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Seedset on Synthetic Haploids of Durum Wheat: Cytological and Molecular Investigations.

HAPLOID PLANTS have half the somatic chromosome number and are powerful cytogenetic and breeding tools. A large number of haploids have been produced in cereal crops including wheat (Kasha et al., 1990; Jauhar et al., 1991; Riera-Lizarazu and Mujeeb-Kazi, 1993). They can be employed in several areas of fundamental research in genetics and cytogenetics, e.g., isolation of aneuploids (Sears, 1954) and elucidation of genetic control of chromosome pairing (Sears, 1976; Jauhar et al., 1991, 1999). The haploid-derived homozygous lines provide a rapid means of achieving homozygosity, thereby accelerating breeding programs (Baenziger, 1996; Khush and Virmani, 1996). By crossing durum wheat (T. turgidum L., 2n = 4x = 28; AABB) with maize, we produced euhaploids (2n = 2x = 14; AB genomes) of seven commercial cultivars (Cappelli, Durox, Langdon, Lloyd, Medora, Monroe, and Renville) of durum wheat (Almouslem et al., 1998) and studied both inter- and intragenomic chromosome pairing (Jauhar et al., 1999). Although the corresponding (homoeologous) chromosomes of the A and B genomes are capable of pairing with one another, a homoeologous pairing suppresser gene, Ph1 (located in the long arm of chromosome 5B), suppresses homoeologous pairing (Sears, 1976). Consequently, only homologous partners pair in tetraploid durum wheat, which ensures diploid-like pairing and disomic inheritance. Even when each chromosome in the durum haploids is present in a single dose, Ph1 does not permit pairing among homoeologues. However, in the absence of Ph1 extensive homoeologous pairing takes place (Jauhar et al., 1999).

In this study, we investigated 101 Ph1-haploids derived from seven commercial durum cultivars. Although all haploids formed mostly 14 univalents, as expected, and showed very irregular meiosis, at least some haploids in each of the seven durum cultivars produced viable seed without colchicine treatment or cross pollination. Reports of seedset on haploid plants are very rare. We investigated the meiotic basis of this interesting phenomenon. Employing fluorescent in situ hybridization (FISH) or [fluorescent genomic in situ hybridization (GISH)], we studied the chromosomal composition of the seed-derived disomic plants. Cytological and molecular investigations on the causes of seedset in synthetic durum haploids are described and the breeding implications of seedset are briefly discussed.

MATERIALS AND METHODS

Haploids of seven durum cultivars, viz., Cappelli, Durox, Langdon, Lloyd, Medora, Monroe, and Renville, which we produced earlier via hybridization with maize (Almouslem et al., 1998) were studied cytogenetically. During the course of these studies, we observed seedset on some haploids of each of these cultivars. The seeds were harvested for further studies.

Production of Plants from Seeds

Thirty-eight of the healthier looking seeds (Cappelli, 1; Durox, 6; Langdon, 7; Lloyd, 3; Medora, 7; Monroe, 4; and Renville, 10) from the durum haploid plants were germinated in petri dishes on moist filter paper. The seedlings were planted in a greenhouse [20-23 [degrees] C, with an 8-h dark period, a 16-h light period (supplemental lighting in conjunction with natural lighting)] in 14-cm-diam pots containing Sunshine Mix No. 1 (Sun Gro Horticulture, Bellevue, WA) and fertilized 7 d later with Osmocote Plus (15-9-12) (Scotts-Sierra Hort. Prod., Marysville, OH) and raised to maturity.

Chromosome Studies by Conventional Techniques

Somatic chromosomes were studied from root-tips taken from the sprouted seed according to the techniques described earlier (Jauhar, 1993; Almouslem et al., 1998). For meiotic studies, immature spikes at the appropriate stage were fixed in Carnoy's fluid containing 95% (v/v) ethanol:chloroform:glacial acetic acid (6:3:1). Anthers were squashed and chromosomes stained with acetocarmine or carbol fuchsin according to procedures described earlier (Jauhar and Almouslem, 1998).

Fluorescent In Situ Hybridization (FISH) Studies on Somatic and Meiotic Chromosomes

Both somatic and meiotic chromosome spreads were obtained by squashing appropriately fixed root-tips and anthers in 45% (v/v) acetic acid. The chromosome spreads were covered with a glass cover slip and observed under a phase contrast microscope. Chromosome numbers were recorded. Slides containing well-spread metaphase chromosomes were transferred to a -80 [degrees] C freezer and kept for up to one month, if necessary.

