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International Maize and Wheat Improvement Center - CIMMYT

Lisboa 27, Colonia Juárez, Apdo. Postal 6-641, 06600, México, D.F., México.

Progress in exploiting Aegilops tauschii for wheat improvement. [p. 79-89]

Abdul Mujeeb-Kazi, Roman Delgado, Alejandro Cortes, Silverio Cano, Victor Rosas, and Jesus Sanchez.

Introduction. In conventional improvement of bread wheat, breeders normally have made crosses between cultivars. Such crosses have few constraints and invariably all associations of parental traits and segregation are based on genetic recombination. The next step in bread wheat improvement is to tap the varied gene pools of other Triticum species. For swift output from such crosses, breeders utilize the numerous alien accessions of species whose genomes are similar to the A, B, or D genomes of bread wheat. These crosses allow for relatively easy alien gene transfers, are compatible with normal field research, and set the stage for the successful introgression of several genes simultaneously by homologous exchange. Elucidated here is the interspecific hybridization area where the focus is on the D-genome diploid Ae. tauschii.

An important component of CIMMYT's strategy for exploiting genetic diversity in order to improve wheat production constraints globally is on disease and stress tolerance research using durum-based synthetic hexaploids (T. turgidum subsp. durum/Ae. tauschii). This approach is aimed at protecting yield potential in farmers' fields, the environment, and farmers' incomes by reducing dependence on pesticides for disease and pest control. Screening work for biotic and abiotic stresses was conducted over the years in various locations in Mexico using primary synthetic hexaploids (SH) developed at CIMMYT. A wide diversity of resistance and tolerance in the various synthetics was observed when subjected to different biotic and abiotic stress pressures. Results indicated that such wheats were, in most instances, significantly superior for biotic/abiotic resistances/tolerances when compared to their durum progenitors. These results unequivocally showed that the synthetics possessed the genetic diversity for disease and stress tolerances that were contributed by the diploid donor species used in their synthesis. They are spring types, highly crossable to advanced bread wheats, and may be used easily in breeding. Hence, the transfer of this resistance/tolerance of the SH wheats to elite, bread wheat cultivars followed. Presented in this article is the data for SH wheats and their prebred wheat derivatives that show improved biotic and abiotic tolerances and are a conduit for global wheat improvement around novel germ plam.

We also are adding brief additional information that elucidates the use of other species that have been used for development of different genetic stocks that could impact wheat improvement and assist molecular diagnostics.

Materials and methods. Germ plasm development. Elite, durum wheat cultivars were crossed with several hundred Ae. tauschii accessions. Embryos were rescued, plated in artificial media, differentiated, and yielded F1 hybrids (2n = 3x = 21, ABD) that produced 2n = 6x = 42, AABBDD synthetic hexaploids upon colchicine treatment. Accession acquisition and procedures for SH production have been earlier described (Mujeeb-Kazi et al. 1996a). These SH wheats are maintained by increasing seed of each combination under controlled conditions by bagging at least 50 spikes/combination with glassine envelopes at each increase cycle. Based upon growth performance in two locations in Mexico (Ciudad Obregon and El Batan), an elite set of 95 SH entries has been assembled for global distribution. Distribution of this elite SH set is handled by CIMMYT's Genetic Resources Group. Further promising SHs for various stresses have been assembled into subsets.

Germ plasm screening. The SH germ plasm was screened for H. sativum, F. graminearum, S. tritici, and T. indica in Poza Rica, Toluca (Scab and Septoria), and Obregon, Mexico, respectively, over 3 to 5 years, which forms the basis of conclusions made in this paper. All evaluations were under field conditions. The synthetics were planted in hill plots. Evaluation protocols were similar to those earlier reported by Mujeeb-Kazi et al. (1996b) for H. sativum, Mujeeb-Kazi et al. (2000b) for Fusarium, Mujeeb-Kazi et al. (2000a) for S. tritici, and Mujeeb-Kazi et al. (2001) for T. indica.

