ITEMS FROM INDIA

BHARATHIAR UNIVERSITY

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

Incorporation of specific rust resistance-genes into some Indian bread wheats.

V.R.K. Reddy and K.M. Gothandam.

Six leaf rust-resistance genes (Lr9, Lr19, Lr24, Lr28, Lr32, and Lr37); three stem rust-resistance genes (Sr24, Sr25, and Sr38); and one stripe rust-resistance gene (Yr17), present singly or in combination (in linked condition), were transferred from alien hexaploid wheat stocks into four Indian hexaploid wheat cultivars (HUW 318, PBW 226, HI 1077, and WH 542). Near-isogenic lines in each BC2F5 and BC5F5 were made from all the 24 cross combinations. The immune to moderately resistant reaction at the seedling stage and highly resistant reaction at the adult-plant stage provided by these genes strongly advocate the use of specific rust-resistance genes (or gene complex) for durable resistance. The constituted lines with specific genes for stem, leaf, and stripe rust (Sr24, Lr32, and Yr17) had a variable rust reaction pattern, so the resistance provided by these genes may be due to a combination or interactive effect with genes already present in the recurrent parent. Constituted lines with the rust-resistance genes Lr9, Lr28, and Lr32 also have reduced severity to a different rust, indicating that the donor parents with these resistance genes also carry unknown resistance genes for other rusts.

In general, the agronomic performance of plants of the BC2F5 was superior to that of the BC5F5 for plant height, tiller number/plant, spike length, number of spikelets/spike, 100-grain weight, and grain yield. The NILs constituted at the BC2F5/BC5F5 had reasonably very good agronomic characteristics, although seed quality characteristics (shrivelling, weight, size, and color) were very poor. Therefore, final selections were made based on seed quality coupled with good agronomic characters. Among the NILs in the BC5F5 population, lines with rust-resistance genes Lr19 + Sr25, Lr24 + Sr24 and Lr32 produced seeds of good quality, and lines with Lr28 and Sr38 + Lr37 + Yr17 produced seeds of inferior quality. A total of 17 lines finally were selected for commercial purposes, and the remaining seven lines were grouped as genetic stocks for use in further breeding programs. The constituted NILs gave significantly higher grain yield than the untreated control plants. Constituted NILs gave 7-38 % more grain yield than the untreated control. However, when compared to the chemical treated control, both increases and decreases in grain yield were noticed. The increase was in the range of 11 to 19.5 %, whereas the reduction was in the range of -0.68-7.45 %. The hexaploid wheat stock Cook*6/C80-1 (Lr19 + Sr25) followed by Sonalika *7/Abe (Lr9) and Darf *6/3Ag/Kite (Lr24 + Sr24) were determined to be the best donor parents.

Thirty four specific rust-resistance genes Lr9, Lr19, Lr24, Lr25, Lr26, Lr28, Lr29, Lr35, Lr36, Lr37, Lr38, Lr39, Lr40, Lr41, Lr42, Lr43, Lr44, Lr45, Sr24, Sr25, Sr26, Sr27, Sr28, Sr31, Sr38, Sr39, Yr9, Yr11, Yr12, Yr13, Yr14, Yr15, Yr16, and Yr17 were transferred to the four Indian hexaploid wheats HS 240, HD 2402, HD 2009, and HP 1102. Gene transfers were confirmed on the basis of morphological, genetical, biochemical, and molecular markers. Morphological markers for awnless spikes (Eagle-Sr26), lax spikes (RL 8061-Sr38+Lr37+Yr17), reduced yellow pigment in the flour (Cook*6/C 80-1-Lr19+Sr25), club spikes (W 3353-Sr27), waxiness (Veery 'S'-Sr31+Lr26+Yr9), and white glumes (TR 380-14 7/3 Ag#14-Sr24+Lr24) were used. Representative NILs were crossed with the universally susceptible wheat variety Agra Local. All F1 hybrid plants were rust resistant. The F2 segregation was 3 resistant:1susceptible; BC1 hybrids were in a 1 resistant:1 susceptible ratio. F2 plants derived from F1 monosomic and disomic hybrids between monosomic Chinese Spring and the representative rust-resistant NILs for Lr38 (HD 2009), Lr44 (HP 1102), Sr28 (HS 240) and Yr17 (HD 2402) segregated in a 3:1 ratio of resistant to susceptible for respective rust genes except for lines 6D, 1B, 2B, and 2A, confirming the successful incorporation of the rust-resistance genes into their respective chromosomes of the Indian wheats.