FISH was conducted on well spread chromosome preparations with total genomic Triticum urartu Tumanian DNA as the probe (100 ng/slide, labeled with biotin-14-dATP), and Aegilops speltoides Tausch genomic DNA as the blocker (2000 ng/slide). Hybridization protocols standardized earlier (Jauhar et al., 1999) were followed. Propidium iodide (PI) was used as a counterstain and fluorescein isothiocyanate (FITC)-conjugated avidin DCS was used to detect the probe.

Slides were observed under a Zeiss Axioskop epi-fluorescent microscope with a Zeiss AttoArc 100 watt adjustable UV light source. To visualize the FITC signal of the labeled probe a Zeiss set code 09 excitation filter with a Chroma D535/ 40 m (Chroma Technology Corp., Brattleboro, VT) emission filter was used. For the PI counterstain a Zeiss set code 00 excitation filter with a Chroma D605/55 m emission filter was used. Images were captured with a SPOT II digital camera (Diagnostic Instruments, Inc., Sterling Hts., MI) connected to an IBM compatible computer using the camera software supplied by the manufacturer. Images were oriented to the proper layout using Paint Shop Pro v. 5 (JASC Software Inc., Minnetonka, MN). Final images were printed with a color printer.

RESULTS

Using the maize technique, we earlier produced 101 durum haploids (2n = 2x = 14) (Fig. 1A) with Ph1 (Almouslem et al., 1998) and studied their meiosis in pollen mother cells (PMCs) (Jauhar et al., 1999). These Ph1-haploids formed mostly 14 univalents at metaphase I (average of 1350 PMCs = 0.2 II + 13.6 I, Jauhar et al., 1999), and consequently showed numerous abnormalities at anaphase I and telophase I. It is remarkable that despite the meiotic abnormalities, at least some haploids of each of the durum cultivars produced seed (Fig. 1B; Table 1). We investigated the cytological basis of this seedset on haploids. Using fluorescent in situ hybridization (FISH), we elucidated the chromosomal constitution of the seed-derived disomic plants. Some interesting meiotic abnormalities that led to seedset are listed below.

[Figure 1 ILLUSTRATION OMITTED]
Table 1. Seedset on haploid plants of seven durum cultivars.

           Total number     Number euhaploid     Number of
Cultivar    euhaploids    plants with set seed   seeds set

Capelli          2                  1                2
Durox           25                  3               22
Langdon          4                  4               11
Lloyd           28                  2               38
Medora          23                  3               12
Monroe           3                  2                4
Renville        16                  3               10
Total          101                 18               99

           Mean number    SE of
Cultivar   seed/plant    the mean

Capelli       1.00         1.00
Durox         0.88         0.80
Langdon       2.75         1.44
Lloyd         1.36         1.01
Medora        0.52         0.33
Monroe        1.33         0.88
Renville      0.63         0.45
Total         0.98         0.36


First Division Restitution (FDR): Meiotic Non-Reduction

Pollen mother cells of all haploids had mostly 14 univalents and showed FDR to varying degrees (5-10% of the PMCs analyzed) (Fig. 2A-D) that led to the formation of unreduced gametes. At metaphase I, the 14 univalents organized themselves on the equatorial plate (Fig. 2A). As anaphase I approached, the univalents divided into chromatids (Fig. 2B) but failed to move to the poles, resulting in restitution nuclei (Fig. 2C). Thus, the first meiotic (reductional) division was bypassed. These restitution nuclei sometimes went through a normal second (equational) division and formed dyads (Fig. 2D) instead of tetrads. The dyads then produced apparently functional unreduced gametes with 14 chromosomes.

[Figure 2 ILLUSTRATION OMITTED]

Anaphase Movement of Chromosomes

During anaphase I in the haploids, varying numbers of chromosomes moved to the two poles. Distributions of 7:7, 8:6, 9:5, 10:4, 11:3, 12:2, and 13:1 were observed (Fig. 3A-H). FISH analyses of PMCs at anaphase I showed the random movement of the A- and B-genome chromosomes (Fig. 4A-D). In extreme cases, all chromosomes moved to one pole (Fig. 3H) which, in essence, resulted in nonreduction of chromosome number at the first meiotic division (Fig. 3I). These nuclei underwent a normal second (equational) division and gave rise to unreduced gametes with 14 chromosomes as shown in Fig. 2.

