Chromosome Number Variations in Newly Synthesized Hexaploid

Cereal Research Communications 38(4), pp. 449–458 (2010) DOI: 10.1556/CRC.38.2010.4.1

Chromosome Number Variations in Newly Synthesized Hexaploid Wheats Spontaneously Derived from Self-fertilization of Triticum carthlicum Nevski / Aegilops tauschii Coss. F1 hybrids

K. NIWA*, H. AIHARA,A.YAMADA and T. MOTOHASHI

Department of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 243-0034, Japan

(Received 21 October 2009; accepted 17 February 2010)

This study was aimed at elucidating numerical variation of chromosomes in newly synthesized hexaploid wheats. We carried out artificial crosses between Triticum carthlicum (2n =4x = 28, AABB) as female parent and Aegilops tauschii (2n =2x = 14, DD) as male parent, obtain- ing intergeneric F1 hybrids (2n =3x = 21, ABD). After self-fertilization of the F1 hybrids hav- ing 21 somatic chromosomes, we obtained F2 seeds (synthetic hexaploid wheats), and deter- mined their somatic chromosome number. Of the expected 150 cross combinations of F1 hybrids between six strains of T. carthlicum and 25 strains of Ae. tauschii, 67 cross combinations of synthetic hexaploid wheats were obtained. Compared to strains of Ae. tauschii ssp. tauschii, those of Ae. tauschii ssp. strangulata produced synthetic hexaploid wheats showing euploidy with a high frequency. In addition, among strains of Ae. tauchii ssp. tauschii, those from Iran contributed more to the production of synthetic hexaploid wheats showing euploidy than those from Afghanistan, Pakistan, Turkey or the former USSR.

Keywords: Aegilops tauschii, aneuploid, euploid, polyploid, synthetic hexaploid wheats, Triticum carthlicum

Introduction Common wheat, Triticum aestivum L. (2n =6x = 42, AABBDD), is a representative polyploid crop species. As suggested by Kihara (1944) and McFadden and Sears (1944), common wheat has been proven to be derived from tetraploid Emmer wheat (2n =4x = 28, AABB) and Aegilops tauschii Coss. (2n =2x = 14, DD). Spontaneous self-fertilization of unreduced female and male gametes in F1 hybrids between Emmer wheat and Ae. tauschii have also given rise to F2 hybrids with AABBDD (Kihara 1946), indicating that hybridization promotes the formation of cytologically unreduced gametes (de Wet 1980). Induced chromosome doubling in F1 hybrids between Emmer wheat and Ae. tauschii obtained by colchicine treatment have yielded allopolyploids with AABBDD (McFadden and Sears 1946). These are called synthetic hexaploid wheats with AABBDD, and resemble com-

* Corresponding author; E-mail: [email protected]

0133-3720/$20.00 © 2010 Akadémiai Kiadó, Budapest 450 NIWA et al.: Chromosome Polymorphism in Synthetic Wheats mon wheat morphologically. Synthetic hexaploid wheats serve as bridge plants to transfer useful genes of either tetraploid Emmer wheat or Ae. tauschii to common wheat by means of artificial crosses and selection (Lange and Jochemsen 1992a; Mujeeb-Kazi et al. 1996). Among several tetraploid Emmer wheats (T. carthlicum Nevski, T. dicoccoides Körn, T. dicoccum Schübl., T. durum Desf., and T. orientale Perc.), when T. carthlicum is crossed with Ae. tauschii, the F1 hybrids tend to frequently produce unreduced female and male gametes (Tanaka 1959; Xu and Dong 1989; Fukuda and Sakamoto 1992b). T. carthlicum is cultivated in limited regions: the Caucasus, Asia Minor, and western Iran (Tzvelev 1976), and is a useful genetic resource for cytogenetic study on the formation of spontaneous unreduced gametes and on the characterization of synthetic hexaploid wheats. Ae. tauschii has recently been considered to contain two subspecies, namely ssp. tauschii and ssp. strangulata (Eig) Tzvel. (Tzvelev 1976; Hammer 1980; Dudnikov 2000; Dudnikov and Kawahara 2006), although van Slageren (1994) did not recognize the two subspecies. Subspecies tauschii is widely distributed in Europe, the Caucasus, Central Asia, the Mediterranean, Asia Minor, Iran, the Himalayas, and China, whereas ssp. strangulata is only found in the Caucasus, Central Asia, and Iran (Tzvelev 1976; van Slageren 1994). The purpose of this study was to clarify numerical variation in chromosome number in synthetic hexaploid wheats spontaneously derived from F1 hybrids between T. carthlicum and Ae. tauschii.