Abiotic stress screening for salinity was conducted under greenhouse conditions at El Batan, Mexico, according to the hydroponic protocol of Gorham et al. (1987) and Shah et al. (1987) based upon K:Na discrimination after 21-day growth of the seedlings in 50 mM NaCl. Screening for drought and waterlogging was done in Obregon, Mexico.

Identification and utilization of resistant SH germ plasm. From the stress test data, resistant SHs were identified and hybridized with elite but stress-susceptible bread wheat cultivars according to a global priority. The hybrids were advanced by the pedigree method, with a focus on making selections for plant type, maturity, height, and specific plus multiple disease resistance . Protocols for evaluation and the test sites were similar to those described for the SH germ plasm above. The SH/BW or BW/SH entries from all biotic stress related germ plasm were field planted in 2- to 3-m double rows, tested, and led to desired selections that were stabilized routinely using the maize-based, DH protocol (Mujeeb-Kazi 2000) before international seed distribution.

Germ plasm distribution strategy. For each of the four biotic stresses, SH wheats and their advanced derivatives from crosses onto BW have been distributed to wheat breeding programs upon request and also registered as germ plasm stocks in Crop Science with samples of each entry reported deposited in the U.S. germ plasm bank and the Gene Bank of CIMMYT in Mexico. Small amounts (3 g) of the samples can be obtained from the author on a one-time basis only.

Produciton of additional stocks. Similar to the production of D-genome hexaploids, the protocols and strategy for the development of new hexaploid stocks utilizing the A- and B-genome diploid accessions have been described (Mujeeb-Kazi 2003). In addition, to facilitate gene pyramiding, new tetraploids also have been produced involving A- and D-genome diploids. The current status of all these germ plasms are in Tables 1 and 2.

Development of mapping populations. The main biotic stress priority currently is F. graminearum head scab in bread wheat. Some superior advanced BW/SH derivatives have been identified that are being used by breeders at CIMMYT, the U.S., and some national breeding programs for wheat improvement. One such line (Mayoor//TKSN1081/Ae. tauschii 222) possesses FHB resistance across all four categories; type I (penetration), type II (spread), type III (toxin-DON), and type IV (test weight loss). This line also possesses multiple disease resistance to all three rusts, H. sativum, S. tritici, and N. indica. The bread wheat cultivar Flycatcher is susceptible to all scab types and the other six stresses. This line has formed the basis of developing a mapping population that involves crossing the resistant and the susceptible line. The F1 derivatives were crossed with maize and 170 DHs were produced that form a mapping population for molecular study and phenotyping currently underway in Mexico. Listed in Table 10 are other populations produced for scab and drought tolerance.

Results and discussion. Interspecific combination stocks consist of amphiploids of the A-, B-, and D-genome diploid accession hybrids with durum wheat cultivars. These stocks are all hexaploid (2n = 6x = 42) and genomically AAAABB, AABBBB, and AABBDD. The A-genome sources are T. monococcum subsps. aegilopoides and monococcum and T. urartu. Aegilops speltoides accessions of the Sitopsis section were involved in the B-genome products, whereas Ae. tauschii accessions provided the D-genome accessional diversity as elucidated in Fig. 1 and Fig. 2. A- and B-genome stocks are targeted mainly for durum improvement, whereas the D-genome hexaploid contribution is principally for bread wheat. For a specific stress resistance, A- and D-genome accessions have led to new tetraploids (2n = 4x = 28, AADD) being produced, facilitating gene pyramiding for bread wheat improvement.

Focusing mainly by on the D genome, we report that practical outputs have emerged from interspecific combinations for numerous biotic/abiotic stresses. Currently, the most significant in practical usage are the D-genome SH stocks for FHB, H. sativum, Karnal bunt, S. tritici, drought, waterlogging, and salinity tolerance. Once these resistances/tolerances are identified in the D-genome SH stocks, their combinations with elite but trait susceptible bread wheat cultivars have yielded derivatives that are free threshing, resistant/tolerant, and in good agronomic plant type. Data of some of these promising synthetics and their derivatives are presented in Tables 2 to 9.

