ITEMS FROM INDIA

BHABHA ATOMIC RESEARCH CENTRE

Nuclear Agriculture and Biotechnology Division, Mumbai-400085, India.

Identification, validation, and use of molecular markers for combining quality with durable rust resistance in Indian wheat. [p. 33]

B.K. Das and S.G. Bhagwat (Nuclear Agriculture & Biotechnology Division) and A. Saini and N. Jawali (Molecular Biology Division).

Genetic improvement of wheat for quality and rust resistance is continuing. The HMW-glutenin subunits are being used as a criterion for selection. Rust resistance genes such as Sr31 and Sr24/Lr24 are being combined with high yielding ability. Selections made on the basis of good agronomic characters are being advanced.

Marker-assisted selection for pyramiding stem rust resistance genes Sr31 and Sr24 in Indian wheat is being carried out. SCAR markers that identify the Sr31 gene in homozygous or heterozygous condition were developed. DNA markers reported in literature for the Sr24/Lr24 gene (SCS73719) was validated in Indian wheat cultivars and segregating populations. Phenotypic scoring for stem rust reaction and a cosegregation study with the marker was made in an F2 population from the cross 'Kalyansona (-Sr24/Lr24)/Vaishali (+Sr24/Lr24)'. The SCAR marker for the Sr24 gene, along with the SCAR markers for Sr31, are being used for pyramiding these two genes.

Selections for Glu-D1d (coding for HMW-glutenin subunits 5+10), Sr24, and Sr31 are being made from intercultivar crosses by use of molecular markers.

Identification of DNA markers for stem rust resistance gene Sr26. [p. 33]

Ruchi Rai, B.K. Das, and S.G. Bhagwat (Nuclear Agriculture and Biotechnology Division).

The stem rust-resistance gene Sr26 has the potential to provide durable resistance to stem rust if it is pyramided with other Sr gene(s). Two markers, one based on a RAPD marker and the other based on an AP-PCR marker associated with stem rust resistance conferred by Sr26, were identified. Linkage analysis was done by studying cosegregation of the markers with resistant phenotype in a F2 population from a cross between Kalyansona and the Australian cultivar Kite (Sr26).

Thermotolerance in wheat. [p. 33-34]

Suman Sud and S.G. Bhagwat (Nuclear Agriculture and Biotechnology Division).

High temperature stress is a major environmental constraint that lowers wheat productivity in warmer areas. In India, wheat cultivars are developed for different agroclimatic zones and, thus, show wide variability. We explored the cultivated germ plasm to mine genes for thermotolerance for further utilization. Fifty-six Indian bread wheat genotypes were assayed for acquired thermotolerance at the seedling stage. Ten-day-old seedlings were hardened and then subjected to membrane thermostability (MTS) and cell viability (TTC reduction) assays. Six thermotolerant genotypes identified from the assays and one relatively nontolerant genotype were grown in the field till maturity. Flag leaf area was estimated 10 days after emergence, and flag leaf senescence was recorded 17, 24, and 31 days after flag leaf emergence. Daily maximum and minimum air temperatures were recorded throughout the crop season. Variability was detected among the 56 genotypes for acquired thermotolerance. Significant correlation was observed between MTS and TTC values. The TTC assay, which measured cell viability after heat shock treatment, showed significant positive association with grain yield/plant and yield/meter. Variation among the thermotolerant genotypes for yield and yield components was observed. Because the TTC assay showed positive correlation with yield under high temperature stress, it can be used as a selection criterion in breeding for warmer areas. To improve the productivity in warmer areas selection for heat stress tolerance accompanied by superior yield components will be needed. The tolerant genotypes identified in this study can serve as parents in improvement of thermotolerance of wheat.

Agronomic characterization, Rht genotyping, and high-molecular-weight glutenin subunit profiling of oligo-derived lines. [p. 34]

Suman Sud and S.G. Bhagwat (Nuclear Agriculture & Biotechnology Division).

Wheat is cultivated world wide in cooler environments. There is increasing interest in cultivation of wheat in nontraditional areas, including warmer environments. Also, a growing problem concerns the rise and fluctuation in temperature at the time when wheat is cultivated. Higher temperature during early phase of growth affects tillering, number of spikelets/spike, and biomass production. Oligoculm wheat is known to have high vigor and Gigas features. The vigor of oligoculm wheat expressed as long culms, thick stem, large leaves, long spikes with a high number of spikelets, and large grains. A set of 14 oligoculm derivatives obtained from the cross 'oligoculm wheat/Kundan//selection 212' was evaluated for their field performance under high temperature stress conditions. Grain yield/spike were higher in 10 derivatives, whereas on area basis the derivatives were poorer. Biomass/plant and biomass/meter were significantly higher in many of the oligoculm derivatives indicating their superior ability to accumulate biomass. The spikelet number/spike and flag leaf area were significantly higher than the checks, hence the derivatives can be used as a source of these yield components for improvement in warm environment. Harvest index in the derivatives was generally lower. The oligoculm derivatives lacked major dwarfing gene as observed by their responsiveness to gibberellin and by use of perfect markers, as a result these were taller than the semidwarf check cultivars. The oligo derivatives were found to vary in their HMW-glutenin subunit composition. Two derivatives, 8-44-1 and 8-44-2, had the subunit composition N, 7+9, and 2+12 (Glu-1 score 5). Ten derivatives showed subunit composition N, 13+16, 2+12 (Glu-1 score 6). One line each showed subunit composition 1, 7+8, 2+12, and 1, 7+9, 2+12 (Glu-1 scores 8 and 7, respectively). The variability in Glu-1 scores offers scope to select for strong or weak dough. The oligo derivatives lacked tolerance to heat stress as indicated by their rapid leaf senescence. The performance of the derivatives may be improved by introduction of semidwarfing gene(s) and by improving tolerance to high temperature.

Identification of Rht genes and pin a and pin b status of Indian wheat cultivars. [p. 34]

E. Nalini (Molecular Biology Division), S.G. Bhagwat (Nuclear Agriculture & Biotechnology Division), and N. Jawali (Molecular Biology Division).

The semi dwarfing genes Rht1 and Rht2 are common among Indian wheat cultivars. Forty-five cultivars were analyzed using PCR-based, allele-specific perfect markers. Thirteen had Rht-B1b and 24 had Rht-D1a. Grain hardness is an important quality related trait. Sixty bread wheat cultivars were analyzed for their status of pin a and pin b genes. The null form of pin a was found to be most frequent among the hard Indian bread wheats. Mapping of wheat genome using a intervarietal cross between cultivars Kalyansona and Sonalika is in progress.

Artificial neural network for identification of wheat grains. [p. 34-35]

B.P. Dubey (Reactor Control Division), S.G. Bhagwat (Nuclear Agriculture & Biotechnology Division), S.P. Shouche (Computer Division), and J.K. Sainis (Molecular Biology Division).

Observing the shape, size and color of grains is normally employed for identification of a wheat cultivar. Use of computer based image analysis may be a good alternative to visual identification. Grain shape and size are considerably influenced by changes in environment. The Artificial Neural Network (ANN), when combined with digital imaging, may have the potential for cultivar identification. Three bread wheat varieties were grown in different environments to create variation in the grain shape and size. Morphometric features of these grains were quantified using Comprehensive Image Processing Software. Data on 45 parameters were used to train ANN with different combinations of nodes and iterations. Similar samples were used for testing. A commercial and an in-house developed ANN software packages were used in this study. Best results were obtained with the resilient back propagation architecture for both. The success of correct identification was about 88 % for all the grains together and ranged from 84-94 % for individual varieties. The results showed that ANN, combined with image analysis has excellent potential for wheat cultivar identification.

Publications. [p. 35]

• Das BK, Sharma A, Marathe S, Rao VS, and Bhagwat SG. 2006. Evaluation of bread and chapati making quality of wheat selections and parents differing in HMW subunits of glutenin. J Food Sci Technol 43(1):69-72.
• Das BK, Saini A, Bhagwat SG, and Jawali N. 2005. Development of a SCAR marker linked to a stem rust resistance gene Sr31 in bread wheat. In: DAE-BRNS Life Sciences Symposium on Molecular Biology of Stress Response and its Applications, BARC, Mumbai, India. 19-21 December 2005, Book of Abstracts, p. 47.
• Nalini E, Bhagwat SG, and Jawali N. 2005. Validation of allele specific primers for identification of Rht genes among Indian bread wheat varieties. Cereal Res Comm 33:439-446.
• Rai Ruchi, Das BK, Bhagwat SG, and D'Souza SF. 2005. RAPD analysis of wheat genotypes carrying stem rust resistance gene for identification of a marker. In: DAE-BRNS Life Sciences Symposium on Molecular Biology of Stress Response and its Applications, BARC, Mumbai, India. 19-21 December 2005, Book of Abstracts, p. 64.
• Sud S and Bhagwat SG. 2005. Acquired thermotolerance in bread wheat seedlings and its relationship with plant performance in field. In: National Seminar on Plant Physiology, Navsari Agricultural University, Navsari (Gujarat) and the Indian Society for Plant Physiology, New Delhi, India. 23-25 Nov, Book of Abstracts, p. 198.