Inoculated, rust-resistant NILs had a higher peroxidase activity compared to the healthy controls. The polyphenol oxidase activity showed an increase in both susceptible wheat parents and rust-resistant NILs 24 hours after inoculation. The activity remained higher in the resistant plants during later stages of infection. Increases in catalase activity in both the susceptible wheat parent and the rust-resistant NILs 1 day after inoculation were noticed. The activity remained higher during later stages of infection in both susceptible and resistant plants. However, the overall activity was higher in susceptible wheat parents than in the resistant NILs. Lipoxygenase activity increased in both the susceptible wheat parents and their rust-resistant NILs 2 days after inoculation, but subsequently declined 7 days after inoculation with the respective rust pathogen in resistant plants. A consistent increase was noticed 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 increased time postinoculation. The percent decrease was greater in susceptible parents than the resistant NILs. The total soluble protein content in susceptible plants decreased as the time after inoculation increased, whereas in resistant NILs, the protein increased up to 48 hours postinoculation and subsequently showed a decrease in the later stages of infection. However, the percent decrease was more in susceptible than resistant plants 7 days after inoculation.

A significant decrease in the specific activities of RNase-I and combined RNase-II Nuc-I were observed in resistant NILs compared to the susceptible parents 10 and 15 days after inoculation with the rust spores. Resistant NILs had a relatively higher chlorophyll content than the susceptible wheat parents. Resistant NILs were able to retain a relatively steady level of chlorophyll content, whereas the chlorophyll content in the susceptible wheat parents decreased at a faster rate until reaching a level of less than 50 % of that in resistant NILs. The total free amino acid content increased until the 8th day after inoculation in both the susceptible parent and in the resistant NILs, after which there was a slight reduction occurred in both. The increase was more pronounced in the resistant lines than in the susceptible parents. During the second week after inoculation, resistant plants were able to maintain a level of total free amino acids significant increases in total free phenols and tannin content were observed in the constituted NILs compared to their respective recurrent wheat parents. The constituted NILs and had significantly higher nuclear DNA than their respective susceptible wheat parents.

The NILs for leaf rust-resistance gene Lr9 together with their parents were screened for polymorphism at the molecular level using the previously identified RAPD primer OPA-01 (Schachermayr et al. 1994), which has complete linkage to Lr9. The RAPD primer OPA-07 amplifies a 1,500-bp fragment and resulted in an additional band in the resistant NILs. Similarly, primer OPJ-13 distinguished resistant and susceptible lines and amplified a fragment of 1,100 bp as weak band and in NILs with the Lr49 gene in the wheat NILs. A polymorphism between the NILs for gene Lr24 was identified using the previously identified RAPD primer OPJ-09. This primer amplifies a fragment at 550 bp in all the NILs with Lr24.

Genes for hybrid necrosis and chlorosis have been identified in approximately 100 hexaploid and tetraploid wheats.

Publications.