[Figures 3-4 ILLUSTRATION OMITTED]

Investigation of Seeds Produced by Durum Haploids

The synthetic haploid plants were vigorous and produced 5 to 12 tillers each. When grown to maturity (Fig. 1A) they set seed in several spikes. There was variation among haploids in their capacity to set seed (Table 1). The Langdon haploids, for example, produced on an average 2.75 seeds per haploid.

The seed on haploid plants was generally shriveled, compared to the plump seed on normal durum cultivars (Fig. 1B). Shriveling was due to poor development of endosperm, presumably caused by the haploid nature of the plant; less nutrition was available for endosperm formation. However, the seeds were viable. The 38 seeds selected from the seven cultivars showed 100% germination in petri dishes under laboratory conditions. It is possible that with double fertilization, the gametes that produced the endosperm might not be balanced and may have led to shriveling.

Production of Disomic (2n = 28) Plants

Plants obtained from seed from haploids were disomic and normal, with a typical phenotype of the parental durum cultivar from which they were derived. They were vigorous and tillered as well as the parental cultivar and were fully fertile. No height or leaf measurements were taken.

FISH Analysis of Somatic Chromosomes of Seed-Derived Plantlets

Chromosome counts from root-tips of seed-derived plantlets confirmed their disomic status (Fig. 5A and C). Fluorescent GISH, using total genomic probe of T. urartu (the A-genome donor), showed the complete duplication of the A-genome and B-genome chromosomes (Fig. 5B and D). The chromosomes involving the 4A [multiplied by] 7B translocation, the evolutionary signature of durum wheat, were easily observed (Fig. 5B and D).

We found that the distal segment translocated from chromosome 7B constitutes about 24% of the long arm of 4A.

FISH Analysis of Meiotic Chromosomes of Seed-Derived Plants

At meiosis, all 38 haploid-derived disomic plants studied formed 14 bivalents as in parental durum cultivars (Fig. 5E). FISH analysis of the meiotic chromosomes showed that seven bivalents belonged to the A genome and seven to the B genome (Fig. 5F). This confirmed the precise duplication of the A- and B-genome chromosomes in the seeds obtained from haploid plants, as a result of functioning of unreduced gametes.

DISCUSSION

Chromosome doubling during meiosis is believed to contribute significantly to the widespread occurrence of polyploids in nature (Harlan and de Wet, 1975; Veilleux, 1985; Jauhar, 1993), even though mitotic chromosome doubling may also occur spontaneously (Jauhar and Singh, 1969). Meiotic nonreduction and functioning of unreduced male and female gametes leads to chromosome doubling in intergeneric hybrids of grasses (see for reference, Xu and Joppa, 1995). The unreduced gametes generally arise through two types of meiotic restitutions: the first division restitution (FDR), or second division restitution (SDR) (Peloquin et al., 1989a, b; Tai, 1994). Haploids have been produced in numerous species of grasses including cereals: rice (Oryza sativa L., Gosal et al., 1997), wheat (e.g., Jauhar et al., 1991; Riera-Lizarazu and Mujeeb-Kazi, 1993; Baenziger, 1996; Hu, 1997), maize (Buter, 1997), barley (Hordeum vulgare L., Kasha et al., 1990; Pickering and Devaux, 1992; Forster and Powell, 1997), oat (Avena sativa L., Rines et al., 1997), and rye (Secale cereale L., Deimling and Flehinghaus-Roux, 1997). However, reports of seedset on haploids are extremely rare. We observed seedset on haploids of all seven commercial durum wheat cultivars and investigated the cytological basis of this interesting phenomenon. We discovered that two types of meiotic abnormalities in our durum haploids played a major role in producing fertile seeds: (i) FDR, and (ii) anaphase I movement of all chromosomes to one pole.