BHARATHIAR UNIVERSITY

Cytogenetics Laboratory, Department of Botany, Coimbatore-641 046, India.

Breeding for rust resistance in some Indian hexaploid wheat cultivars. [p. 35-38]

Specific genes for rust (stem, leaf, and stripe) resistance were transferred from hexaploid wheat stocks into the Indian hexaploid wheat cultivars HW 2084, PDSN-32, and K 9107. Among the three recipient wheat parents, HW 2084 and K 9107 were free from leaf rust and stem rust, respectively, however, both were susceptible to the other two wheat rusts. The third cultivar, PDSN-32, was susceptible to all the three rusts. Thus, we improved these Indian wheat cultivars by incorporating the respective rust-resistance genes for which they are susceptible. The donor parents included 10 hexaploid wheat stocks carrying a total of five leaf rust-resistance genes (Lr19, Lr24, Lr26, Lr28, and Lr37), six stem rust-resistance genes (Sr24, Sr25, Sr27, Sr31, Sr36, and Sr38), and seven stripe rust-resistance genes (Yr8, Yr9, Yr11, Yr13, Yr15, Yr16, and Yr17), present either singly or in combinations (linked condition). Transfers were made using a simple backcross-breeding method followed by selection.

Selection was made in the BC2/BC3 and/or BC5 generations, and one NIL each in the BC2F5/BC3F5 and BC5F5 was selected from all 29 cross combinations. All the lines were screened against individual rust races at the seedling stage in the glasshouse with a mixture of rust races and at the adult-plant stage in natural and artificial conditions in the field. An immune to moderately resistant reaction at the seedling stage and a highly resistant reaction at the adult-plant stage provided by the incorporated genes strongly advocate the use of specific rust-resistance genes for durable resistance.

All the rust resistance genes used provided a moderate to high degree of resistance under field conditions (Table 1). Specific rust resistance from single genes included Lr19, Lr24, Lr28, Lr37, Sr27, Sr38, Yr9, Yr11, Yr13, Yr15, and Yr16. Other rust-resistance genes, Lr26, Sr24, Sr25, Sr31, Yr8, and Yr17, were useful in combination with other resistance genes already present in the genetic background of recurrent parents.

Yield performance of the NILs was tested under rust-free conditions. Many of the NILs were significantly higher in grain yield than the chemically treated control plants. In general, the agronomic performance of the plants at the BC2F5/BC3F5 was superior to those of BC5F5-selected plants for plant height, tiller number/plant, spike length, number of spikelets/spike, 1,000-kernel weight, and grain yield. However, the various agronomic characters recorded in the BC5F5 also were comparatively superior to those of the untreated recurrent parents (Table 2). The NILs of the BC2F5/BC3F5 had very good agronomic characteristics, but the seed quality (plumpness, weight, size, and color) was not improved in many lines. Based on seed quality coupled with good agronomic characters and yield, 20 lines were finally selected for commercial purposes, and the remaining nine lines were grouped as genetic stocks for use in breeding programs.

Confirming the transfer of rust-resistance genes to Indian wheat cultivars. Transfer of rust-resistance genes into Indian wheats was confirmed through morphological, genetical, biochemical, and molecular markers. The presence of morphological markers of the donor parents, such as awnless spike (Darf*6/3Ag/Kite, Sr24+Lr24; Joss Chambier, Yr11; Longbow, Yr13; and Cap-5BL-7BL, Yr16), lax spike (RL 6081, Sr38+Lr37+Yr17), reduced yellow pigment in the seed flour (Cook*6/C 80-1, Lr19+Sr25+Sr36), clubby tip (G-25, Yr15), waxy color (Veery'S', Sr31+Lr26+Yr9), and powdery mildew severity in the F1 hybrid derivatives of different crosses between Indian wheats and donor parents suggests the successful transfer of these morphological characters along with rust-resistance genes from the donor parents to recipient Indian wheats.

Inheritance studies in the NILs for Yr9; Lr24 (HW 2084); Yr11, Yr13 (PDSN-32); and Lr19, Sr27 (K 9107); which involved crossing each of the NILs with the universally susceptible wheat cultivar Agra Local, showed that rust resistance in the NILs was due to a single, dominant gene. The F1 hybrids exhibited complete rust resistance, whereas the F2 plants segregated as 3 resistant : 1 susceptible to the respective rusts. Similarly, the BC1 hybrids segregated 1 resistant : 1 susceptible to respective rusts. These results confirm the transfer of rust-resistance genes into Indian wheats.

The F2 segregation data of the monosomic and disomic F1 hybrids of crosses between the complete set of CS monosomics and the NILs for Sr27 (HW 2084), Lr24 (PDSN-32), and Lr19 (K 9107) were studied for their respective rust resistance. A segregation ration of 3:1 (resistant:susceptible) except for lines 3A, 3D, and 7D in HW 2084, PDSN-32, and K 9107, respectively, confirmed the successful incorporation of these genes on to the respective chromosomes of the recipient wheat parents.

We studied changes in enzymatic activities of peroxidase, polyphenol oxidase, catalase, and lipoxygenase in the leaves of 25-day-old plants of rust-susceptible wheat parents and rust-resistant NILs inoculated with respective rust pathogens and found altered activity. The constituted lines had a higher peroxidase activity compared to healthy controls 2-7 days after inoculation. Polyphenol oxidase activity increased in all NILs 3-7 days post inoculation, whereas activity declined in the susceptible parents. Catalase activity was higher in susceptible wheat parents than the resistant NILs. Lipoxygenase activity increased in both the susceptible wheat parents and their NILs 2 days after inoculation but subsequently declined 7 days after inoculation in resistant plants. A consistent increase was noticed in plants of the susceptible parents. Esterase activity increased in all the NILs 3-7 days after inoculation but declined activity was observed in the susceptible parents.

The total lipid content of the leaves increased in both susceptible and rust-resistant NILs 2 days after inoculation but subsequently decreased with an increase in post inoculation time. The percent decrease was greater in the susceptible parents than in the resistant NILs. Soluble protein content increased in resistant NILs 24 hours after inoculation but decreased during later stages of infection. The percent decrease was more in susceptible than resistant lines 7 days after inoculation. Specific activities of ribonuclease-I and combined ribonuclease-II and nuclease-I was high at day15 compared to day 10 in susceptible and resistant lines. Resistant NILs had relatively higher chlorophyll content than that of the susceptible parents. Total free amino acid content increased up 8 days after inoculation in both susceptible and resistant lines followed by a slight reduction in both the cases. Respiration rate increased to a greater extent in the resistant NILs compared to susceptible parents. On the third day after inoculation, the reduction in respiration rate was drastic in the susceptible parents, whereas in resistant NILs, respiration was more or less constant. A significant increase in total free phenols and tannin content was observed in the NILs over their respective recurrent parents. The NILs had significantly higher nuclear DNA than their respective susceptible parents.

A change in the intensity of isoenzyme bands of polyphenol oxidase, peroxidase, esterase, and superoxide dismutase in susceptible and their resistant NILs were noted with Rf values. Isozyme analysis of polyphenol oxidase shows some specific bands only in NILs with different rust-resistance genes. Esterase activity of some specific esterase activity bands was present only in rust-resistant NILs, whereas these specific bands were absent in the susceptible lines. Specific SOD activity bands were observed only in resistant lines and were missing in susceptible lines. SDS-PAGE analysis of soluble protein did not show any major qualitative difference in the protein profiles in the leaves of susceptible parents and its resistant NIL. However, quantitative changes were observed in the intensity of the banding pattern; some of the major protein bands were found to increase in intensity in NILs with rust-resistance genes Yr9, Sr27, Yr17 (HW 2084); Lr19, Sr27, Yr9 (PDSN-32); and Lr19, Lr28, Yr16 (K 9107) over their susceptible (recurrent) parents. The intensity of specific protein bands with Rf values of 0.13, 0.26, 0.29, 0.36, 0.74, 0.78, 0.82, and 0.85 decreased in the leaves of susceptible plants. SDS-PAGE analysis of seed-storage proteins, under reduced conditions for the presence to Sec-1, revealed that all Veery 'S' derivatives, Veery'S', and Kavakaz (aºknown standard for the T1B·1R translocation) showed a Sec-1 band with an Rf value of 0.56, which is a characteristic feature of T1B·1RS lines. This result confirmed the successful incorporation of rye segment carrying the gene complex Sr31+Lr26+Yr9.

Molecular screening of NILs for Yr15 with the RAPD primers OPA-19 and OPB-13 confirmed the transfer of Yr15. The RAPD primers OPA-19 and OPB-13 amplified the diagnostic fragments at 1,420 bp and 1,500 bp and resulted in one additional band in the resistant NIL. This band was absent in susceptible parent.