• Arumugam S and Reddy VRK. 1999. Molecular confirmation of leaf rust resistance gene Lr9 in hexaploid wheat. J Cytol Genet (Accepted).
• Arumugam S and Reddy VRK. 1999. Confirmation of transfer of rust resistance gene complex Sr31+Lr26+Yr9 into Indian wheats through C-banding technique. J Cytol Genet (Accepted).
• Dhamayanthi KPM and Reddy VRK. 1999. Induced autotetraploidy by colchicine in Capsicum annuum L. var Byadagi Dabba and KAU-cluster. In: Proc Natl Symp Recent Trends in Plant Cytogenetics and Biotechnology, 21-22 Feb, 1999. Dept of Botany, Andhra University, Visakhapatnam, India. Abst. No. 16.
• Gothandam KM, Reddy VRK, Arumugam S, Subhashini A, and Murugesan GS. 1999. Hybrid necrosis and hybrid chlorosis in Indian hexaploid and tetraploid wheats. Crop Res (Accepted).
• Reddy VRK. 1999. Evaluation of aneuploidy and fertility in natural population of triticale. Ad Plant Sci 12(I): 93-95.
• Reddy VRK. 1999. Genetics of leaf rust resistant mutant in hexaploid wheat variety WH 147. In: Proc 86th Indian Sci Cong, 3-7 Jan, 1999. Anna University, Chennai, India (Agric Sci Sect). p. 1.
• Reddy VRK. 2000. Induced slow-rusting mutants in hexaploid wheat WH 147. J Indian Bot Soc (Proof).
• Reddy VRK and Arumugam S. 1999. Wheat cytogenetics - Transfer of alien rust resistance genes into Indian hexaploid wheat. In: Proc Natl Symp Recent Trends in Plant Cytogenetics and Biotechnology, 21-22 Feb, 1999. Dept of Botany, Andhra University, Visakhapatnam, India. Abst. No. 1.
• Reddy VRK and Arumugam S. 1999. Role of genome combination on stability in wheat and triticale. In: Proc State Level Sem 'Modern Trends in Plant Science' (UGC, New Delhi and TNSCST, Chennai sponsored), 4-6 March, 1999, Department of Botany, AVC College, Mannampandal, Mayiladuthurai, Tamil Nadu, India. pp. 63-64.
• Reddy VRK and Arumugam S. 1999. Transfer of specific rust resistance genes to hexaploid wheat. In: Proc State Level Sem 'Modern Trends in Plant Science' (UGC, New Delhi and TNSCST, Chennai sponsored), 4-6 March, 1999, Department of Botany, AVC College, Mannampandal, Mayiladuthurai, Tamil Nadu, India. p. 107.
• Reddy VRK, Arumugam S, Subhashini A, Gothandam KM, and Murugesan GS. 1999. Cytogenetics of triticale. III. Meiotic stability and agronomic characters. J Cytol Genet 34(1):15-20.
• Reddy VRK, Arumugam S, Subhashini A, Gothandam KM, and Murugesan GS. 1999. The effect of nuclear DNA and heterochromatin on meiotic stability in hexaploid triticale. J Nat Conserv 11(1):41-45.
• Reddy VRK, Arumugam S, Subhashini A, and Viswanathan PV. 1999. Transfer and characterization of rust resistance genes in Indian hexaploid wheat cultivars. Ann Wheat Newslet 45:68-69.
• Reddy VRK and Binumol M. 2000. Radiosensitivity in wheat and triticale - effect of ploidy level. Adv Plant Sci 13(II):accepted.
• Reddy VRK and Dhamayanthi KPM. 1999. Mutation studies in Capsicum annuum L. In: Proc State Level Sem 'Modern Trends in Plant Science' (UGC, New Delhi and TNSCST, Chennai sponsored), 4-6 March, 1999, Department of Botany, AVC College, Mannampandal, Mayiladuthurai, Tamil Nadu, India. p. 67.
• Reddy VRK and Gajalakshmi K. 1999. Genetics of some induced mutants in wheat and triticale. J Cytol Genet (Accepted).
• Reddy VRK and Gothandam KM. 1999. Effect of fungicide Tilt on meiosis and fertility characters in bread wheat cv. HW 741. Ad Plant Sci 12(I): 259-262.
• Reddy VRK and Manju S. 2000. Hybrid weakness in Indian hexaploid and tetraploid wheats. Adv Plant Sci 13(I):accepted.
• Reddy VRK and Rajeswari S. 1999. Lead induced cytotoxicity in Allium cepa L. Adv Plant Sci 12(II):411-416.
• Reddy VRK and Subhashini A. 1999. Confirmation of transfer of rust resistance genes into hexaploid wheat. In: Proc State Level Sem 'Modern Trends in Plant Science' (UGC, New Delhi and TNSCST, Chennai sponsored), 4-6 March, 1999, Department of Botany, AVC College, Mannampandal, Mayiladuthurai, Tamil Nadu, India. pp. 61-63.
• Reddy VRK and Subhashini S. 1999. Wheat cytogenetics. II. Confirmation of incorporation of rust resistance genes into India wheats. In: Proc Natl Symp Recent Trends in Plant Cytogenetics and Biotechnology, 21-22 Feb, 1999. Dept of Botany, Andhra University, Visakhapatnam, India. Abst. No. 2.
• Reddy VRK and Sundaravadivelu K. 1999. Improvement of cotton (Gossypium hirsutum L.) - Induced mutagenesis. In: Proc Natl Symp Recent Trends in Plant Cytogenetics and Biotechnology, 21-22 Feb, 1999. Dept of Botany, Andhra University, Visakhapatnam, India. Abst. No. 22.
• Reddy VRK and Vishnu Priya S. 1999. Hybrid necrosis and chlorosis genes in some hexaploid and tetraploid wheats. J Cytol Genet (Accepted).
• Reddy VRK and Viswanathan PV. 1999. Genetics of leaf rust resistant mutant WH 147-LM-1 in hexaploid wheat variety WH 147. Acta Agron Hung 47(1): 63-64.
• Reddy VRK and Viswanathan PV. 1999. Induced rust resistant mutants in hexaploid wheat WH 147. Crop Res (Accepted).
• Reddy VRK, Viswanathan PV, Arumugam S, Asir R, and Dhamodaran S. 1999. Induction of morphological mutants in triticale. Adv Bio-Sci 18(1):67-72.
• Reddy VRK and Viswanathan PV. 1999. Effect of transferred rust resistance genes on yield performance in hexaploid wheat. Acta Agron Hung 47(1): 65-68.
• Reddy VRK and Zereena V. 1999. Rye chromosome composition and kernel characters in hexaploid triticales. Crop Res 17(1):75-79.
• Viswanathan PV and Reddy VRK. 1998. Induced desirable agronomical mutants in wheat and triticale. J Ind Bot Soc 77:67-69.
• Viswanathan PV and Reddy VRK. 1999. Effect of ploidy level on chlorophyll mutations in wheat and triticale. J Ind Bot Soc (Proof).
• Viswanathan PV and Reddy VRK. 1999. Genetics of grain mutants in hexaploid triticale TL 419. Crop Res (Accepted).
  