Materials and Methods Plant materials Six strains of T. carthlicum were used as female parents (Table 1). Seventeen strains of Ae. tauschii ssp. tauschii and eight of Ae. tauschii ssp. strangulata were used as male parents (Table 1); most of their characteristics are described in Kihara and Tanaka (1958). All the materials were kindly provided by the Plant Germ-plasm Institute, Kyoto University, Ja- pan. Detailed information is available within Kawahara (1997). Cytogenetic analysis

In order to obtain F1 hybrids with 21 somatic chromosomes, we carried out artificial crosses between T. carthlicum (2n =4x = 28) as the female parent and Ae. tauschii (2n =2x = 14) as the male parent. Before anthesis of the female parent, young anthers were re- moved and each spike bagged in a glassine bag. When emasculated florets became recep- tive, they were pollinated and bagged again. To obtain F1 hybrids, we did not use treatment with plant growth regulators after pollination or embryo culture after fertilization. Em- bryos were excised from the crossed spikes giving rise to F1 hybrid seedlings. After check- ing that the chromosome number of F1 seedlings was 21 (2n =3x = 21, ABD) by the aceto-carmine squash method and growing F1 hybrids without chemically induced chromosome doubling, self-pollinated F2 seeds were obtained by covering each spike of F1 hybrids with a glassine bag before flowering. The somatic chromosome number of root-tip

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cells from two to six F2 seeds for each cross combination was counted by the aceto-carmine squash method.

Results Among the expected 102 cross combinations between six strains of T. carthlicum and 17 strains of Ae. tauschii ssp. tauschii, 102 were successfully crossed (16–127 florets per cross combination). We obtained 66 cross combinations of F1 seeds and 32 cross combinations of F2 seeds. The ratio of F2 seeds showing aneuploidy was 21.0% (Table 2). F2 seeds with less than 42 chromosomes (hypoploids) were occasionally observed (Table 1, Fig. 1). Furthermore, of the expected 48 cross combinations between the six strains of T. carthlicum and eight strains of Ae. tauschii ssp. strangulata, 46 were successfully crossed (10–32 florets per cross combination). We obtained 43 cross combinations of F1 hybrids and 35 cross combinations of F2 seeds. Most of the F2 seeds (92.3%) had 42 chromosomes (Table 3).

Table 1. A list of Aegilops tauschii and Triticum carthlicum strains used in this study and their origin

Strain no. Geographical origin1 Strain no. Geographical origin1 Ae. tauschii ssp. tauschii Ae. tauschii ssp. strangulata KU20-1 Derbent, Dagestan, USSR KU20-9 Behshahr, Iran KU20-2 Unknown KU2073 Behshahr, Iran KU20-10 Ramsar, Iran KU2075 Behshahr, Iran KU2001 Quetta, Pakistan KU2076 Gorgan, Iran KU2006 Quetta, Pakistan KU2077 Darkalah, Iran KU2010 Kandahar, Afghanistan KU2078 Aliabad, Iran KU2021 Kabul, Afghanistan KU2088 Sari, Iran KU2068 Tehran, Iran KU2090 Behshahr, Iran KU2100 Ramsar, Iran T. carthlicum KU2108 Hashtpar, Iran KU138 Unknown KU2131 Van, Turkey KU139-1 Unknown KU2138 Van, Turkey KU139-2 Unknown KU2142 Maku, Iran KU1632 Erevan, Armenia, USSR KU2158 Ramsar, Iran KU1800 Borzhormi, Georgia, USSR KU2160 Ramsar, Iran KU1801 Borzhormi, Georgia, USSR KU2801 Baku, Azerbaijan, USSR KU2810 Erevan, Armenia, USSR 1 After Kawahara (1997)

