A. Mujeeb-Kazi, A. Cortés, V. Rosas, S. Cano, J. Sánchez, L. Juárez, and R. Delgado

Introduction

The CIMMYT Wheat Wide Crosses program has been exploiting accessions of the primary gene pool diploid (2n=2x=14, DD) wheat relative Aegilops tauschii (syn. Aegilops squarrosa, Triticum tauschii) for the past 10 years. Because of their wide diversity, global distribution, and genetic proximity to the D genome of bread wheat, the accessions provide a unique opportunity for bread wheat improvement.

Ae. tauschii accessions have been indiscriminately hybridized with T.turgidum to produce 800 synthetic hexaploids (SH) so far. All SHs have a spring habit, which has accelerated screening without having to deal with vernalization and other constraints. Synthetics resistant to scab have been crossed to susceptible bread wheats in an attempt to transfer the Ae. tauschii resistance to superior bread wheat cultivars. To diversify the resistance available in Ae. tauschii and its accessions, perennial Triticeae species of the tertiary gene pool have also been hybridized to bread wheat, leading to amphiploids and backcross derivatives. The above two groups of materials form the present objective of alien germplasm screening for Fusarium graminearum using artificial inoculation in the field in Atizapan, Toluca, Mexico. Test germplasm is distributed over all three Triticeae gene pools.

Materials and Methods

• 800 SH wheats, derived from crosses of 34 T. turgidum and 460 of the 490 Ae. tauschii accessions.
• Advanced progenies from resistant synthetic/susceptible bread wheats.
• Amphiploids of Thinopyrum bessarabicum and Th. elongatum with bread wheat, and some BCI self-fertile intergeneric hybrid combinations.
• Disomic 44 chromosome addition derivatives from bread wheat/Leymus racemosus//bread wheat combinations.
• Disomic addition lines of Th. bessarabicum.
• 190 A genome hexaploids derived from durum x A genome diploid combinations (2n=6x=42, AAAABB) and 50 B genome hexaploids from durum/Ae. speltoides accessions (2n=6x=42, AABBBB).

• CIMMYT station, Toluca, Mexico (19°17'N, 99°39'W, 2640m above sea level).
• Plot size: (a) Unreplicated hill plots of all genetic stocks comprised of SH wheats, amphiploids, BCI self-fertiles and disomic addition lines. b) two 2-m rows, 15 cm between rows in 90 cm beds.

• Fusarium head scab isolates were obtained from Toluca, Patzcuaro, and El Tigre, Mexico. A concentration of 50,000 spores/ml of water was used for the inoculum.*
• Cotton inoculation method: A tiny tuft of cotton permeated with inoculum suspension is placed in a floret by opening the glumes of a spikelet in the middle part of the spike with a pair of tweezers. The spike is then covered with a glassine bag to prevent damage. Five to ten random spikes were inoculated per entry.

Disease evaluation

Fusarium head scab. Disease was scored 30-35 days after inoculation. Inoculated spikes were harvested, percent Fusarium-infected spikelets evaluated, and scab scores of inoculated spikes averaged.

• Mitosis and Giemsa C-banding. Standard protocols based on aceto-orcein staining for mitosis and 4% Giemsa staining used in CIMMYT's wide crosses laboratory were followed.
• The meiotic procedure utilized alcoholic carmine and aceto-carmine combination of staining (Mujeeb-Kazi et al. 1994). The fluorescent in situ hybridization (FISH) meiotic protocol was adapted from Islam-Faridi and Mujeeb-Kazi (1995).

Results and Discussion

Resistance in SH wheats
The SH wheats (T. turgidum x Ae. tauschii) most resistant to F. graminearum during field screening at Toluca, Mexico, are presented in Table 1. Only those entries with less than 15.0% infection scores (Type II) are shown. Resistant bread wheat (BW) check Sumai scored less than 15%, while the susceptible BW check cultivar 'Flycatcher' ranged between 24.6 and 45.5% with a cross year mean of 33.8%. The susceptible durum wheat 'Altar 84' had a mean score of 40.8%. Figure 1 shows a susceptible durum wheat, and a F. graminearum-resistant SH using artificial inoculation in the field. Resistance in BW/SH advanced derivatives

The most advanced and promising entries from the BW/SH combinations were further tested for the other three scab resistance categories (Types I, III, IV). Four were found to possess combined resistance to all four types of scab (Table 2). These are currently being used in bread wheat breeding at CIMMYT and in collaborative activities with the US Scab Initiative (Fig. 2a,b) (Mujeeb-Kazi et al. 1998).

Figure 1. Fusarium type II testing showing in (a) durum wheat susceptibility, and (b) resistance in the synthetic hexaploid (SH).

Figure 2. Fusarium type II testing showing in (a) bread wheat (BW) susceptibility, and (b) resistance of a derivative from the susceptible BW/resistant SH with the Mayoor//TK SN 1081/Ae. tauschii (222) pedigree.

Figure 3. The Mayoor//TK SN 1081/Ae. tauschii (222) scab resistance line with multiple disease resistance showing in (a) high Helminthosporium sativum resistance at the dough grain fill stage, and (b) resistance at maturity. The susceptible BW check is on the left.