Distribution of necrosis genes and evaluation of resistance to rusts in some bread wheats. [p. 38-42]

S. Premalatha, V.R.K. Reddy, K. Thamayanthi, and R. Kannan.

Introduction. Hybrid necrosis are frequently met with inter and intraspecific wheat crosses and are serious barriers to the transfer of genes in a planned hybridization program. Hybrid necrosis, causing gradual death or debility of F1 hybrids is often noticed in wheat crosses and this kind of weakness can be clearly distinguished phenotypically. The genetic trait of hybrid necrosis is governed by two complementary genes, Ne1 and Ne2 (Caldwell and Compton 1943; Hermsen 1963), located on chromosomes 2BS and 2BL (Tsunewaki 1960; Nisikawa et al. 1974), respectively. Hybrid necrosis proves to be a hurdle in the process of gene transfers from alien or other diverse sources of resistance to disease. Thus, documenting information on the different genes for resistance to diseases relevant to India as well as the information about the genes in wheat stocks is important. The present study determines the genes for necrosis. Stocks also were e valuated for resistance to rusts in wheats.

Materials and methods. Eighty-two T. aestivum cultivars were crossed to two T. aestivum subsp. aestivum testers, C306 (Ne1ne2) and Klein Lucero (ne1Ne2). The F1 hybrids and parents were raised in field as well as in greenhouse under optimal conditions for the expression of Ne genes. The genotypes of the parents with respect to Ne were determined from the phenotype of the F1 hybrids. The stocks were also evaluated for resistance to rusts under high incidence of natural infection.

Results and discussion. Study of hybrid necrosis. Eighty-two wheat stocks were tested for the presence or absence of necrosis gene(s). The results are presented in Table 3 and indicate that out of 82 cultivars, eight stocks were Ne1 carriers, 35 stocks were Ne2 carriers, and the remaining 39 were noncarriers for the necrosis genes (ne1, ne2). Tsunewaki (1971) tested two strains of T. aestivum subsp. sphaerococcum and reported on of them as Ne1 carrier, whereas Zeven (1971) recorded one strain that had the gene Ne1. Cultivars of T. turgidum subsp. dicoccum, like other tetraploid species of wheat, are either Ne1 carriers or noncarriers (Nishikawa 1967; Tsunewaki 1969). The Ne2 gene, found restricted to the western 6X wheats, is presumed to have originated by mutation at the hexaploid level in Europe (Tsunewaki and Kihara 1962). Zeven (1966) and Tsunewaki and Nakai (1973) reported high frequencies of Ne1 and Ne2 carriers in the hexaploid wheats of Asian and Western populations, respectively.

Similarly, eight Ne1 carriers, 35 Ne2 carriers, and 39 noncarriers of the necrosis genes were found among the 82 stocks of T. aestivum subsp. aestivum. Pukhal'skii (1980) found that Ne2 occurred especially in winter wheat and Ne1 in spring wheat, but Zeven (1981) suggested that spring and winter wheats were possibly derived from two different groups, one carrying the necrosis gene Ne1 and the other Ne2. Narula et al. (1971) and Kochumadhavan et al. (1980) have reported that the Indian bread wheat cultivars predominantly have Ne1, whereas the western European and North American wheats are mainly Ne2 carriers. Tsunewaki (1971) studied 22 accessions of compactum wheats from the U.S. and reported 5 % Ne1 carriers, 18 % Ne2 carriers, and 77 % noncarriers, whereas 34 strains of T. aestivum subsp. compactum from Asia were either Ne1 carriers (32 %) or noncarriers (68 %). Similarly, (9.76 %) Ne1 carriers, (42.68 %) Ne2 carriers, and (47.56 %) noncarriers were found among the 82 stocks of T. aestivum subsp. aestivum.

Disease resistance. This study reveals that the alien genes Lr9, Lr28, Lr32, and Lr37 and Lr19, Lr24, Sr24, Sr25, Sr26, Sr27, Lr25, and Sr31 condition a high degree of resistance. The gene Lr37 showed a high degree of resistance at the adult-plant stage, and this resistance gene, linked with Sr38 and Yr17, provides a high degree of resistance. The genes Lr21 and Sr33 conferred moderate resistance. Sr30 and Sr37 showed moderate resistance to stem rust. Sr36 and SrAgi conferred high degree of resistance to stem rust. The gene Yr9 is associated with Lr26 and Sr31, Yr9 has resistance to stripe rust, and Lr26 was found to be susceptible to leaf rust. Genes Lr19, Lr24, Lr25, and Lr28 confer effective seedling resistance to different pathotypes of leaf rust (Sawhney and Goel 1983) and also APR. Gene Lr24 is known to be linked with Sr24 (McIntosh et al. 1977), and this linkage confers a high level of resistance to both leaf and stem rusts in India. Gene Lr25 is effective against 10 important pathotypes of leaf rust in seedling stage (Sawhney and Goel 1983). The gene Lr37 showed a high degree of resistance in adult-plant stage, and this resistance gene is linked with Sr38 and Yr17, both providing high degree of resistance (Bariana and McIntosh 1993). The gene Lr21 from Ae. tauschii confers a moderate degree of resistance to leaf rust at adult-plant stage. This gene was reported to be susceptible to most of the virulent pathotypes of leaf rust in seedling (Rajendra Kumar et al. 1988) but exhibited adult-plant resistance. The gene SrAgi is the only stem rust-resistance gene present in the partial amphidiploid TAF 46 (2n = 56) generated by Cauderon et al. (1973). SrAgi is highly effective against stem rust. Gene Sr36 derived from T. timopheevii subsp. timopheevii and SrAgi conferred high degree of resistance, whereas Sr30 from common wheat and Sr37 derived from alien gene of T. timopheevii subsp. timopheevii was moderately resistant to stem rust. The genes Sr26, Sr27, Sr31, Sr36, and SrAgi confer a high degree of resistance to stem rust, and Sr25, Sr30, Sr37, and Sr38 confer a moderate degree of resistance. Gene Sr24 is completely ineffective to stem rust. Sr26, Sr27, and Sr31 confer seedling resistance to 19 pathotypes occurring in India and effective in adult-plant stage as well (Sawhney and Goel 1981). Genes Sr26, Sr27, Sr31, Sr32, Sr36, and SrAgi confer a high degree of resistance, whereas Sr25, Sr30, Sr33, Sr37, and Sr30 are moderately resistance. The genes Sr24, Sr20, and Sr34 were reported to be ineffective (Menon and Tomar 2001).

References.

• Bariana HS and Mclntosh RA. 1993. Cytogenetic studies in wheat XIV. Location of rust resistance genes in VMP1 and their genetic linkage with other disease resistance genes in chromosome 2A. Genome 36:476-482.
• Caldwell RM and Compton LE. 1943. Complementary lethal genes in wheat causing a progressive lethal necrosis of seedling. Heredity 34:66-70.
• Cauderon Y, Saigne B, and Dauge M. 1973. The resistance to wheat rusts of Agropyron intermedium and its use in wheat improvement. In: Proc 4th Internat Wheat Genet Symp (Sears ER and Sears LMS Eds). Bull Missouri Agric Exp Sta, Columbia, MO. Pp, 401-407.
• Hermsen JGTh. 1963. The genetic basis of hybrid necrosis in wheat. Genetica 33:245-287.
• Kochumadhavan M, Tomar SMS, and Nambisan PNN. 1980. Investigations on hybrid necrosis in bread wheat. Ind J Genet 40:496-502.
• Kumar R, Sawhney RN, Sharma JB, and Chopra VL. 1988. The influence of plant development stage on expression of resistance to leaf rust (Puccinia recondita) in near-isogenic lines of wheat. Plant Breed 100:225-227.
• Mclntosh RA, Dyck PLD, and Green GJ. 1977. Inheritance of leaf rust and stem rust resistance in wheat cultivars Agent and Agatha. Aust J Agric Res 28:37-45.
• Menon MK and Tomar SMS. 2001. Aegilops-derived specific genes in common wheat and their introgression into Indian bread wheat cultivars. Ind J Genet 61:92-97.
• Narula PN, Srivastava OP, and Srivastava PSL. 1971. Genetics of hybrid necrosis in breadwheat. Ind J Genet 31:136-139.
• Nishikawa K. 1967. Identification and distribution of necrosis and chlorosis genes in tetraploid wheat. Seiken-Ziho 19:37-42.
• Nishikawa K, Mori T, Takami N, and Furuta Y. 1974. Mappping of progressive necrosis genes Ne1 and Ne2 of common wheat by the telocentric method. Jap J Breed 24:277-281.
• Pukhal'skii VA. 1980. Genes for hybrid necrosis and some aspects of genetic theory of breeding. Plant Breed Abstr 50 (6108).
• Sawhney RN and Goel LB. 1981. Race-specific interaction between wheat genotypes and Indian cultures of stem rust. Theor Appl Genet 60:161-166.
• Sawhney RN and Goel LB. 1983. Effectiveness of newly described leaf rust resistance genes against Indian culture of standard races and biotypes of leaf rust in wheat. Wheat Inf Serv 56:34-36.
• Tsunewaki K and Nakai Y. 1973. Considerations on the origin and speciation of four groups of wheat from the distribution of necrosis and chlorosis gene. In: Proc 4th Internat Wheat Genet Symp (Sears ER and Sears LMS eds). Bull Missouri Agric Exp Sta, Columbia, pp. 123-129.
• Tsunewaki K. 1960. Monosomic and conventional gene analysis in common wheat. III. Lethality. Jap J Genet 35:71-75.
• Tsunewaki K. 1969. Origin and phylogenetic differentiation of common wheat revealed by comparative gene analysis. In: Proc 3rd Internat Wheat Genet Symp (Shepherd KW Ed), Adelaide, Australia. Pp. 71-85.
• Tsunewaki K. 1971. Distribution of necrosis genes in wheat. V. Triticum macha, T. spelta and T. vavilovi. Jap J Genet 46:93-101.
• Tsunewaki K and Kihara H. 1962. Comparative gene analysis of common wheat and its ancestral species. I. Necrosis. Jap J Genet 37: 474-484.
• Zeven AC. 1966. Geographical distribution of genes causing hybrid necrosis in wheat. Euphytica 15:281-284.
• Zeven AC. 1971. Fifth supplementary list of wheat varieties classified according to their genotype for hybrid necrosis and geographical distribution of Ne genes. Euphytica 20:239-254.
• Zeven AC. 1981. Eighth supplementary list of wheat varieties classified according to their genotype for hybrid necrosis. Euphytica 30:51-21-539.