In order to compare ssp. tauschii with ssp. strangulata, we constructed a two-way con- tingency table including ssp. tauschii vs. ssp strangulata and the number of F2 seeds showing euploidy vs. aneuploidy, and carried out a c2 test of independence, according to Zar (2010). Statistical analysis (df = 1, c2 = 7.1308, 0.001 < P < 0.01) significantly sup- ported our observation that ssp. strangulata tends to produce euploid F2 individuals and that ssp. tauschii tends to give rise to aneuploid F2 individuals. Thus, this study showed

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Figure 1. Variation in chromosome number in root-tip cells of synthetic hexaploid wheats derived from F1 hybrids between Triticum carthlicum and Aegilops tauschii ssp. tauschii. (a) 2n = 39, (b) 2n = 42, and (c) 2n =43 that synthetic hexaploid wheats from F1 hybrids between T. carthlicum and Ae. tauschii ssp. tauschii have variable chromosome number. Among 57 F2 seeds from Ae. tauschii ssp. tauschii from Iran, 54 (94.7%) had 42 somatic chromosomes, while of 63 F2 seeds from ssp. tauschii from Afghanistan, Pakistan, Turkey and the former USSR, 45 (71.4%) showed 42 somatic chromosomes (Table 2).

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Table 2. Somatic chromosome numbers in F2 seeds originating from crosses between Triticum carthlicum and Aegilops tauschii ssp. tauschii

Cross combination No. of F2 No. of F2 seeds with Frequency of seeds F2 seeds observed with 42 39 40 41 42 43 45 chromosomes (%) T. carthlicum × Ae. tauschii ssp. tauschii from Iran KU138 × KU2158 3–––3–– 100 KU138 × KU2160 3–––3–– 100 KU139-1 × KU20-10 3–––3–– 100 KU139-1 × KU2100 3––12–– 66.7 KU139-1 × KU2108 3–––3–– 100 KU139-1 × KU2158 3–––3–– 100 KU139-2 × KU2108 3–––3–– 100 KU1632 × KU2100 3–––3–– 100 KU1632 × KU2160 3–––3–– 100 KU1800 × KU2100 3–––3–– 100 KU1800 × KU2108 3–––3–– 100 KU1800 × KU2158 3–––3–– 100 KU1801 × KU20-10 3–––3–– 100 KU1801 × KU2068 6–114–– 66.7 KU1801 × KU2100 3–––3–– 100 KU1801 × KU2108 3–––3–– 100 KU1801 × KU2158 3–––3–– 100 KU1801 × KU2160 3–––3–– 100 Subtotal 57 – 1 2 54 – – 94.7 T. carthlicum × Ae. tauschii ssp. tauschii from Afghanistan, Pakistan, Turkey and the former USSR KU138 × KU2001 6–––6–– 100 KU139-1 × KU2001 6––15–– 83.3 KU1632 × KU2010 3–––3–– 100 KU1800 × KU2001 6––15–– 83.3 KU1800 × KU2006 6––15–– 83.3 KU1800 × KU2010 6––141– 66.7 KU1800 × KU2810 6–1221– 33.3 KU1801 × KU2001 6–123–– 50.0 KU1801 × KU2010 6–––6–– 100 KU1801 × KU2021 6––231– 50.0 KU1801 × KU2810 6––1311 50.0 Subtotal 63 – 2 11 45 4 1 71.4 T. carthlicum × Ae. tauschii ssp. tauschii from unknown regions KU139-1 × KU20-2 61–23–– 50.0 KU1800 × KU20-2 6––33–– 50.0 KU1801 × KU20-2 6––24–– 66.7 Subtotal 18 1 – 7 10 – – 55.6 Total 138 1 3 20 109 4 1 79.0

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Table 3. Somatic chromosome numbers in F2 seeds originating from crosses between Triticum carthlicum and Aegilops tauschii ssp. strangulata