The combination Mayoor//TK SN 1081/Ae. tauschii (222) and several of its sister lines exhibit superior scab resistance across its four categories and also possess resistance to S. tritici, Tilletia indica, and H. sativum (Fig. 3a,b). One line was crossed with 'Flycatcher' (susceptible to all the above stresses), and the F₁ seed was used to produce 160 doubled haploids (DH) for molecular mapping/phenotyping.

The tertiary gene pool for bread wheat improvement
New genetic diversity. Tertiary pool species hold promise for providing additional genetic diversity for scab resistance. Of high priority at this stage are crosses of wheat x Th. bessarabicum and their backcross derivatives, where the ph locus is involved to promote the introgression of alien genes. Several disomic additions of Th. bessarabicum in wheat have been identified as low scoring type II infection stocks. These are being exploited for achieving genetic introgressions in addition to the priority use of the amphiploid (T. aestivum/Th. bessarabicum) (Mujeeb-Kazi 1998).

Figure 4. Two meiocytes from the cytogenetically manipulated derivatives showing low chromosomal pairing (a) and high chromosome pairing (b) involving wheat and alien chromosomes as a consequence of the Ph and ph genes.

The functioning of the cytogenetic manipulation process, where ph ph plants are first detected by the PCR technique, exhibits high meiotic pairing (Fig. 4a,b) and demonstrates wheat/Th. bessarabicum chromosome associations identified by FISH. The ph based manipulation protocol is anticipated to permit multiple exchanges and may short-cut the transfer process where several chromosomes control resistance.

Scab resistance from Leymus racemosus. A wheat x L. racemosus F₁ hybrid was first produced in CIMMYT in 1981. Its C-banded profile was reported later (Mujeeb-Kazi et al. 1983) as was its potential for scab resistance. More recently Chen et al. (1997) reported that three of the L. racemosus addition lines developed by them demonstrated scab resistance. We thus re-examined our earlier uncategorized 44 chromosome stocks (Chinese Spring/L. racemosus//CS/3/Pvn (n) ) in the MV-2000 Toluca cycle. Currently six disomic addition lines have been identified with low scab scores based upon C-banding. Three ditelocentric lines with low type II scores have also been identified. Each entry is targeted for subsequent ph based manipulation.

• Several diploid (2n=2x=14, AA) accessions combined with elite durum cultivars yielded AAAABB hexaploids, after their AAB F₁ hybrids were colchicine doubled. In the initial screening only five of the 174 hexaploids exhibited Type II promise with mean infection scores between 13.5 to 15.0%. These will be evaluated further. Novel B genome hexaploids (2n=6x=42, AABBBB) have been produced that may have potential for scab resistance.
• Another strategy in place is attempting to incorporate resistant D genome diversity into the A genome via homoeologous exchange facilitated by the ph1c genetic durum stock 'Capelli'. Cytological evidence from F₁ hybrids validate A and D genome chromosome pairing.

Conclusions

• Synthetic hexaploid wheats derived from T. turgidum x Ae. tauschii crosses express moderate but satisfactory levels of scab resistance.
• Resistance from SH wheats has been transferred to elite-but-susceptible bread wheat cultivars.
• One promising linethe multiple disease resistant Mayoor//TK SN1081/Ae. tauschii (222) has been crossed with Flycatcher (susceptible) and a DH population developed for molecular mapping.
• Tertiary pool diversity for scab identified in some Thinopyrum and Leymus species is being introgressed into bread wheat.
• Durum improvement for scab is being addressed via AAAABB, AABBBB hexaploid genetic stocks and by the scab resistant D genome to A genome homoeologous transfers.

References

Chen, P., D. Liu, and W. Sun. 1997. New countermeasures of breeding wheat for scab resistance. In: Dubin, H.J., L. Gilchrist, J. Reeves, and A. McNab, eds. Fusarium Head Scab: Global Status and Future Prospects. Mexico, D.F.: CIMMYT. p. 59-65.

Islam-Faridi, N.I., and A. Mujeeb-Kazi. 1995. Visualization of Secale cereale DNA in wheat germplasm by fluorescent in situ hybridization. Theor. Appl. Genet. 90:595-600.

Mujeeb-Kazi A. 1998. An analysis of the use of haploidy in wheat improvement. In: Application of Biotechnologies to wheat breeding. M.M Kohli & M. Francis (ed.). CIMMYT-INIA, La Estanzuela, Uruguay. p. 33-48.

Mujeeb-Kazi, A., L.I. Gilchrist, and R. Delgado. 1998. Potential of alien genetic diversity for improving resistance in wheat. Agron. Abst. p.:78.

Mujeeb-Kazi, A., Q. Jahan, and A.A. Vahidy. 1994. Application of a somatic and meiotic cytological technique to diverse plant genera and species in the Triticeae. Pak. J. Bot. 26:353-366.

Mujeeb-Kazi, A., M. Bernard, G.T. Bekele, and J.L. Miranda. 1983. Incorporation of alien genetic information from Elymus giganteus into Triticum aestivum. Proc. 6^th. Int. Wheat Genetics Symp., Kyoto Japan, p. 223-231.

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