FDR in pollen mother cells resulted in the inclusion of all 14 univalents (or 28 chromatids) in one nucleus, which on equational division led to the production of unreduced, functional gametes. It is reasonable to assume that this phenomenon also occurred during megasporogenesis and produced unreduced female gametes. And the fusion of unreduced male and female gametes produced normal seed which gave rise to disomic durum plants. FISH analysis showed the complete duplication of the A- and B-genome chromosomes. Fertility of the derived disomics and the presence of two of the marker chromosomes involving the 4A[multiplied by]7B translocation, a typical evolutionary signature of durum wheat, further testified to the precise duplication of all chromosomes. Meiotic nonreduction is of common occurrence in nature. As early as 1930, Aase stated: "Haploidy may be changed to diploidy in the meiotic division through non-reduction of univalents." Meiotic restitution is known to induce chromosome doubling and hence fertility in several interspecific and intergeneric hybrids of grasses (Maan and Sasakuma, 1977; Jauhar, 1993; Xu and Joppa, 1995). It is important to note that in all these wide hybrids, there is very little chromosome pairing, if any. We observed this phenomenon mostly in our durum haploids which had Ph1 and hence no pairing. It is possible that lack of pairing may be a prerequisite for the occurrence of meiotic restitution and hence chromosome doubling.

Seedset was observed on maize and oat haploids; both sets of haploids had little or no chromosome pairing. In a population of 282 maize haploids (monoploids), Chase (1949) found that 139 shed pollen, 68 formed kernels, and 34 yielded self-pollinated progeny. This self-fertility was attributed to spontaneous chromosome doubling in cells giving rise to reproductive tissue (Chase, 1949). Rines and Dahleen (1990) found that haploid oat plants recovered from oat X maize crosses were partially self-fertile with up to 23% seedset. However, they found that many of the plants grown from this seed were aneuploids.

Another interesting meiotic abnormality observed in durum haploids was the movement of all 14 univalents to one pole at anaphase I. This phenomenon has essentially the same consequences as the FDR described above. When all univalents move to one pole, they, in essence, bypass the reductional division of meiosis and then, on equational division, produce unreduced gametes. In spontaneous haploids of pearl millet, Jauhar (1970) and Powell et al. (1975) observed that all seven univalents moved to one pole in some PMCs and possibly contributed to the development of unreduced gametes.

The phenomena described above could have breeding implications. In both phenomena, the diploid chromosome complement is first restored and then the diploid complement undergoes mitotic (equational) division resulting in unreduced gametes (microspores during male meiosis and megaspores during female meiosis). During the equational division, sister chromatids of each chromosome (univalent of durum haploids) move to opposite poles, and therefore the two resultant nuclei are essentially similar to each other and to the parental meiocyte. The haploid-derived homozygous lines or doubled haploids (DH) may prove useful in basic cytogenetic studies, in mapping, and in practical plant breeding. Thus, DH can be employed in developing comprehensive maps and analysis of quantitative trait loci (Hayes et al., 1996; Cadalen et al., 1998). The instant homozygosity derived through chromosome doubling of haploids can accelerate breeding programs (Baenziger, 1996; Khush and Virmani, 1996). To be useful, however, an efficient method of DH production is necessary. Our durum haploids produced a low frequency of DH. However, there was a genotypic variation for DH production in our durum cultivars and it may or may not be possible to select for this trait. It is nevertheless significant that we obtained doubled haploids in all seven commercial cultivars of durum wheat without colchicine treatment or cross pollination.

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Abbreviations: DH, doubled haploid; FDR, first division restitution; FISH, fluorescent in situ hybridization; FITC, fluorescein isothiocyanate; GISH, genomic in situ hybridization; PI, propidium iodide; PMCs, pollen mother cells; SDR, second division restitution.

P. P. Jauhar,(*) M. Dogramaci-Altuntepe, T. S. Peterson, and A. B. Almouslem

P.P. Jauhar, M. Dogramaci-Altuntepe, and T.S. Peterson, USDA-ARS, Northern Crop Science Lab., Fargo, ND 58105; A.B. Almouslem, Dep. of Botany, Faculty of Sciences, Univ. of Aleppo, P.O. Box 12252, Aleppo, Syria. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA or imply approval to the exclusion of other products that also may be suitable. Received 1 March 2000. (*) Corresponding author (pjauhar@badlands.nodak.edu).

Published in Crop Sci. 40:1742-1749 (2000).
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Date:Nov 1, 2000
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