Publications. [p. 42]

• Gajalakshmi K and Reddy VRK. 2005. Quality analysis of some Indian hexaploid wheat cultivars. Ann Wheat Newslett 51:50-51.
• Kalaiselvi G and Reddy VRK. 2005. Yield performance of the near-isogenic lines carrying different rust resistance genes in three Indian wheat cultivars. Research on Crops 6(1):148-151.
  

CH. CHARAN SINGH UNIVERSITY

Department of Agricultural Botany, Meerut - 250 004, India.

P.K. Gupta, H.S. Balyan, R. Bandopadhyay, J. Kumar, A. Mohan, N. Kumar, P.L. Kulwal, S. Rustgi, R. Singh, A. Goyal, A. Kumar, N. Girdharwal, V. Kumar, and R. Rouf Mir.

Development and use of molecular markers for wheat genomics and breeding. [p. 43-46]

Updating and construction of framework linkage map(s) using trait specific intervarietal RIL populations. Three framework linkage maps using three mapping populations are being prepared in our laboratory for QTL interval mapping of various agronomically important traits. These three mapping populations were originally prepared for the following three traits: (i) grain protein content (GPC), (ii) grain weight (GW), and (iii) preharvest sprouting tolerance (PHST).

Updating the framework linkage map of GPC population. We have prepared a framework linkage map for GPC population using 171 SSR markers (Prasad et al. 2003). The map spanned a genetic distance of 3,272.4 cM and had large gaps in certain regions, which adversely affected the precision of QTL mapping studies. In view of this, following two exercises were undertaken.

(a) The genotypic data used was on a set of 39 markers (including ISSR, SSR, and RAPD markers) recorded at NCL, Pune (India) under a network project, which allowed integration of 18 new markers to the available genetic map, giving a total of 189 markers on the map that is available with us at present.
(b) An additional set of 75 SSRs was used to study polymorphism between parents of GPC population (WL711 and PH132). Twenty-nine of the above 75 SSRs showed polymorphism. Genotyping of RILs with these 29 polymorphic markers is in progress, and the data generated will be used for filling the gaps in the available genetic map. Additional markers will be subsequently used to increase further the density of markers on the map.

Framework maps for GW population. A framework linkage map for four different chromosomes (1A, 2A, 2B, and 7A) was initially prepared using genotyping data for 453 molecular markers (34 SSR, 299 AFLP, and 120 SAMPL) on 100 RILs of GW population. Only 68 of the above 453 markers could be assigned to the above four chromosomes, and an average genetic distance of 13.37 cM to 19.74 cM between any two markers was observed (Kumar et al. 2006).

With a view of developing the whole-genome framework linkage map of GW population, an additional set of 259 SSRs was screened for polymorphism between the parents of GW (Rye Selection 111 and Chinese Spring) population. One hundred thirty (50.19 %) of the above 259 SSRs were found polymorphic. Genotyping of 100 RILs with a total of 53 of 130 polymorphic SSRs has already been completed and for the remaining 80 SSRs is in progress. As soon as the genotyping is complete, a framework map will be prepared and used for QTL interval mapping.

Framework maps for PHST population. A framework linkage map for a solitary chromosome (3A) was earlier prepared for PHST population, using genotyping data for 124 molecular markers (11 SSR, 76 AFLP and 37 SAMPL) on 100 RILs of the above population. Only 13 of the above 124 marker could be assigned to 3A, and an average genetic distance of 21.47 cM between any two markers was observed (Kulwal et al. 2005). A map of 3A was prepared for QTL interval mapping, since 3A was known to carry genes for PHST.

To develop the whole-genome framework linkage map of PHST population, additional 362 (149 gwm and 213 wmc) SSRs were screened for polymorphism between the parents of the PHST mapping population (HD2329 and SPR8198). One hundred thirty-four (37.02 %) SSRs of the above 362 SSRs were found to be polymorphic. Genotyping of 100 RILs with a total of 56 polymorphic SSRs has been completed and genotyping with the remaining 78 SSRs is underway. The genotypic data will be used for construction of a framework map for QTL interval mapping.

Single locus QTL analyses for growth and yield traits (using grain weight mapping population). QTL analysis for agronomically important traits in bread wheat was conducted following SMA, SIM, and CIM using an intervarietal RIL population derived from a cross between Rye Selection 111 (high GW) and CS (low GW). The parents and the above RILs were grown in six different environments, and the data on different agronomic traits were recorded in each case. For conducting single-marker regression analysis, genotypic data of 419 markers (299 AFLP and 120 SAMPL) were used with phenotypic data for different agronomic traits recorded in each of the six different environments, and also with the data pooled over different environments (total seven environments). QTL interval mapping also was conducted using framework linkage maps prepared for chromosomes 1A, 2A, 2B, and 7A (see above for map details).

Single-marker analysis (SMA) for growth and yield traits. Using the intervarietal RIL population for GW, single-marker analysis was conducted for four growth related traits (days to heading (DH), days to maturity (DM), early growth habit (EGH), and plant height (PH)) and seven yield and yield-contributing traits (tillers/plant (TPP), biological yield (BY), grain yield (GY), harvest index (HI), spike length (SL), spikelets/spike (SPS), and grains/spike (GPS)). For three growth traits, 28 markers showed significant association (4 with DM, 7 with DH, and 17 with PH). Similarly, for yield traits, 38 markers showed significant association with five of the seven yield and yield-contributing traits (2 with BY, 3 with SL, 10 each with GY and TPP, and 13 with GPS), in at least four of the above seven environments. The PV explained by the associated markers for individual growth related traits ranged from 3.86­16.81%, and for yield and yield-contributing traits, it ranged from 3.90­14.53 %.

QTL interval mapping for growth related traits. A total of 64 QTL were detected following SIM and CIM, which included 24 QTL for four growth related traits (DH, DM, EGH, and PH) and 40 QTL for seven yield contributing traits (TPP, BY, GY, HI, SL, SPS, and GPS). All these QTL were detected above a threshold LOD of 2.00-4.50 following both SIM and CIM. The PV explained by an individual QTL ranged from 7.42­32.16%.

Two-locus QTL analysis for yield and yield-contributing traits.
Population for grain protein content (GPC). Two-locus QTL analysis was conducted (QTLNetwork V2.0) for seven different yield and yield contributing traits (TPP, BY, GY, HI, SL, SPS, and GPS) using phenotypic data of 100 RILs (GPC population) recorded at six different environments. As many as 35 QTL (including, main effect QTL (M-QTL) and interacting QTL (QE, QQ, and QQE)) were detected for five (TPP, BY, GY, HI, and SL) of the above seven yield and yield-contributing traits, with minimum and maximum number of QTL detected for TPP/HI and SL, respectively. Out of the above 35 QTL, 10 M-QTL were distributed on nine different chromosomes (1A, 1D, 2B, 4A, 5A, 6A, 6B, 7A, and 7B); four M-QTL were involved in 'Q x E' interactions. The remaining 25 QTL were E-QTL, which were either involved in digenic interactions (Q x Q) or digenic environmental interactions (Q x Q x E).