MARATHWADA AGRICULTURAL UNIVERSITY

Wheat and Maize Research Unit, Parbhani 431 402 Maharashtra, India.

K.A. Nayeem.

Current activities.

The Wheat and Maize Research Unit at MAU-Parbhani received a sanction for a project entitled 'Use of Nuclear Techniques in Genetic Enhancement of Wheat Quality' for 1999-2001. The main subject is the improvement of quality traits of T. durum by nuclear techniques. High quality HMW and LMW glutenins and gliadins present in the D genome of the wheat PBNS-l666 are to be transferred to the A or B genomes of T. durum. The improved Parbhani durum PBND-527 will be used for breeding and the progeny subsequently screened by molecular and cytogenetic techniques.

News.

A Kisan Melawa on the cultivation of T. durum wheats was organized on 19 February, 2000. Vice-chancellor Dr. C.D. Mayee, president of the program, stressed the need to grow T. durum for export purposes. Currently, the best durums are available through the All-India Wheat Project Program. Dr. N.D. Raut, joint director of Agriculture Aurangabad stated that the area under rain-fed wheat should be increased because of suitable climatic conditions. Dr. K.A. Nayeem gave details on the use of T. durum for industrial purposes, and noted that small industries are needed for the preparation of end products.

Publications.

• Nayeem KA, Devkule SN, and Bhagwat SG. l999. Seed protein variation in radiation induced mutant of wheat. Ind J Genet 59(3):363-369.
• Nayeem KA and Veer MV. 1999. Gene effects for thermo tolerance parameters and some yield components in bread wheat (T. aestivum L.) under normal and late sowing. Crop Improv 25(2):2229-231.
• Veer MV and Nayeem KA. 1997. Heterosis of heat tolerance and agronomic characters in bread wheat. J Maharashtra Agric Univ 22(1):112-115.

Correlation and path analysis in morphological characteristics above the flag-leaf node at flowering stage in Triticum durum.

P.J. Kulkarni and K.A. Nayeem.

Evaluation of genetic variability in the available crop species is the first step toward selecting better performing genotypes among divergent groups in plant breeding programs. Wheat is one of the most important cereal grains in the world. The analysis of various morphological characters in addition to chlorophyll content at flowering can help to identify desirable genotypes before maturity.

Significant differences among the genotypes were observed for all the 15 characteristics, suggesting the presence of sufficient variability among the genotypes for these traits (Table 1).

PBND-850 was observed to be the earliest flowering (62.50 days), PBND-4279 had the longest ear (9.99 cm), PBND-4277 had the shortest peduncle (25.5 cm), and DWR-1006 had the highest number of spiklets/spike. For photosynthetic capacity, PBND-4250 (1.53) was the top ranking line, and PBND-785 had the highest spike density (3.12). The highest total chlorophyll content was observed in PBND-4260 (3.13 mg/g). We can utilize the above genotypes as potential donors in an appropriate breeding program for improving durum wheat. Table 2 lists the desirable genotypes for various selected characteristics.

