Gross morphology of plants and spikes of wheat/ Ae. peregrina 3774 interspecific hybrids with and without 5B chromosome ( a ) Plants ( b ) Spikes 

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... ( Triticum aestivum L.) is one of the most important food crops of the world, which serves as a staple food of about two billion people (36 % of the world population). Among cereals, wheat alone provides 28 % of world ’ s edible dry mat- ter and 60 % of daily calories intake in several developing countries (Distelfield et al. 2007). Wheat production will have to be doubled to 1200 mMT by the year 2025 in order to meet the increasing world demands and future needs of wheat. This increase must be brought about by improving productivity on land that is already under cultivation and not by bringing new land into use by destruction of forests, grasslands, etc. (Vasil 2003). The related wild species are still a valuable source of useful agronomic traits for the continued improvement of cultivated wheats. Wide hybridization of wheat with related wild species, coupled with cytogenetic manipulation of the hybrid material, has been instrumental in the genetic improvement of wheat. Chromosome engineering methodologies based on the manipulation of pairing control mechanisms and induced translocations have been employed to introgress into wheat specific disease and pest resistance genes. Several related wild progenitor and non-progenitor species have been used for de- velopment of alien addition, substitution and translocation lines and transfer of useful variability in wheat for disease resistance and quality improvement (Qi et al. 2007; Rawat et al. 2009; Schneider et al. 2008). Although a large number of wheat-alien addition/substitution lines carrying useful variability have been developed but only a very few have been exploited commercially. Most of the introgressed alien segments from wild relatives into wheat either do not compensate well for the loss of wheat chromatin (causing duplications deficiencies) or show linkage drag causing depression in yield and performance of the plant (Jiang et al. 1994). In hexaploid wheat ( Triticum aestivum L.) the meiotic pairing among its homoeologous chromosomes and in interspecific hybrids is suppressed by the dominant gene of Ph1 (pairing homoeologous) locus on the long arm of chromosome 5B ( R il ey an d C ha p m an 19 5 8 ) . T h e p ai r i n g a m o n g homoeologous chromosomes is possible only in the absence of the Ph1 gene through the absence of chromosome 5B, or in the presence of its recessive deletion mutation ph1b (Sears 1977) or in the presence of the dominant inhibitor Ph I (Riley et al. 1961). The advantage of induced homoeologous pairing is the compensating transfer of desired alien chromosome segments reducing the unwanted alien chromatin (Baum et al. 1992). Induced homoeologous pairing has been used to transfer gene conditioning resistance to the leaf rust ( Lr24 ) from two different Agropyron elongatum chromosomes to wheat chromosomes (Riley et al. 1968; Sears 1972). The gene for stem rust resistance from Ae. caudata (CC) was also transferred to the common wheat using monosomic 5B stock (Dyck et al. 1990). The present article deals with the characterization of interspecific hybrids of monosomic for chromosome 5B Triticum aestivum cv. Pavon x Aegilops species without 5B chromosome at cytological, molecular and morphological levels and end use for induced homoeologous pairing between Triticum aestivum and Aegilops species chromosomes for precise transfer of useful variability into wheat cultivars. The monosomic 5B line of Triticum aestivum cv. Pavon (2n-1) obtained from Dr. Adam J. Lukaszewski, Professor of Genetics, Department of Botany & Plant Sciences, University of California Riverside was used as female parent. Non- progenitor wild species Aegilops peregrina and Aegilops kotschyi (UUSS, 2n=28), maintained at the Eternal University were used as the male parent for interspecific hybridization. After 2 – 3 days of emasculation, the stigmas of emasculated florets were pollinated twice with fresh pollen from Aegilops species. A total 153 F 1 hybrid seeds (ABDUS) were grown in the following year 2013 – 14. Seeds were surface sterilized with 0.2 % Bavistin and sown in the seedling tray for 21 – 25 days and then transplanted in experimental fields of Eternal University. The wheat ph1b deletion of Ph1 locus generated by Sears (1977) spans ~70 Mb. To screen for deletion lines with different breakpoints and thereby define more closely