International Triticeae Mapping Initiative population (ITMIpop). ITMI population was also used for two-locus QTL analysis (QTLNetwork V2.0) using data for the above seven yield and yield-contributing traits, recorded at four different environments. As many as 41 QTL (including M-QTL and interacting QTL (QE, QQ, and QQE)) were detected for all the seven yield and yield-contributing traits, with a minimum number of QTL (one) detected for BY/HI and maximum number of QTL (four) detected for GY. Out of the above 41 QTL, 16 QTL were M-QTL distributed on seven different chromosomes (1A, 2A, 2D, 4A, 4B, 5A, and 6D); eight M-QTL were involved in 'Q x E' interactions. Twenty-eight of the above 41 QTL (including both M-QTL and E-QTL) were either involved in digenic interactions (Q x Q) or digenic environmental interactions (Q x Q x E).

High resolution mapping of the genomic regions containing important QTL for GPC and PHST.
QTL for high GPC (QGpc.ccsu-2D.1) on 2DL. A population of about 2,000 F2 plants was derived from a cross between the two extreme RILs for high GPC versus low GPC. By analyzing the above population with markers flanking the QTL (QGpc.ccsu-2D.1) of interest 40 F2/F3 homozygous recombinants were identified.

In order to develop more markers to enrich the interval (11.4 cM) carrying QGpc.ccsu-2D.1, 64 AFLP primer combinations (8 Eco RI x 8 Mse I) were used, to identify chromosome arm (2DL)-specific AFLP markers (using nullisomic tetrasomic and ditelosomic lines of 2D), which could detect polymorphism among extreme RILs, representing parents of the above mapping population. Only seven AFLP markers specific to 2DL detected polymorphism among the parental genotypes and will be used for genotyping of the above 40 F2/F3 plants. An additional set of 960 AFLP primer combinations (unmapped) and 17 SSR primers (known to be mapped in the region of interest) are being used to identify an additional set of 2DL specific polymorphic markers. All these markers, identified as above, will be used for high resolution mapping of the region carrying the QTL of interest.

QTL for PHST (QPhs.ccsu-3A.1) on 3AL. A population of about 1,600 F2 plants was derived from a cross between SPR8198 (PHS tolerant) and HD2329 (PHS susceptible), with the objective of enriching the region carrying the QTL of interest (QPhs.ccsu-3A.1). The above population is being screened for the presence of recombinants using flanking markers of the region carrying QPhs.ccsu-3A.1. The recombinants identified as above will be genotyped using chromosome arm specific markers showing polymorphism between the genotypes representing parents of the above population. For this purpose, a set of 144 markers (19 SSRs and 125 STS) was developed using a chromosome arm (3AL)-specific genomic DNA library of bread wheat (see Gupta et al. 2005 for details). In addition to the above markers, a set of 1024 AFLP primer (16 Eco RI x 64 Mse I) combinations will be utilized to identify chromosome arm specific polymorphic markers.

Physical mapping of SSRs on 21 chromosomes of bread wheat. In bread wheat, a total of 485 SSRs including 325 gSSRs and 160 EST-SSRs were tried with 192 deletion lines, leading to successful mapping of 228 loci including 161 gSSR loci and 67 EST-SSR loci covering all the 21 chromosomes. A maximum number of 92 (40 %) loci were assigned to B genome followed by 68 (30 %) loci to A-genome and 68 (30 %) loci to D-genome. Except for eight discrepancies, a high degree of collinearity of gSSRs was observed between the physical and genetic maps. As many as 75 loci (including 32 gSSR and 43 EST-SSRs) were never used earlier for genetic mapping.

Marker-assisted selection for high GPC. In order to transfer a segment carrying a QTL for high GPC, 10 Indian cultivars were crossed with Yecora Rojo (seeds of Yecora Rojo was procured from J. Dubcovsky), a high grain protein content genotype. The F1 plants were backcrossed with their corresponding recurrent parents to obtain BC1 seeds during Rabi, 2004-05. The above BC1 seed were used to raise BC1F1 population in phytotron (IARI, New Delhi) during summer of 2005. Genomic DNA was extracted from the above BC1F1 plants to perform three-step selection (see below) using molecular markers, which will give plants carrying desirable allele for high GPC along with a genomic background similar to their respective recurrent parents. Different steps involved in the selection process are as follows:

Step-one selection. Selection of BC1F1 plants was exercised using molecular markers (SSR (gwm193) and an allele specific amplification (ASA) marker) flanking the QTL of interest. Eleven BC1F1 plants showed the presence of the desirable alleles at both the marker loci that flank the QTL of interest.
Step-two selection. Background selection on the above, 11 BC1F1 plants was exercised using 20 SSR markers specific to chromosome 6B, carrying the QTL of interest. All 11 BC1F1 plants showed the presence of recipient parent alleles at 5 to 14 marker loci.
Step-three selection. All of the above 11 BC1F1 plants were subjected to the whole-genome background selection using 80 SSR markers, scattered throughout the genome. This selection identified seven of the above 11 BC1F1 plants that better represent the genomes of their recurrent parents. These plants were backcrossed with their recurrent parents and the backcross hybrid seeds were used for raising BC2F1 population in 2005­06.

Development and use of EST-SNPs in bread wheat. Under the aegis of the international Wheat SNP Consortium (WSC), we undertook discovery, validation, and genotyping of SNPs in a set of 48 wheat EST contigs (each having 20-89 EST from 2 to 11 different genotypes), assigned to our group. Contigs were classified into subcontigs belonging to three individual subgenomes (A, B, and D) of the hexaploid wheat, to avoid confusion between homoeologous sequence variants (HSVs) among related genomes and SNPs. As many as 230 putative SNPs were detected in 155 such subcontigs (representing homoeoloci) belonging to 42 EST contigs. STS primers were designed for the above 42 EST-contigs and were used to amplify genomic DNA from 30 elite wheat genotypes. Only 10 of the above 42 primers were used for resequencing and allowed validation of seven SNPs (1 SNP/520 bp) out of 30 putative SNPs detected in silico in the amplifiable region (amplicon) of the above 10 primers in corresponding EST contigs. Allele-specific primers also were designed for the seven validated SNPs and were used for genotyping of 50 elite wheat genotypes (including 30 genotypes used for validation of SNPs) to study the occurrence of these SNPs. Of the above seven validated SNPs, four belonged to a solitary locus (PKS37), thus allowing discrimination of haplotypes at this locus. Altogether, the results suggested that EST-SNPs constitute an important source of molecular markers and could be developed and used in large number for studies on wheat genomics.

Identification of natural variants of the Agp-L gene through EcoTILLING. In order to identify variants of genes involved in starch biosynthesis and to develop functional markers (FMs) for early identification of desirable genotypes through MAS, we initiated an EcoTILLING program in bread wheat, involving large subunit of ADP-glucose pyrophosphorylase (Agp-L), a key enzyme in starch biosynthesis pathway. For this purpose, genome specific primers developed for B and D sub-genomes of bread wheat (Blake et al. 2004) are being used on a set of 52 elite wheat genotypes available with us.

Use of C0t fractionation (CF) and methyl filtration (MF) for genomics research in bread wheat. Gene-enrichment strategies of methyl-filtration and reassociation kinetics were used to generate and analyze 2,000 hypomethylated (MF), 1,026 high C0t (HC), and 1,253 reassociated DNA (RD) sequences of bread wheat, which together constituted 1.61 Mb of genomic sequences. Comparison of each of the above fractions with each of the other two fractions revealed large compositional/structural variations among the three groups of sequences. About 30 % of sequences from MF and 17 % of sequences from HC still represented repeat elements. SSRs also were detected in all the above three (MF, HC, and RD) fractions. A density of 1SSR/3.11 kb in MF was comparable to known SSR density in wheat genomic sequences. A density of 1SSR/2.27 kb in HC was comparable to that of SSRs in ESTs and a density of 1SSR/2.45 kb in RD was intermediate. In MF and RD sequences, the frequency of noncoding (nc) RNA genes was higher than that of protein coding genes but in HC sequences, the frequency of protein coding genes was relatively higher. Altogether, the frequencies of protein-coding genes relative to ncRNA genes varied from high to low in the following order HCÆRDÆMF, and those for ncRNA genes relative to protein coding genes varied in the following order RDÆMFÆHC. When matching was done with ESTs, large proportion of matching ESTs in the databases was found to represent transcripts encoded by transposable elements (TEs) and rRNA sequences; this was true with each of the three groups of sequences analyzed. Putative functions were also assigned to genes predicted from MF, HC and RD fractions. Genes encoding proteins conferring resistance against biotic stresses (mostly represented by gene families) were more frequent in MF sequences, and the genes encoding proteins involved in different metabolic pathways (perhaps housekeeping genes) were more frequent in HC sequences. However, the genes predicted in RD sequences largely matched proteins encoded by TEs. Matching with physically mapped wheat ESTs, allowed assignment of 58 MF sequences to 238 loci on 21 bread wheat chromosomes, of which 145 (60.9 %) loci reside in the distal halves of chromosomes. Also, while looking for synteny with rice sequences, synteny of 140 MF sequences was detected with rice genome sequences spread over all the 12 rice pseudochromosomes, with maximum number of loci on chromosome 3 and minimum number of loci on chromosome 12. A small proportion of MF and HC sequences also showed similarity with maize MF and HC sequences, suggesting that these sequences were conserved during evolution of cereals. Altogether, the above study suggests that the two gene-enriched fractions of wheat genome (MF, HC) differ significantly from each other and also differ from wheat ESTs, so that MF and HC sequences (along with wheat ESTs and RD sequences) should be used together to study the composition of wheat genome.