In the present study, spikelet density had the highest coefficient of variation (5.28 %) followed by photosynthetic capacity (4.75 %) and extruded peduncle length (4.46 %). The lowest coefficients of variation were observed for total chlorophyll content (1.54 %) followed by days-to-50 % flowering (1.58 %).

Genotypic and phenotypic coefficients of variation were lowest for days-to-50 % flowering, as also was observed in studies by Takawale et al. (l989) and Getchew et al. (1993). The highest genotypic and phenotypic coefficients of variation were observed for extruded peduncle length. In general, estimates of the phenotypic coefficient of variation were higher than the corresponding estimates of genotypic coefficient of variation (Yadav and Mishra 1993).

Very high, broad sense estimates of heritability were observed for all characteristics studied except spikelet density. High heritability percents were observed for total chlorophyll content (98.90 %), extruded peduncle length (97.90 %), and awn length (97.30 %). High heritability along with sufficient genetic advance estimates are useful as selection criteria. Among all the characteristics studied, the estimated highest genetic advances were for extruded peduncle length (10.93 %) and peduncle length (9.37 %).

Correlation studies. The genotypic, phenotypic, and environmental correlation coefficients among the 15 characteristics were calculated. A negative and significant correlation was observed for photosynthetic capacity with flag leaf area at all levels. The number of spikelet/spike had a significant positive correlation at the environment level with photosynthetic capacity. Total chlorophyll content had a significant positive correlation with chlorophyll a content, chlorophyll b content, and carotenoid content at both the genotypic and phenotypic levels.

Path coefficient analysis. A path coefficient analysis was worked out taking the number of spikelets/spike as a dependent variable and the remaining 14 characters as independent variables. The greatest direct effects on the number of spikelets/spike was for flag-leaf length (1.798), spikelet density (1.323), and spike length (1.206).

Among the estimates of indirect effects, no positive indirect effect was observed for the number of spikelet/spike. For selection criteria, emphasis should be placed on the characteristics of flag-leaf length, spikelet density, and spike length.

References.

• Dewey DA and Lu KH. 1959. A correlation and path analysis of crested wheat grass seed production. Agron J 51:151-158.
• Getchew B, Testaye T, and Deminssie M. 1993. Variability and correlation studies in durum wheat. Rachis 12(1/2):30-41.
• Hiscoxx JD. and Israel-Slam GF. 1979. A method for extraction of chlorophyll from leaf tissues without maceration. Can J Bot 57:1332-1334.
• Singh PK and Chaudhary BJ. 1977. Biometrical methods in quantitative genetic analysis. Kalyani Publishers, New Delhi, India. 223 pp.
• Takawale PS, Thete RY, and Dumbre AD. 1989. Analysis of genetic variability and association of grain yield and other economic character in wheat. J Maharashtra Agric Univ 14(3):366-367.
• Yadav RK and Mishra RK. 1993. Genetic analysis of wheat varieties for yield and its conponants under rainfed conditions. Agric Sci Digest (Karnal) 13(1):6-8.

CH. CHARAN SINGH UNIVERSITY

Department of Agricultural Botany, Meerut (U.P.), India.

Molecular markers for gene tagging and genetic diversity studies at Meerut, a center for the Wheat Biotechnology Network in India.

P.K. Gupta, H.S. Balyan, M. Prasad, R.K. Varshney, and J.K. Roy.

The Department of Biotechnology and the Government of India, under the Wheat Biotechnology Network Programme, had sanctioned a project entitled 'Characterizing and using the Quality Traits in Wheat Aided by Molecular Markers.' The other network partners include Punjab Agricultural University (PAU), Ludhiana; G.B. Pant University of Agriculture & Technology, Pantnagar; Directorate of Wheat Research, Karnal; National Chemical Laboratory, Pune; and Agharkar Research Institute, Pune. The major objective of the project at our center is the development of molecular markers for three grain quality traits including grain protein content (GPC), grain size (GS), and preharvest-sprouting tolerance (PHST).