Cross combination No. of F2 No. of F2 seeds with Frequency of seeds F2 seeds observed with 42 38 41 42 43 chromosomes (%) KU138 × KU2076 2 – – 2 – 100 KU138 × KU2077 3 – – 3 – 100 KU138 × KU2078 3 – 1 2 – 66.7 KU138 × KU2088 3 – – 3 – 100 KU138 × KU2090 3 – – 3 – 100 KU139-1 × KU20-9 3 – – 3 – 100 KU139-1 × KU2073 3 – – 3 – 100 KU139-1 × KU2075 3 – 1 2 – 66.7 KU139-1 × KU2076 3 – – 3 – 100 KU139-1 × KU2077 3 – – 3 – 100 KU139-1 × KU2088 3 – – 3 – 100 KU139-1 × KU2090 3 – – 3 – 100 KU139-2 × KU2073 3 – – 3 – 100 KU139-2 × KU2075 3 – – 3 – 100 KU139-2 × KU2078 3 – – 3 – 100 KU1632 × KU20-9 3 – – 3 – 100 KU1632 × KU2075 3 – – 3 – 100 KU1632 × KU2076 3 – – 3 – 100 KU1632 × KU2078 3 – – 3 – 100 KU1632 × KU2088 3 – – 3 – 100 KU1632 × KU2090 3 – 1 2 – 66.7 KU1800 × KU20-9 3 1 1 1 – 33.3 KU1800 × KU2073 3 – – 3 – 100 KU1800 × KU2076 3 – – 3 – 100 KU1800 × KU2077 3 – – 3 – 100 KU1800 × KU2078 3 – 1 2 – 66.7 KU1800 × KU2090 3 – – 3 – 100 KU1801 × KU20-9 3 – – 3 – 100 KU1801 × KU2073 3 – – 3 – 100 KU1801 × KU2075 3 – – 3 – 100 KU1801 × KU2076 3 – – 3 – 100 KU1801 × KU2077 3 – – 3 – 100 KU1801 × KU2078 3 – – 3 – 100 KU1801 × KU2088 3 – 1 1 1 33.3 KU1801 × KU2090 3 – – 3 – 100 Total 104 1 6 96 1 92.3

This value for ssp. tauschii from Iran is as high as that of ssp. strangulata from Iran, which indicates that Iranian strains of Ae. tauschii ssp. tauschii also tend to produce synthetic euhexaploid wheats when crossed with T. carthlicum

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Discussion

We obtained 67 F2 lines of synthetic hexaploid wheats among the expected 150 cross combinations in this study. This result indicates easy production of synthetic hexaploid wheats between T. carthlicum and Ae. tauschii by spontaneous formation of unreduced gametes in F1 hybrids, without the need to use techniques for chromosome doubling or embryo res- cue of F1 hybrids, and is in accordance with the findings of Tanaka (1959), Xu and Dong (1989) and Fukuda and Sakamoto (1992b). If the Ae. tauschii has useful genetic traits that can be detected, they could be transferred to T. aestivum through newly synthesized hexaploid wheats. Therefore, the F2 lines we produced have potential importance as genetic resources. The occurrence of aneuploids in newly synthesized hexaploid wheats has been reported often, for example by Kihara and Lilienfeld (1949), Nishikawa (1964), Xu and Dong (1989), Matsuoka and Nasuda (2004) and Zhang et al. (2008a). The F2 seeds in this study presumably occurred by the fertilization of unreduced female and male gametes that spontaneously formed in F1 hybrids (ABD) with 21 chromosomes. Although we did not ob- serve the process of meiosis or mitosis during gametogenesis in our F1 hybrids, it is likely that synthetic hexaploid wheats with less than 42 chromosomes (hypoploids) or over 42 chromosomes (hyperploids) are, respectively, produced by chromosome elimination or by unequal segregation of chromosomes during the formation of unreduced gametes at meiosis. The hypoploids caused by the former mechanism are the predominant ones that we presume took place in our F1 hybrids (Tables 2 and 3). This study showed the occurrence of numerical chromosome variants in newly synthesized hexaploid wheats from F1 hybrids with 21 somatic chromosomes. This phenomenon occurred in F1 hybrids from wide hybridization. A likely possibility is that the occurrence of variants is dependent on the genotype of the parents or environment in which F2 seeds were formed. Another possibility is suggested by the following two findings. Ozkan et al. (2001) found that elimination of genome-specific sequences is initiated in F1 plants between T. turgidum and Ae. tauschii. Kashkush et al. (2002) showed gene loss or methyla- tion occurred in an F1 intergeneric hybrid between Ae. sharonensis and T. monococcum.It is worthwhile knowing whether our findings of numerical chromosome variants in newly synthesized hexaploid wheats are affected by parental genotypes, environmental factors, changes in genomic structure or alteration in gene expression in the intergeneric F1 hybrids. In terms of spontaneous origin of T. aestivum, we speculate that it requires several criti- cal cytogenetic events; first to accomplish crosses between tetraploid Emmer wheat and Ae. tauschii, second to make viable F1 hybrids, third to produce unreduced female and male gametes in F1 hybrids, fourth to produce fertile F1 hybrids, and fifth to produce euploid F2 seeds from the F1 hybrids. The first event was reported by Matsuoka et al. (2007), who carried out 74 cross combinations between T. turgidum ssp. durum cv. Langdon and 74 accessions of Ae. tauschii, and obtained F1 hybrid seeds in 66 cross combinations, and by Zhang et al. (2008b), who carried out 372 cross combinations between T. turgidum and Ae. tauschii and obtained seeds in 49.7% of cross combinations. These

