chinese Spring was used for blocking. Slide preparation, hybridization, washing conditions and probe detection were carried out as described by Han et al. (2003) and rarely modified. Slides were examined with a Zeiss fluorescence microscope and photographs were taken on AxioCam HRm (Carl Zeiss, Canada).

Results

The somatic chromosome constitution of six progenies from hybrids between amphiploid 8801 and triticale strain T182 was monitored from the F₂ to F₆ generation using multicolor GISH. The results are shown in Table 1. Chromosome numbers in F₂ progenies were only studied in three combinations; lines 1, 3 and 5. The chromosome numbers were 42 in all plants studied. Two of the progenies had 7 R genome and 7 E genome chromosomes, while the third had 8 R and 6 E genome chromosomes.

Table 1. McGISH analysis of segregating populations derived from triticale T182 (ABR) × partial amphiploid 8801(ABE).

Generation	F2				F3				F4				F5				F6
Line	Ch	R	E	Tr	Ch	R	E	Tr	Ch	R	E	Tr	Ch	R	E	Tr	Ch	R	E	Tr
1	42	7	7		36–42	5–9	7–8	1–3E/R	40–43	3–4	2–5	-
3	42	8	6		42	11	2	2E/R	38–40	10–12	0–1	0–1	42	14
4a					42–46	4–11	5–9	1–2E/R	30–45	1–10	2–10	1–3	42	3	10	1	36	2	8	1E/R+1W/R
4b									41–43	10–14	1–3		41–42	12–14	1–3		42	12	2
5	42	7	7		32–38	1–9	6–10	1	29–38	1–7	1–9	0–1	28
6					28–29	0–1	0–1	0–1	28–29	0–1	0–1		28

Abbreviations: Ch, total chromosome number; R, number of R genome chromosomes; E, number of E genome chromosomes; Tr, number of E/R translocations; W/R, translocations between wheat and R genome chromosomes.

The chromosome constitution of each line shown in Table 1 was different as generations were advanced from F₃ to F₅ or F₆. The progeny of plant 4b presented a relatively uncomplicated stream beginning with a total chromosome number of 41–43 in F₄ with variable numbers of R and E genome chromosomes. Somewhat similar numbers of total chromosomes, R and E genome chromosomes were observed in F₅. The final chromosome number in F₆ was 42 including 12 R and two E genome chromosomes as shown in Figure 1.

Figure 1. An F₆ mitotic cell of line 4b showing a total of 42 chromosomes consisting of 28 wheat plus 12 R genome chromosomes (green) and 2 E genome chromosomes (red).

Progenies of plants in line 4a showed a more complex lineage. Beginning in F₃, all categories of chromosomes showed wide ranges of numbers. Total chromosome numbers ranged from 42 to 46 while R and E chromosomes ranged from 4 to 11 and 5 to 9, respectively. E/R translocations were detected in F₃; either one per cell (Figure 2a) or two per cell as shown in Figures 2b, 2c, and 2d.

Figure 2. E/R translocated chromosomes: (a) An F₄ cell of line 5 with 28 chromosomes including one R (green) genome chromosome plus an E/R translocated chromosome (arrowed); (b) an F₄ cell of line 4a showing four E genome chromosomes, 10 plus two telo R genome chromosomes plus two E/R translocated chromosomes (arrowed); (c) an F₄ cell of line 4a showing eight E genome chromosomes, seven R genome chromosomes plus two E/R translocated chromosomes (arrowed); (d) an F₄ cell of line 4a showing 10 E genome chromosomes, five plus telo R genome chromosomes plus two E/R translocated chromosomes (arrowed).

Wide ranges in chromosome numbers and constitutions were also detected in F₄, with 30–45 total chromosomes and ranges of 1–10 R and 2–10 E genome chromosomes. The numbers were more stable in F₅ with 42 chromosomes, including three R and 10 E chromosomes. The F₆ chromosome constitution was a total of 36 comprised of two R plus eight E and two translocated chromosomes, an E/R and one wheat/R translocation (Figure 3).

Figure 3. An F₄ cell of line 4A showing eight E genome chromosomes, two from the R genome plus one E/R and one wheat/R translocated chromosomes (arrowed).

Line 3 exhibited 42 chromosomes in F₂, consisting of 28 wheat, eight R and six E. The total chromosome number in F₃ was 42 with a preponderance of R genome chromosomes (11) and only two E and two E/R translocations. The situation was similar in F₄; however the F₅ constitution was a total of 42 with 14 R genome chromosomes. Presumably the E genome and translocated chromosomes had been eliminated by this stage.