Development of molecular markers. Both hybridization-based and PCR-based markers have been tried. These included (i) oligonucleotide in-gel hybridization, (ii) RAPD, (iii) DNA-amplification fingerprinting (DAF), (iv) STS, (v) microsatellite-primed PCR (MP-PCR), (vi) sequence-tagged microsatellite sites (STMSs), (vii) AFLP, and (viii) selective amplification of microsatellite polymorphic loci (SAMPL). However, the most useful approaches for detection of relatively high level of polymorphism included STS, STMS, AFLP, and SAMPL. For enrichment of the number of STMS molecular markers, we also entered into an international collaboration, which led to the availability of 500 locus-specific STMS primer pairs. This international effort is under the auspices of the Wheat Microsatellite Consortium, of which we are the only member from India. Subsequently, we identified five molecular markers including two each for GPC and PHST and one marker for GW. Of these five markers, four are STMS (STR or microsatellite) markers and one is an STS marker. Using nullisomic-tetrasomic and ditelocentric stocks, the loci for these markers also were assigned to specific chromosome arms (Table 1). Recombinant-inbred lines for all these three traits were developed by Drs. H. S. Dhaliwal and Harjit-Singh at PAU, Ludhiana.

Table 1. Summary of successful tagging of different quality traits already achieved using microsatellite/STS markers in bread wheat (*Superscript containing primers are STS primers and rest are the STMS primers).

Trait	Primers tried	Primers scored	Polymorphic primers	Linked markers	Chromosome arm	QTLs identified	Contribution (%)	Reference
Grain protein content (GPC)	232	167	57	Xwmc41	2DL	QGpc1.ccsu-2D	18.73	Prasad et al. 1999
	114	95	30	Xwmc415	5A	QGpc1.ccsu-5A	6.21	Singh et al. 2000
Preharvest sprouting tolerance (PHST)	346	262	87	Xwmc104	6BS	major gene		Roy et al. 1999
	138*	133*	30*	Xmst101*	7DL
Grain weight (GW)	346	267	63	Xwmc333	1AS	QGw1.ccsu-1A	15.09	Varshney et al. 2000

Marker validation of two identified STMS markers for GPC using NILs. In order to validate the two STMS markers linked with GPC, three sets of 10 NILs for high GPC were derived using two genotypes with low GPC (WL711 and HD2329) as recipient parents and another two genotypes with high GPC (PH132 and PH133) as donor parents (these NILs were developed by Dr. Harjit-Singh at PAU, Ludhiana). One NIL with high GPC (12 %) derived from HD2329 when analyzed with WMC41 gave a characteristic amplification profile similar to that of its donor parent PH132. Another NIL 2215 with high GPC (12 %) derived from WL711 when tried with WMC415 also gave an amplification profile resembling that of its donor parent PH133. The remaining eight NILs with high GPC gave patterns similar to those of their corresponding recipient parents with both the markers, suggesting that QTLs other than those associated with the above markers actually were transferred from the donor parents and contributed to high GPC in these NILs. Thus, marker validation using NILs with high GPC, although demonstrating the utility of these two microsatellite markers for their use in marker-assisted selection in plant breeding, also suggested that many more QTLs could be present and need to be identified using molecular markers (Harjit-Singh et al. 2000).

Preparation of integrated physical maps of 2DL, 6BS, and 7DL with loci for grain-protein content and preharvest-sprouting tolerance in bread wheat. With the use of nullisomic-tetrasomic and ditelocentric lines, the marker Xwmc41 (linked with GPC) was located on 2DL, and two other markers, Xwmc104 and Xmst101 (linked with PHST), were localized on 6BS and 7DL, respectively. With the use of overlapping deletion lines for the long arm of chromosome 2D, the marker locus Xwmc41 was assigned to the terminal 0.24 fraction of 2DL. Also, from the two molecular markers associated with PHST, WMC104 (an STMS marker) was assigned to the terminal 0.76 fraction of the 6BS satellite and MST101 (an STS marker) was assigned to the proximal centromeric 0.10 fraction of 7DL. Thirteen STMS markers, previously mapped genetically on 6BS and 7DL, also were assigned to specific physical regions of these two arms. The markers that were physically mapped during our study were integrated with those previously mapped in a similar manner, and integrated into physical maps with 27 markers on 2DL, 16 on 6BS, and 54 on 7DL (Varshney et al. 2000c).