Synthetics for each stress also have been grouped into subsets and are being utilized for DNA fingerprinting via collaborators who are applying the D-genome microsatellites for establishing polymorphisms, which are abundant in the germ plasm. Based on this molecular input, molecular mapping populations for some attributes have been produced by crossing susceptible BW/resistant SH F1s with maize leading up to 100/170 DH/trait (Table 10). Mapping populations produced are for drought and FHB. Precaution was taken to use parents for these populations that possess multiple disease resistances.

Selection criteria for resistant/tolerants SHs and their derivatives. The criteria set for identifying resistance or tolerance to biotic/abiotic stress for traits of this study were set from data generated in our tests over several years of evaluation at sites in Mexico. The limits set were stringent for the germ plasms to be advanced for prebreeding objectives, not to exceed a 3-3 double digit score for H. sativum and 2-2 for S. tritici, 15.0 % or less type-11 infection score for F. graminearum, and less than 3.0 % infected kernels for N. indica. For salinity, tolerant germ plasms were to possess K:Na discrimination values of greater than 2.0 where values close to 1.0 were associated with non-tolerance to the stress. For drought tolerance the parameters comprised of grain yield, above ground biomass and 1,000-kernel weight, whereas chlorosis was the principal factor that discriminated entries tolerant to waterlogging conditions.

Synthetic related germ plasm for biotic stress resistances.
Fusarium graminearum. The SH wheats (T. turgidum/Ae. tauschii) most resistant (less than 15 % infection) to F. graminearum (type II) are presented in Table 3a. The resistant BW check Sumai 3 scored around 15 % or slightly less, whereas the moderately susceptible BW check Flycatcher always had over 20 % infection and the durum wheat Altar 84 over 40 %. After several cycles of testing some advanced BW/SH scab-resistant entries were selected for type-II resistance (Table 3b). These derivatives also generally possessed resistance to leaf rust, stripe rust, and S. tritici. Each scab-resistant entry selected had a disease score of less than 15 % across each test year. Sumai-3 averaged 12 % over the test years.

The most promising entries from the BW/SH combinations were further tested for the other three scab-resistance categories (types I, III, and IV). Three were found to possess combined resistance to all four types of scab. These are currently being used in bread wheat breeding at CIMMYT and in the collaborative activity with the U.S. Scab Initiative.

The combination 'Mayoor//TK SN 1081/Ae. tauschii (222)' and several of its sister lines exhibited superior scab resistance across the four categories and also have resistance to S. tritici, N. indica, and H. sativum. One line was crossed with Flycatcher (susceptible to all the above stresses), and the F1 seed used to produce 170 DHs for molecular mapping/phenotyping. Various other lines also were utilized for additional mapping populations (Table 10).

Septoria tritici. Septoria leaf blotch limits wheat production in high rainfall areas across 10.4 x 10^6^ ha globally. Septoria tritici-resistant SHs crossed with the S. tritici-resistant wheat cultivars Seri M82, Yaco, Borlaug M95, Opata M85, Kauz, Papago M86, and the moderately resistant cultivar Bagula, gave advanced lines with good leaf blotch resistance.

Ratings of S. tritici resistance were based upon leaf damage recorded at water, milk, and dough growth stages using a double-digit modified scale. The disease ratings of each of the germ plasms indicated their superiority over the bread wheat check cultivars (scores of 2.1 or 1.1 versus 4.1 to 8.9). All germ plasms have a good agronomic plant type and were high yielding under optimum disease free environments. Pedigrees, disease scoring, and agronomic phenotype descriptor details of some of the most promising lines are in Tables 4a and 4b.

Helminthosporium sativum. Spot blotch effects wheat crops across several environments from Latin America, Africa, Asia, and southeast Asia with Bangladesh being represented as a major disease location. Our Mexican screening site is the most severe. Several SH/BW germ plasms were evaluated at this location based upon damage recorded progressively (79 to 96 days) on leaves and grain. All lines possessed superior C. sativus resistance as compared with Mayoor a resistant check and Ciano 79 the susceptible check.