Cereal Research Communications 38, 2010 456 NIWA et al.: Chromosome Polymorphism in Synthetic Wheats and our results indicate that artificial crosses between the two are easy. The second was reported by Nishikawa (1964) and Matsuoka et al. (2007), who described the occurrence of normal as well as dwarf and necrotic F1 hybrids between the tetraploid Emmer wheat and Ae. tauschii. The third was shown by Kihara (1946), Fukuda and Sakamoto (1992a), Lange and Jochemsen (1992b), Xu and Dong (1992), Xu and Joppa (1995, 2000), Matsuoka and Nasuda (2004), Zhang et al. (2007) and Zhang et al. (2008a). The fourth was demonstrated by the observation of a cluster of Ae. tauschii accessions involved in crosses that produce highly fertile F1 hybrids between T. turgidum ssp. durum cv. Langdon and 74 accessions of Ae. tauschii found in the southwestern Caspian region and Transcaucasus (Matsuoka et al. 2007). The fifth was addressed and confirmed by our results. This study showed a high frequency of occurrence of euploid F2 individuals between T. carthlicum and Ae. tauschii ssp. strangulata from Iran. Although ssp. strangulata has limited distribution areas in the Caucasus, Central Asia, and Iran (Tzvelev 1976), it will be also needed to characterize those except from Iran. Regardless, this study has shown that Ae. tauchii, including ssp. tauschii and ssp. strangulata from Iran is involved in the production of synthetic hexaploid wheats showing euploidy when crossed with the tetraploid wheat T. carthlicum. Molecular analysis using RFLP with 254 accessions of T. aestivum and 172 of Ae. tauschii revealed that the D genome of T. aestivum is most closely related to the genepool of ‘strangulata’ in Transcaucasia, Armenia in particular and southwestern Caspian Iran (Dvorak et al. 1998). These results may support Ae. tauschii from Iran hav- ing also played an important role in spontaneous establishment of T. aestivum. Recent studies on the origin of hexaploid wheats have shown that they are of recurrent origin. Caldwell et al. (2004) suggested that T. aestivum originated recurrently in at least two genetically distinct progenitors contributing to the formation of the D genome in 564 lines of T. aestivum and 203 of Ae. tauschii. Using 169 accessions of T. aestivum and 100 of Ae. tauschii, Giles and Brown (2006) concluded that there were at least two Ae. tauschii sources that contributed germplasm to the D genome of T. aestivum. As suggested by Matsuoka et al. (2007), who proposed that T. aestivum speciation occurred at multiple sites by observing natural variation in fertile F1 hybrids between T. turgidum ssp. durum cv. Langdon and Ae. tauschii, our results may support multiple origins of T. aestivum.

Acknowledgement We are grateful to the Plant Germ-plasm Institute, Graduate School of Agriculture, Kyoto University, Japan, for supplying the seeds used in this study.

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