Line 1 showed a total chromosome number of 42 in F₂ with seven E and seven R. Variable numbers of all three categories were observed in F₃ and F₄.

Lines 5 and 6 had highly variable numbers of E and R genome chromosomes in F₃ and F₄; however, by F₅ only 28 chromosome plants were recovered.

Line 3, although it showed small numbers of E and translocated chromosomes in F₄, returned to 28 wheat and 14 rye chromosomes in F₅.

Lines 5 and 6 although they had variable numbers of E and R genome chromosomes in F₃ and F₄ both reverted to 28 chromosomes by F₅.

Some common features were observed in progenies of all lines. E/R translocations were observed in all lines, beginning in F₃; however by F₅ and F₆ only line 4a contained translocations. In most but not all lines in all generations, R genome chromosomes were more numerous than E genome chromosomes. Line 4a was an exception, where the proportion of E genome chromosomes was high in all generations, culminating with eight E genome chromosomes in F₆.

Line 4a showed other unusual characteristics compared to the other lines. Most lines contained 42 chromosomes with 28 being of wheat origin. This was the case for line 4a in F₅, however in F₆ the chromosome constitution was somewhat unusual with a total chromosome number of 36, consisting of two R, eight E and two translocated chromosomes. That means that there were only 24 wheat chromosomes in that line.

Meiotic behavior was studied in F₄ and F₅ plants of line 4b containing a total of 42 chromosomes consisting of 28 wheat plus 12 R and two E chromosomes. Meiosis was very regular in these lines and similar in both generations. The total number of bivalents per cell was about 20 with an average univalent frequency of slightly in excess of one per cell (Table 2).

Table 2. Meiotic characteristics of F₄ and F₅ progenies of line 4 derived from intercrosses of triticale T182 (ABR) × partial amphiploid 8801(ABE).

		F4	F5
I		1.8 (0–4)	1.3 (0–4)
II	Ring	9.5 (6–14)	9.1 (4–14)
	Rod	10.6 (7–12)	11.3 (8–16)
Xta		29.6	29.4

Abbreviation: Xta, average chiasmata frequency per cell.

Discussion

The multicolor GISH technique was very efficient in determining the numbers of R and E genome chromosomes in various progenies at all generations. The ratio of R:E genome chromosomes through succeeding generations varied for all the lines. The ratio varied from 10:2 to 2:8 in various lines. In general, the number of alien chromosomes decreased with later generations in most populations. In some lines, such as 5 and 6, the alien chromosomes were eliminated entirely. Generally, the ratios of alien chromosomes varied with each line. From the patterns observed, it can be assumed that if sufficient numbers of lines were studied, it would be possible to identify lines with desired ratios of R and E genome chromosomes.

Intergenomic chromosome translocations have been previously reported in various types of intergeneric hybrids and partial amphiploids, etc. Intergeneric chromosome translocations were detected in progenies of wheat–Elymus repens hybrids (Zeng et al. 2013). Numerous intergenomic translocations were reported in partial amphiploids involving E1E2J and Js genomes (See Fedak and Han 2005, plus references contained therein). Spontaneous Robertsonian translocations (Ropiquet et al. 2008), are the most common in these types of amphiploids, but terminal, interstitial and trigenomic types have also been reported. Robertsonian translocations probably occur spontaneously between unpaired chromosomes, such as E and R in this study. There is not likely any affinity between these two genomes since no multivalents were observed in meiosis in this study (Table 2). Therefore the E/R translocations must occur spontaneously as do the 1B/1R translocations in wheat rye studies. In this study they were detected as early as F3.

Alloploidy includes irreversible structural changes at the genome and chromosome levels (Leitch and Bennett 1997), DNA sequence levels (Song et al. 1995), gene expression (Scheid et al. 1996; Comai et al. 2000), or elimination of highly repetitive sequences (Salina et al. 2000). In this study, the only visible signs of structural changes in the aneuploids were the spontaneous translocations. These cytogenetic stocks might reveal additional alterations mentioned above, if the lines were so analyzed.

The parental amphiploid 8801 (ABE genome) possesses several useful agronomic traits that are lacking in triticale line T182. For example, 8801 has excellent resistance to Fusarium head blight (Miller et al. 2011), a trait that is lacking in most lines/cultivars of triticale. In addition, 8801 also has good resistance to Ug99 (the new race of stem rust-unpublished data).

Even after studying this small population of progenies there are numerous indicators of the potential of transferring E genome traits on E genome chromosomes into triticale. Another potential would be the establishment of novel amphiploids with combinations of both E and R genome traits.

Funding

China Scholarship Council 10.13039/501100004543

Disclosure statement

No potential conflict of interest was reported by the authors.