The SHs represent diverse accessional gene pyramiding and were developed by intercrossing several different T. turgidum/Ae. tauschii involving different Ae. tauschii accessions. From segregating F2 populations, spot blotch-resistant plants were selected and hybridized with Zea mays. The resulting haploids (n = 3x = 21, ABD) were colchicine treated to yield homozygous doubled derivatives (2n = 6x = 42, AABBDD). Some of the best lines (SH and advanced lines) are listed in Tables 5a and 5b. The disease scores do not exceed 3-2 and grain finish is less than 2 versus susceptible scores of 9-9 and 4, respectively.

Neovossia indica. Several of the Ae. tauschii accessions in our working collection were identified as sources of Karnal bunt resistance. These accessions were randomly hybridized with T. turgidum cultivars to yield SH wheats and resistant entries identified. The bread wheat germ plasm lines were derived from the Karnal bunt-resistant SHs crossed with Karnal bunt-susceptible bread wheat cultivars Flycatcher, Kauz, Yaco, Borlaug, and Papago M86. Segregating generations of the crosses were advanced by pedigree method. The mean agronomic performance of the germ plasm lines over 5 years of field tests demonstrates an acceptable phenotype; which is an asset for breeding use.

The disease score was based on the number of infected and healthy kernels at maturity in each plot. Synthetic hexaploid and bread wheat germ plasm line infections ranged from 0 % up to 1.97 % compared with a 30 % mean infection of WL711, the susceptible bread wheat check cultivar. The durum wheats in the pedigrees had infection levels from 0.3 to 1.6 %, whereas the SH wheats were immune. These germ plasms offer genetic diversity of the Ae. tauschii accessions as well as the A- and B-genome diversity of the durum cultivars in the SH pedigrees. Data of some resistant SH germ plasm lines is in Table 6.

Drought tolerance. The results of this screening are presented in Table 7. The yield of the synthetics ranged from 638 kg/ha to 4,037 kg/ha with an overall mean yield of 2,098 kg/ha. The highest yielding synthetic was comparable to the highest yielding check cultivar, Nesser (4,065 kg/ha). Compared to the check cultivars, 1,000-kernel weight was significantly heavier on all the synthetic genotypes. Mean 1,000-grain weight ranged from 33.4 g to 51.2 g with an overall average of 42.2 g. Eighteen synthetic lines had biomass yield at maturity better than the highest biomass yielding check Nesser (10.8 t/ha).

Waterlogging tolerance. Test were conducted under flooded basins 12 m wide by 11 m long. Twenty-four genotypes were planted in each basin. Waterlogging treatment commenced at 15 days after emergence for 7 weeks with continuous standing water maintained at 3-8 cm. Assessment of damage was based on degree of leaf yellowing (chlorosis) visually estimated on each plot at the end of the 49-days waterlogged treatment and mas made 1 day after the basins were drained.

Table 8 presents 14 synthetic lines tolerant or moderately tolerant to waterlogging as compared to the check cultivars. Adjusted mean percent chlorosis over 2 years ranged from 7 % to 75 % with an overall mean of 43 %. Five synthetic entries had leaf chlorosis scores of less than 10 % compared to 13.9 % of the most tolerant bread wheat check cultivar Ducula.

Table 8. Percent leaf chlorosis and agronomic characteristics of synthetic hexaploids (Triticum turgidum/Ae. tauschii) tolerant to waterlogging. Ae. tauschii accession number in the wheat wide crosses working collection are in parentheses.