Genetic mapping of Xwmc41 using ITMI mapping population. The molecular marker WMC41 (linked with a QTL for GPC) was mapped genetically using the ITMI population in collaboration with Dr. M. Röder, IPK, Gatersleben Germany. The marker locus Xwmc41-2D was found between the RFLP markers Xfb122 and Xfb068, which matches with the physical map.

Genetic diversity studies using STMS, AFLPs, and SAMPLs. A set of 55 elite wheat genotypes (originating in 29 countries and representing six continents) was used to estimate genetic diversity among wheat genotypes using three types of markers: STMS, AFLPs, and SAMPLs. Twenty wheat STMS (microsatellite) markers were used to examine their utility, first for detecting DNA polymorphism, second for identifying genotypes, and third for estimating genetic diversity among wheat genotypes. A total of 155 alleles was detected at 21 loci using the above microsatellite primer pairs (only one primer amplified two loci; all other primers amplified one locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (six A-genome, five B-genome, and two D-genome chromosomes). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. A dendrogram, prepared on the basis of a similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, which was unique and differed from all other genotypes belonging to cluster I and placed in subcluster Ia. With a set of only 12 primer pairs, a maximum of 48 of the 55 wheat genotypes could be distinguished. Further additions of primers to this set did not prove useful for distinguishing the remaining genotypes. The results demonstrated the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity (Prasad et al. 2000).

An AFLP analysis also was conducted using eight primer-pair combinations with the 55 wheat genotypes. Each of these combinations gave a large numbers of AFLP fragments from the 55 accessions. A total of 287 polymorphic bands (46 %) out of 612 bands were identified (mean = 76 bands per primer pair), confirming the high multiplex ratio of AFLPs. The highest percentage of polymorphism (74 %) was obtained with primer combination EACG x MCAG, whereas the lowest value (22 %) was obtained with primer combination EAAC x MCAT (Table 2). The genetic similarity coefficients for all the possible 1,485 genotypes pairs ranged from 0.44 between genotypes E2401 and E661 to 0.99 between genotypes E3901 and E4328 and averaged to 0.71. The UPGMA cluster analysis was used to prepare a dendrogram, and the genotypes were grouped into two major clusters.

The SAMPL technique, which combines the features of microsatellites and AFLPs, also was used to study genetic diversity among the 55 wheat genotypes. We successfully used two SAMPL primer combinations. Several polymorphic fragments were obtained with each combination. The results of SAMPL are quite encouraging. Out of a total of 87 bands, 43 polymorphic bands (49 %) were identified. The average number of scorable bands per primer-pair combination was 44. The maximum number of bands was obtained with primer SAMPL-7. The highest percentage of polymorphism (52 %) was obtained with primer SAMPL-6 (Table 2). Genetic-similarity coefficients for pairs of genotypes ranged from 0.35 between genotypes E288 and E3389 to 0.96 between genotypes E4229 and E4813 and between E3839 and E3901. The average genetic similarity coefficient was 0.65. Clusters were constructed using Jaccard's similarity coefficients, and the genotypes were grouped further into two major clusters.

The SAMPL and AFLP were also compared. SAMPL gave 52 % polymorphism, whereas AFLP gave 46 %. The maximum genetic similarity coefficients were 0.99 in AFLP and 0.96 in SAMPL, whereas the minimum genetic similarity coefficients were 0.44 in AFLP and 0.35 in SAMPL. Comparing both SAMPL and AFLP markers, SAMPL was considered to be more informative marker system.

AFLP/SAMPL for gene tagging. Keeping in view the potential of AFLP/SAMPL, we also have initiated work involving the use of AFLP/SAMPL approach for gene tagging. At present, we are studying polymorphisms among different parents, PH132 (high GPC) and WL711 (low GPC), Rye Selection 111 (high GW), Chinese Spring (low GW), SPR8198 (tolerant to PHS), and HD2329 (susceptible to PHS). A number of polymorphic bands already have been identified using both AFLPs and SAMPL. Bulk segregant analysis and selective genotyping will be made using these polymorphic marker(s) to study association of the marker(s) with quality traits.