the region containing the ph1 locus, PCR markers for loci dispersed within the ph1b deletion have been developed (Gill et al. 1993). Screening of the F 1 hybrids at molecular level was done using molecular marker Xpsr2120 which has been tagged with the ph1b deletion of PhI gene on chromosome 5BL (Qi et al. 2007). The sequence of the primer pair was; forward (5 ′ -TTA ACG CCA GGG CAT ACT C-3 ′ ) and re- verse (5 ′ -CTG CAG GAG GCG CTG GA-3 ′ ) with amplicon length of 232-bp. DNA was isolated from fresh intact leaf of hybrids using CTAB methods described by Qu et al. (1998). PCR was carried out in Applied Biosystems Thermocycler model ABI Veriti according to the method of Ayala et al. (2013) with some modifications. To study the meiotic chromosome number and homoeologous pairing behaviour, spikes of F 1 hybrids were fixed in Carnoy ’ s fixative for 24 h and transferred to 70 % ethanol solution. Anthers of hybrids at various stages of meiosis-I were crushed in 2 % acetocarmine (Sigma) solution and pollen mother cells (PMCs) were scored for analysis of chromosome number and pairing behaviour with compound microscope (Magnus). Several morphological characteristics such as plant height, number of tillers per plant and spike morphology were also studied in hybrid plants with and without chromosome 5B. Cytogenetic analysis revealed that the hybrid plants had either 35 or 34 chromosomes as expected and were complete- ly male sterile (Fig. 1). Plants with 34 chromosomes showed high level of induced homoeologous pairing due to the defi- ciency of 5B chromosome whereas the plants with 35 chromosomes with 5B chromosome had more number of univalents and reduced homoeologous pairing (Fig. 1). Koebner and Shepherd (1986), Sears (1977) also reported higher level of homoeologous pairing between wheat, rye and barley chromosomes without 5B rather than using the ph1b mutation of wheat. Higher chromosomal pairing and multivalent formation in plants without chromosome 5B have confirmed the effectiveness of absence of 5B for induced homoeologous pairing for introgression of desirable traits from related wild species to wheat cultivars. The induced homoeologous chromosome pairing system of Triticum aestivum has been frequently used for obtaining the hybrid F 1 generation (Liu et al. 2002; Cai et al. 2005). Further screening of the F 1 hybrids using Xpsr2120 revealed that out of a total 126 plants, 98 (77.77 %) were without chromosome 5B while 28 plants were with 5B (Fig. 2, Table 1). The absence of amplification was associated with the absence of chromosome 5B. Sears (1972) also reported that the selfing of monosomic plants in Triticum aestivum developed about 75 % of the female gametes which were without the critical monosomic chromosomes. Results obtained at our cytological level were in agreement with molecular marker results, as the hybrids with 34 chromosomes showed no amplification with Xpsr2120 while hybrids with 35 chromosomes showed amplification of PCR product of approximate- ly 230 bp size indicating the presence of chromosome 5B in those plants. Synchrony in the results of cytology and molecular marker confirms the use of this marker for identification of plants without chromosome 5B at the seedling stage thus avoiding cytological analysis during flowering. Comparison of morphology between the hybrids plants tagged as with (+5B) and without ( − 5B) on the basis of cytological and molecular results revealed that the plants without chromosome 5B had stunted and bushy growth habit with reduced height and more number of tillers per plant while those with 5B plants had normal growth (Fig. 3). All the plants with and without 5B chromosomes were highly male sterile due to aneuploidy, abnormal meiosis and imbalanced gamete formation. These morphological attributes of the interspecific hybrid plants without 5B, like nullisomic 5B plants of Triticum aestivum can be used as the morphological marker for the enrichment of plants without 5B for cytological and molecular characterization. The present investigation clearly indicates that one or more of the molecular, morphological and cytological approaches could be used to screen and confirm the wheat and related wild species interspecific hybrids without chromosome 5B in crosses involving monosomic for chromosome 5B as the female parent for induced homoeologous pairing. The interspecific hybrids without 5B chromosome had high level of induced homoeologous pairing for precise and compensating transfer of useful variability from related non-progenitor ...