Synthetic hexaploid	Chlorophyll (%)	Yield/ spike (g)	Days to flowering	Physiological maturity (days)	Plant height (cm)	Spike length (cm)	1,000-kernel weight (g)	Grains/ spike
Dverd2/Ae. tauschii (221)	7.3	1.59	102	136	77	11.1	49.9	32
Botno/Ae. tauschii (617)	7.6	1.57	106	140	95	11.5	46.1	34
68.111/RGB-U//Ward/3/Ae. tauschii (454)	8.2	1.54	96	129	85	9.5	36.1	43
Ceta/Ae. tauschii (895)	8.6	1.67	102	136	89	10.8	38.0	44
SCA/Ae. tauschii (409)	8.9	1.72	92	128	93	11.5	42.8	40
68.111/RGB-U//Ward/3/Pgo/4/Rabi/5/Ae. tauschii (878)	12.0	0.88	99	131	93	8.4	34.2	26
Altar 84/Ae. tauschii (221)	12.3	2.10	106	139	83	12.6	40.8	51
SCA/Ae. tauschii (518)	12.7	1.47	106	139	84	12.4	46.2	32
68.111/RGB-U//Ward/3/Pgo/4/Rabi/5/Ae. tauschii (878)	13.4	1.55	104	138	89	11.2	48.4	32
68112/Ward//Ae. tauschii (369)	13.7	1.82	100	134	81	12.8	50.5	36
GAN/Ae. tauschii (897)	14.5	1.98	97	132	87	10.5	45.8	43
DOY1/Ae. tauschii (458)	15.1	1.55	103	138	89	12.0	51.8	30
CPI/Gediz/3/GOO//Jo69/CRA/4/Ae. tauschii (409)	16.1	1.75	101	136	88	11.9	52.9	33
68112/Ward//Ae. tauschii (369)	16.8	1.51	100	136	81	12.2	48.7	31
Ducula (tolerant check)	13.9	1.42	99	144	69	7.5	38.4	37
Pato Blanco (tolerant check)	16.9	1.17	102	139	68	8.4	31.9	37
Seri M82 (susceptible check)	74.8	1.62	101	141	66	8.8	37.9	43
LSD (0.05)	7.0	0.27	2	4	10	0.6	2.3	6
CV (%)	9	12	4	2	7	8	5	10

Salt tolerance. From the wide array of synthetic hexaploids produced in CIMMYT and after an initial field screening in Baja, CA (sea water dilution from 8 to 24 dS) we selected a few promising salt-tolerant types from the synthetic germ plasm that were subsequently evaluated in hydroculture (Table 9). The hydroculture screening protocols for salinity levels maintained by NaCl over regimes between 0 and 250 mol/m3 together with growth related parameters and Na and K estimations for establishing K+/Na+ discrimination values were identical to those adopted in the laboratory in Bangor, Wales (Gorham et al. 1987; Shah et al. 1987). K+/Na+ discrimination is a trait that enhances salinity tolerance in bread wheat compared to durum wheat and is present in the wheat ancestor Ae. tauschii. K+/Na+ ratios were lower in the durum parents (close to 1.0) than in the elite synthetics (over 1.0 and greater than 2.0 being selected for breeding use). This data confirmed that the K+/Na+ trait was present in the synthetics and demonstrated its poor expression in durum wheats.

Conclusions. Experts predict that today's worldwide population of just over 6.0 billion people will grow to 8.2 billion over the next two decades. By 2050, 12 billion people will crowd the planet with more than 90 % of the growth occurring in developing nations. Estimates are that the world would require one billion metric tons of wheat over the next 2 decades compared to the current production of slightly over 600 million metric tons. Hence one can extrapolate and conclude that the mean global yield of wheat would have to shift from 2.5 t/ha to over 4 t/ha. These ominous circumstances are placing a formidable task before agricultural scientists and the food management sector. This productivity only can happen around an invigorated research program with super integration of multidisciplinary activities in which germ plasm and genetic resources would remain paramount. Plant breeders involved in crop improvement efforts in order to meet the ever-increasing demand for food are finding less and less appropriate germ plasm with desired traits among cultivated crops themselves with which to make the needed improvements. Fortunately, new and useful genetic resources are being found in wild, uncultivated plants. The challenge is to incorporate this germ plasm into existing food crops, which forms the crux of this article.

The main focus addressed here has been on utilizing novel genetic diversity that resides in the wild wheat progenitor species, with major emphasis on one diploid progenitor only; Ae. tauschii (2n = 2x = 14; DD). Many options are available to utilize this species source and in each case the end product will yield diversity that is beneficial for wheat improvement. The outputs reported here address only a few stresses and, from such a resource, cultivars have already been released in China showing superior rust resistance and in Spain with good quality coupled in both cases with high yield. Hence, the future provides an optimistic avenue for wheat improvement that will be made more efficient due to the allelic diversity of the alien sources that support molecular applications. All germ plasms are freely available for wheat researchers globally by contacting the senior author at the m.kazi@cgiar.org.

References.

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