References.

• Blake NK, Sherman JD, Dvorák J, and Talbert LE. 2004. Genome-specific primer sets for starch biosynthesis genes in wheat. Theor Appl Genet 109:1295-1302.
• Gupta PK, Balyan HS, Bandopadhyay R, Kumar N, Sharma S, Kulwal PL, Rustgi S, Singh R, Goyal A, and Kumar A. 2005. Development and use of molecular markers for wheat genomics. Ann Wheat Newslet 51:51-55.
• Kulwal PL, Kumar N, Gaur A, Khurana P, Khurana JP, Tyagi AK, Balyan HS, and Gupta PK. 2005. Mapping of a major QTL for pre-harvest sprouting tolerance on chromosome 3A in bread wheat. Theor Appl Genet 111:1052-1059.
• Kumar N, Kulwal PL, Gaur A, Tyagi AK, Khurana JP, Khurana P, Balyan HS, and Gupta PK. 2006. QTL analysis for grain weight in bread wheat. Euphytica (submitted).
• Prasad M, Kumar N, Kulwal PL, Röder MS, Balyan HS, Dhaliwal HS, and Gupta PK. 2003. QTL analysis for grain protein content using SSR markers and validation studies using NILs in bread wheat. Theor Appl Genet 106:659­667.
  

Publications. [p. 46-47]

• Balyan HS, Gupta PK, Kulwal PL, and Kumar N. 2006. QTL analyses for three grain quality traits in bread wheat using intervarietal mapping populations. Czech J Genet Plant Breed 41:In press.
• Balyan HS, Gupta PK, Rustgi S, Bandopadhyay R, Goyal A, Singh R, Kumar A, Kumar N, and Sharma S. 2006. Development and use of SSRs of bread wheat for genetic and physical mapping and transferability to the species of Triticum-Aegilops complex. Czech J Genet Plant Breed 41:In press.
• Goyal A, Bandopadhyay R, Sourdille P, Endo TR, Balyan HS, and Gupta PK. 2005. Physical molecular maps of wheat chromosomes. Funct Integr Genomics 5:260-263.
• Gupta PK and Kulwal PL. 2006. Methods of QTL analysis in crop plants: Present status and future prospects. In: Professor CP Malik Commemoration Volume on the occasion of his 70th birthday (Trivedi PC Ed) (in press).
• Gupta PK, Kulwal PL, and Rustgi S. 2005. Wheat cytogenetics in the genomics era and its relevance to breeding. Cytogenet Genome Res 109:315-327.
• Gupta PK, Rustgi S, and Kulwal PL. 2005. Linkage disequilibrium and association studies in higher plants: Present status and future prospects. Plant Mol Biol 57:461-485.
• Gupta PK. 2005. The 2005 Nobel prize in physiology or medicine: Helicobacter pylori and its role in gastritis and peptic ulcer disease. Curr Sci 89:1651-1654.
• Kulshreshtha R, Kumar N, Balyan HS, Gupta PK, Khurana P, Tyagi AK, and Khurana JP. 2005. Structural characterization, expression analysis and evolution of the red/far-red sensing photoreceptor gene, PHYTOCHROME C (PHYC), localized on the 'B' genome of hexaploid wheat (Triticum aestivum). Planta 221:675-689.
• Kulwal PL, Kumar N, Kumar A, Gupta RK, Balyan HS, and Gupta PK. 2005. Gene networks in hexaploid wheat: Interacting quantitative trait loci for grain protein content. Funct Integr Genomics 5:254-259.
• Roy JK, Bandopadhyay R, Rustgi S, Balyan HS, and Gupta PK. 2006. Association analysis of agronomically important traits using SSR, SAMPL and AFLP markers in bread wheat. Curr Sci (in press).
• Singh R, Kumar N, Bandopadhyay R, Rustgi S, Sharma S, Balyan HS, and Gupta PK. 2006. Development and use of anchored-SSRs to study DNA polymorphism in bread wheat (Triticum aestivum L.). Mol Ecol Notes DOI:10.1111/j.1471-8286.2005.01202.x
• Sharma S, Balyan HS, and Gupta PK. 2006. Adaptive methylation pattern of ribosomal DNA in wild barley from Israel. Barley Genet Newslet (in press).
• Varshney RK, Balyan HS, and Langridge P. 2006. Wheat. In: The Genomes: A series on genome mapping, molecular breeding and genomics of economic species, Vol. 1, Cereals & Millets, Genome Mapping and Molecular Breeding I. Cereals (Kole C Ed), Springer, Berlin, Germany (in press).
  

 

CHAUDHARY CHARAN SINGH HARYANA AGRICULTURAL UNIVERSITY

Department of Plant Pathology, Hisar-125004, India.

 

The effect of sowing date on the interaction of loose smut and ear cockle in Indian wheat cultivars. [p. 47-48]

Rajender Singh, M.S. Beniwal, and S.S. Karwasra.

Summary. All cultivars except WH896 and Raj 1555 were susceptible to loose smut having 17.71 to 43.33 % disease incidence. With delayed sowing, all cultivars had reduced disease incidence. Conversely, ear cockle incidence was drastically increased in HD2285 and Raj1555 from 28.35 to 48.88 % and 3.83 to 10.35 %, respectively.

Introduction. The simultaneous occurrence of Ustilago segetum var. tritici with Anguina tritici in wheat has been reported by various workers (Bedi et al. 1959; Pruthi and Gupta 1986). During the croping season of wheat, this type of association between Anguina tritici and Ustilago segetum var tritici has been observed in plant pathology research area. Such type of concomitant association of the fungus and nematode has different level of incidence on different wheat varieties cultivated in India and which among two predominant in different cultivars. Therefore, an attempt has been made to study the effect of concomitant occurrence of loose smut and ear cockle on different varieties at different sowing date.

Materials and methods. A field experiment was conducted at Plant Pathology research area of CCSHAU-Hisar during 1997-2000 crop season. In each plot, 25 cultivars inoculated with loose smut spores in previous crop season were sown in 6 rows 2-m long. Each row received 10 nematode gall. Sowing dates of 25 November, 15 December, and 30 December were used for all treatments. Each cultivar has a subplot separated by 1 m distance by another one. Disease incidence was noticed on tiller basis in each cultivars. Tillers having both diseases were counted separately for both diseases.

Results and discussion. We noticed that two-thirds of the spikes constituting the lower part had almost been totally transformed into black sori of the loose smut fungus. The upper portion of this spike was infected by ear cockle nematode and black gall, which were clearly visible from distended glumes. All cultivars except WH896 and Raj 1555 were susceptible to loose smut with 17.71-43.33 % disease incidence. With a delay in sowing, all susceptible cultivars showed drastic reduction in disease expression (Table 1). In the highly susceptible cultivar Sonalika, disease incidence was decreased from 43.33 to23.66%. A reduction in chlamydospore germination/mycelial inactivation along with fall in temperature may have caused this. Conversely, ear cockle incidence was drastically enhanced in HD2285 (from 28.35 to 48.88 %) and Raj 1555 (3.83 to 10.35 %). Sonalika and HD2285 had the maximum amount of disease. A. tritici had more time for infection and prolonged germination time of seed along with fall in temperature. Pruthi and Gupta (1986) reported that the presence of a fungus with a nematode has an adverse effect on the number, motility, and development of larvae at the normal sowing time.

Table 1. The effect of three sowing dates on the interaction of loose smut and ear cockle of wheat cultivars.

Cultivar	Percent disease incidence
	25 November		15 December		30 December
	Loose smut	Ear cockle	Loose smut	Ear cockle	Loose smut	Ear cockle
C306	37.27	17.47	33.95	28.66	27.69	33.07
Sonalika	43.52	17.05	38.46	23.84	30.83	39.16
WH 147	39.75	18.64	31.43	25.88	28.44	31.11
WH 157	35.33	21.33	29.41	28.57	24.28	40.33
WH 283	38.69	17.39	32.44	24.66	23.71	29.42
WH 291	36.08	18.11	30.22	24.83	21.33	31.42
WH 416	35.00	15.00	28.14	20.37	20.77	26.66
WH 533	31.16	22.93	26.66	29.83	20.66	35.28
WH 542	32.33	20.96	28.14	27.33	21.33	33.33
WH 896	0.00	11.38	0.00	16.16	0.00	21.57
Sonak	31.66	22.77	24.33	30.16	18.83	38.83
HD 2009	38.75	20.91	29.33	27.71	23.37	34.57
HD 2285	36.48	24.83	26.83	30.83	19.77	41.33
HD 2329	30.00	18.33	22.22	24.77	16.66	30.96
HD 2687	36.66	18.16	29.83	23.83	23.66	28.71
PBW 175	31.42	18.77	26.83	26.42	21.66	31.88
PBW 343	39.53	14.83	29.83	22.37	21.77	29.83
PBW 373	33.33	18.66	26.71	24.16	20.16	30.57
PBW 435	37.66	20.00	29.33	26.66	23.66	32.22
Raj 1555	0.00	14.28	0.00	18.22	0.00	23.22
Raj 3077	36.66	16.66	28.83	24.77	22.22	30.33
Raj 3765	34.44	18.16	27.66	23.71	21.33	29.33
Raj 3777	32.33	20.33	25.16	26.66	19.66	32.66
UP 2338	33.83	19.14	26.57	25.66	20.44	31.33
UP 2425	36.66	18.44	28.33	24.33	22.22	29.66
C.D. (0.5 %)	2.54	1.95	1.92	2.32	2.62	2.16

References.

• Bedi KS, Chohan JS, and Chahal DS. 1959. Simultaneous occurrence of Ustilago tritici (Pers) Rest and Anguina tritici in a single ear of wheat. Ind Phytopath 12:187.
• Pruthi IJ and Gupta DC. 1986. Studies on combined occurrence of Ustilago tritici and Anguina tritici. Haryana Agric Univ J Res xvi (4):392-394.

DIRECTORATE OF WHEAT RESEARCH
Post Box 158, Agrasain Marg, Karnal 132001, India.

Participatory varietal selection for high yield and faster spread of wheat genotypes in Indo-Gangetic plains of India. [p. 48-50]

Ravish Chatrath, Gyanendra Singh, Randhir Singh, B.S. Tyagi, S.K. Singh, Jag Shoran, Divakar Rai, Sarvan Kumar, and Surendra Singh.

Introduction. Wheat is the leading grain crop in India and is cultivated under a wide range of climatic conditions. The most extensive production of wheat is in areas where the winters are cool and the summers are comparatively hot. The total area under wheat production in India is around 26 x 10^6^ ha with total production hovering around 70 x 10^6^ tons for the last decade. The bread wheat crop accounts for 84 % of the area; durum wheat occupies ~14 %. The productivity, duration, resistance, end product use, and suitability of bread wheat for the economically viable cropping systems of the farming community are some of the reasons that make durum wheat a second priority in the major wheat-growing zones of India. We now realize that yield gains in the Indo-Gangetic Plains area need to be further improved to meet the increased demand of wheat. Two feasible options are to improve yield potential of the genotypes and at the same time reduce the yield gaps between achieved and achievable. Researchers are making efforts to bridge yield gaps by advocating and demonstrating new technology, including cultivars, along with matching production technology in the farmers' field. The concept, methodology, results, and future thrusts for making use of a participatory varietal selection (PVS) approach in the Indo-Gangetic Plains of India are discussed below.

The concept of PVS for yield improvement and adoption. Participatory varietal selection involves the selection of a nonsegregating, characterized product from plant breeding programs by farmers. Such material includes released cultivars, those in advanced stages of testing, and advanced nonsegregating lines. In contrast, participatory plant breeding (PPB) involves selecting genotypes from genetically variable, segregating material by the farmers. The difference between PVS and PPB may not appear to be great at first sight. However, PPB requires more resources than PVS, and PVS identifies material that can be adopted more rapidly by the formal seed sector. The majority of farmers in India, particularly in the eastern, far eastern, and warmer regions, are still growing very old cultivars, local types, or land races even four decades after the beginning of the green revolution and, hence, fail to reap the benefits of modern technologies such as new, improved cultivars and resource conservation technologies. The wheat crop area, which is nearly half a million hectares in the far eastern state, is believed to have the potential of producing about 3.5 t/ha under optimum conditions, provided suitable genotypes resistant to leaf blight and with a 100-day maturity time with recommended agronomic practice are available.

PVS in wheat for Indo-Gangetic plains of India. To deal with such issues as dominance of a single cultivar in a region, slow rate of cultivar replacement, or low yield, a farmer participatory research project entitled 'Participatory Research to Increase the Productivity and Sustainability of Wheat Cropping systems in the Eastern sub Continents of South Asia' funded by DFID has been initiated at the Directorate of Wheat Research, Karnal, through the South Asia Regional Office of CIMMYT in Kathmandu, Nepal, with objectives of increasing wheat productivity and sustainability in this region. The DWR Karnal was assigned to work on material development for PVS at the Shillongani and Ranchi centers under this project. The following activities were conducted during 2003-04 by DWR, Karnal, and the salient features of the progress under the period are given.

Design of PVS. PVS attempts to cover a large number of farmers, especially the resource-poor. Farmers who are risk-averse or limited due to small land holding can, therefore, still afford large plots. Being poor, they also may not have the capacity to use purchased inputs. PVS targets the existing environment to identify suitable cultivars, which means that the field designs have to be simple and adaptable. The project document specifies three approaches for PVS for farmer-managed participatory research (FAMPAR).

Mother trials. Under the PVS program, researcher-designed and farmer-managed trials having a single replication of mainly farmer-selected cultivars conducted by few farmers representing the target area. In such cases, these selected farmers also serve as replicates for data analysis. These trials also aim to produce quantitative data involving the selected farmers at each site. To evaluate and multiply, a set also was grown at the Research Station of DWR, Karnal. Based on the information generated from these trials, surveys, and opinion polls, ranking for the cultivars and traits of choice were estimated. These data will represent the adaptive research area for fine tuning the breeding program at large.

Baby trials. This set of trials was designed by researchers involved in the program but are managed by the farmers at their sites. These are single-entry trials, in which newly selected and proposed entries are grown alongside the farmer's check cultivar under similar management conditions. The individual farmer may serve as a replicate for analyzing the data from each site. For each site or location, the number of baby trials conducted cover the whole range of prevailing conditions of farmers' field in the target area. At each location, data are collected as perceptions of farmers covering that particular locality, and the results obtained again represent the adaptive area of research for future planning.

PVS as an approach. The PVS approach was thought to be a promising strategy of increasing the rate of adoption for new cultivars among farmers in this area. Under this approach a wide range of recently released, high yielding, short duration, modern cultivars are tested in the farmers' field. The step-wise implementation of the following interlinked activities at various sites improves the productivity of wheat in this area to supplement the ongoing wheat improvement program.

1. Planting promising cultivars in the farmers' field to identify need-based cultivars through PVS.
2. Popularizing the farmer's selected cultivars in the targeted area through demonstration.
3. Educating farmers, extension agencies, and nongovernmental organizations through training, field days, farmers' meals, and roving seminars creates awareness for new technology.
4. Conducting base line and impact assessment surveys to get the feedback on farmer perception and fine tuning the technology to come.

Material and methods. Under this program, we organized 8­9 one-acre sized, PVS demonstrations of improved wheat cultivars to convince and educate farmers about new technological advancements that now are available. Selected experimental sites were Darer, Janeshro (Karnal), Mathana, and Kishangarh (Kurukshtra). Eight mother trials consisting of eight cultivars, PBW 343, HD 2687, WH 711, WH 542, UP 2425, PBW 373, RAJ 3765, and UP2338, were sown in farmers' fields for participatory selection. These cultivars also were sown under different tillage options, such as no-till, raised bed, and conventional tillage. To popularize the cultivars other than PBW 343 in this area, eight 'Farmer Days' were organized in Mathana, Kishangarh, Janeshro, and Darer; four were organized in March 2004, before maturity, and four at maturity, in April 2004.

Results and discussion. The results of PVS experiments at the eight sites in two districts in the state of Haryana were analyzed and are presented in Tables 1 and 2. In addition to recording yield data at each site, the selected farmers were also asked to express their preferences. The economic importance of various parameters was assessed on a three-point continuum, very important (3), somewhat important (2), and not important (1) by the farmers in the Farmers Group Discussion mode. The characteristics-wise economic importance score of all the 18 parameters are given in Table 1.

The parameters assessed were germination, number of effective tillers, days to maturity, plant height, lodging resistance, insect and disease resistance, ear head length, grains per spike, grain yield, straw yield, and grain type. The two traits namely germination and grains/ spike with score three were the first priority of all the farmers surveyed. Days-to-flowering and maturity did not receive much attention as evident by the score less then two. Some yield contributing traits like number of effective tillers, grain type, and 1,000-kernel weight were preferred by the farmers and matter while selecting wheat genotype for commercial cultivation at these sites. This type of ranking also is practiced by the researchers and, hence, are in the similar order. During the farmers' days, the participants were briefed about the objectives of the DFID project. More than 1000 farmers participated in this program. However, for the purpose of perception a total 100 farmers were randomly selected from the adapted sites. O n the basis of farmers' responses, the varieties were ranked. All the cultivars sown were evaluated by individual farmers in the field before and at maturity on a three point continuum, very good (3), good (2), and not good (1). The matrix ranking along with the evaluation score of all the genotypes used for study were estimated and are given in Table 2.
Matrix ranking of wheat cultivars on the basis of evaluation score and economic importance score of parameters. The cultivars were ranked on the basis of economic importance of the parameter and the evaluation score. Thereafter, the scores of all the parameters were summed for a cultivar to arrive at a composite score. On the basis of the composite score, PBW 343 ranked first, followed by HD 2687, WH 711, UP 2338, PBW 373, UP 2425, RAJ 3765, and WH 542.

Conclusion. The Participatory Varietal Selection programs in the high potential production system in India successfully identified new, nonrecommended cultivars that farmers preferred and adopted. The farmer participatory approach could be helpful in at least two ways. First, farmers would benefit by adopting new technologies and, second, a strong linkage would be established between researchers, farmers, and other sources of new technology such as the State Agriculture Department and seed certification agency, which, in turn, would increase production, productivity, and profitability of farmer and also help to meet our estimated demand of 109 x 106 t of wheat production by the year 2020.

Acknowledgment. The authors would like express their gratitude to authorities at ICAR, New Delhi; Project Director, DWR, Karnal; Dr. G.O. Ferrara, CIMMYT, SARO, Kathmandu, Nepal; and the DIFD Agency for technical and financial support provided for the present study. We also thankfully acknowledge the support from the coöperators in India.

References.

• Harris D, Raghuwanshi BS, Gangwars JS, Singh SC, Joshi KD, Rashid A, and Hollington PA. 2001. Participatory evaluation by farmers of on-farm seed priming in wheat in India, Nepal and Pakistan. Exp Agric 3: 403-415.
• Joshi A and Witcombe JR. 1996. Farmer participatory crop improvement. Participatory varietal selection, a case study in India. Exp Agric 32:461-477.
• Shoran J, Chatrath R, Singh G, Singh R, Tripathi SC, Sharma AK, Tyagi BS, and Singh SK. 2003. Participatory Research to increase the productivity and sustainability of wheat cropping system in the state of Haryana, India. Annual progress report for review and planning workshop, CIMMTY, SARO, Nepal. 10-14 June.
• Shoran J, Chatrath R, Singh G, Singh R, Tripathi SC, Sharma AK, Tyagi BS, and Singh SK. 2004. Participatory research to increase the productivity and sustainability of wheat cropping system in the state of Haryana, India. Annual progress report for 2nd review and planning workshop, CIMMTY, SARO, Nepal. 14-19 June.
• Singh G, Shoran J, Tyagi BS, Chatrath R, Tripathi SC, and Nagarajan S. 2004. Participatory varietal selection in wheat an approach to increase the adoption of new technologies. Directorate of Wheat Research, Karnal, India. Research Bulletin No. 17, pp. 28.
• Witcombe JR, Joshi A, Joshi KD, and Sthapit BR. 1996. Farmer participatory crop improvement. I. Varietal selection and breeding method and their impact on biodiversity. Exp Agric 32:445-460.
• Witcombe JR, Joshi KD, Rana RB, and Virk DS. 2001. Increasing genetic diversity by participatory varietal selection in high potential production systems in Nepal and India. Euphytica 122:575-588.
• Witcombe JR and Virk DS. 2001. Number of crosses and population size for participatory and classical plant breeding. Euphytica 122:451-462.

Effect of etherel on seed set, outcrossing, pollen sterility, and yield traits in wheat. [p. 51-56]

S.K. Singh and R.M. Singh, A.K. Joshi, and Ram Dhari (Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, India).

Abstract. We studied the effect of etherel at different concentrations on various floral and yield traits, the crop growth stage for chemical spray, and sowing time for their possible use in development of wheat hybrids. Two genotypes, HD 2285 and Raj 3765, were included in the study and data were recorded on reduction in seed number/spike as % of control, outcrossing %, pollen sterility %, plant height (cm), spike length (cm), and spikelet length (mm). Etherel at 12,000 ppm was observed effective for maximum reduction in grain number/spike with increased pollen sterility and outcrosing (%). The reduction in plant height, spike length and spikelet length was observed due to phytotoxic effects of chemicals. Late sowing and chemical spray at early boot stage were found more effective for etherel treatment.

Introduction. Wheat is a strictly self-pollinated crop (Percival 1921). Conventional breeding methods have contributed significantly in increasing wheat production. We need to explore new approaches to break the yield barriers and make wheat cultivation more remunerative. Among the possible alternatives, an important approach has been to exploit hybrid vigor at commercial level. Hybrids in wheat may cause a quantum jump in production at the global level, but the main bottleneck is the floral structure of wheat, which has very small florets, necessitating hand emasculation and pollination, labor oriented and tedious work, which ultimately results in higher cost for hybrid seed. The limitations of genetic and/or cytoplasmic male sterility, i.e., unstable nature, undesirable linkages, and need for use of maintainers, have prompted breeders to develop simple and more efficient methods to create male sterility by other means like use of chemical hybridizing agents (CHAs) and mutagens. Male sterility induced by CHAs (Moore 1950; Naylor 1950) is relatively more convenient to use because there is no need to maintain it (McRae 1985). These chemicals induce male sterility by inhibiting the early stages of sporogenous cell formation to the inhibition of anther dehiscence (McRae 1985). Law and Stoskopf (1973) and Hughes et al. (1974) suggested the use of etherel for creating male sterility in plants. The present investigation was aimed at the effect of different doses of etherel, the stage of crop for spray, and sowing time of genotypes on various floral and yield parameters.

Materials and methods. The wheat cultivars HD 2285 and Raj 3765 were used as experimental material. The field experiments were conducted in randomized block design with three replications during 1998-99 crop season at Agricultural Research Farm at Banaras Hindu University, Varanasi, India (25^o^18' N latitude and 82^o^03' longitude), which falls under semiarid to subhumid climate. The annual average rainfall is 1,081.4 mm, and mean relative humidity is 62 %. The maximum and minimum temperatures range between 23.2-46.4 C and 9.3-24.5 C, respectively. Wheat cultivars were sown on two dates to evaluate timely and late-sown conditions. Each plot had six 2.5-m rows with inter- and intrarow spacing of 25 cm and 10 cm, respectively. The inner two rows were treated as females and the outer four rows as males. A gap of 1 week was kept between the male and female rows to synchronize the pollen release and stigma receptivity; female rows were sown first. Etherel (6,000, 9,000, and 12,000 ppm) was sprayed at two crop growth stages. First spraying was done when spike length was 11­13 mm and was still inside the whorl. This stage of spike length was achieved at around 35 days after germination when the plant height was slightly less than one foot and the second at boot stage prior to spike emergence. Control plants were sprayed with water. Chemicals were sprayed in the evening to give sufficient time for their absorption by the wheat plant. At flowering, the male rows were shaken with the help of a rope pulled across the field. Control plants were also grown and sprayed with water. The data was recorded on reduction in grain number/spike as percent of control, out crossing (%), pollen sterility (%), plant height (cm), spike length (cm), and spikelet length (mm). The data were recorded as per the procedure followed by Mahajan et al. (1997). Analysis of variance was done using factorial randomized block design analysis. The computer program INDOSTAT was used for this purpose.

Results and discussion. The ANOVA (Table 3) revealed that variation in sowing dates, genotypes, chemical concentrations, and spray stages were highly significant for the traits of reduction in grain number/spike (%), plant height (cm), spike length (cm), and spikelet length (mm). Out crossing (%) and pollen sterility (%) had highly significant differences due to genotype and chemical concentrations, indicating that these two traits were unaffected due to change in sowing time as well as spray of chemical in different stages.

Effect on seed set, out crossing, and pollen sterility. The results (Table 4) indicated that the seed setting in the genotype HD 2285 was more adversely affected due to etherel treatment as it showed more than 93 % reduction in seed setting compared to control at higher concentrations. Maximum reduction (93.91 %) was observed at 12,000 ppm concentration in HD 2285 when etherel was sprayed at boot stage in late sown crop. A similar trend also was observed in the Raj 3765. The range of out crossing in the control (untreated) in both the genotypes was 1.35 to 1.63 %. An increase in the outcrossing percent was noticed when these genotypes were treated with etherel and was higher with increased concentration. More than 28 % outcrossing was observed at higher concentrations in both the genotypes. Significant differences were observed between various combinations of sowing time and spray stage within the genotype. Both the genotype showed about 7-8 % pollen sterility in the untreated population. Because etherel works as chemical hybridizing agent that causes male sterility in plant system, it induced more than 97 % pollen steril