Retrotransposon-based techniques (REMAP and IRAP) for gene tagging. Another novel, microsatellite-based approach makes use of one retrotransposon primer with one SSR primer and is described as REtrotransposon Microsatellite Amplified Polymorphism (REMAP). We successfully used by REMAP to study DNA polymorphisms among parents PH132 (high GPC) and WL711 (low GPC), rye selection III (high GW) and Chinese Spring (low GW), and SPR8198 (tolerant to PHS) and HD2329 (susceptible to PHS). Primers based on a single retrotransposon also can be used to amplify the inter-retrotransposon spacer region. This technique, described as Inter-Retrotransposon Amplified Polymorphism (IRAP), also is being tried by us for gene tagging.

Genetic diversity and genomic relationship in tribe Triticeae using in-gel hybridization and STMS markers. Four SSR probes in combination with four restriction enzymes were used for in-gel hybridization in a set of 25 wild accessions representing 15 species to study genetic diversity and its relationship with genomic constitution. Similarly, 15 STMS primer pairs also were used in a PCR study of genetic diversity in 19 accessions representing 14 wild species having different genomic constitutions. Based on the above studies, dendrograms were prepared, and the results are being analyzed.

Publications.

• Towards development of molecular markers for tagging genes for quality traits in bread wheat. In: Proc 9th Internat Wheat Genet Symp (Slinkard AE ed). University Extension Press, University Saskatchewan, Saskatoon, Canada. 3:84-88.
• Gupta PK and Varshney RK. 1998. Molecular markers for genetic fidelity during micropropagation? Curr Sci 76:1308-1310.
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SKUAST REGIONAL AGRICULTURAL RESEARCH STATION

Sher-E-Kashmir, University of Agricultural Sciences & Technology, R.S. Pura-181 102, Jammu, India.

Standard heterosis for grain yield and yield component in wheat.

J.S. Bijral, S.K. Mondal, and Kuldip Singh.

Although the yield advantage of rice hybrids is being assessed on a commercial scale, the anticipated yield increase from wheat hybrids has not been realized. Several workers (Briggle 1963, Johnson and Schmidt 1968, Zeven 1972) have reviewed the literature on heterosis in wheat, and only in a few instance have wheat hybrids been shown to be significantly superior to the best conventionally bred wheat varieties.

In order to obtain realistic estimates of standard heterosis, a set of 212 crosses was produced during 1997-98. Of the 212 F1 hybrids, 141 were 'winter/spring' wheat crosses. The remaining 71 were spring wheat combinations. The hand-produced hybrids, along with the best inbred wheat variety PBW-343, were grown in randomized complete block design with two replications during the 1998-99 season. Each entry was planted in single, 3.5-m row with spacings of 25 cm between the rows and 10 cm between plants. Observations on grain yield/spike (in grams), number of fertile tillers/plant, number of grains/spike, and 1,000-kernel weight (in grams) were recorded on five random competitive plants from each row. Heterosis (standard) was estimated over the check PBW-343.

Of the 212 hybrids, 68 exhibited more than 15 % standard heterosis for grain yield/plant, and their heterotic values for the other yield components were estimated. None of the top-ranking hybrids were heterotic for all the traits simultaneously (Table 1). Standard heterosis for grain yield/plant in the 212 crosses ranged from -59.0-101.5 % with an average of 4.9 %. The cross WW-21/Raj3077 exhibited the highest performance for grain yield/plant (29.8 g) with a corresponding, highly significant positive standard heterosis for grain yield/plant (101.5 %) and number of fertile tillers/plant (96.1 %). The cross WW-21/RSP 303 was the second best cross with standard heterosis values of 86.7, 92.3, and 52.1 %, for grain yield/plant, number of fertile tillers/plant, and number of grain/spike, respectively. Interestingly, the third best cross, WW-21/PBW-373, also involved WW-21 as the female parent and exhibited significant positive standard heterosis values for grain yield/plant (77.9 %), number of fertile tillers/plant (27.6 %), number of grains/spike (22.6 %), and 1,000-kernel weight (5.8 %). The lowest, negative, standard heterosis of -59.0 % for grain yield/plant was recorded in the cross PBW-175/JOB-2028.

Based on a comprehensive 4-year study comprising 36 wheat hybrids, Livers and Heyne (1968) reported that the F1 hybrids collectively exceeded the parental lines with an average hybrid superiority of 32.0 %. Wilson and Driscoll (1983) in their review of literature on heterosis in wheat observed that the yield advantage of wheat hybrids over the best check varieties over different locations and years ranged from 4.1-31.0 %.

In summary, the results of this study showed that varieties WW-21, Raj-3077, RSP-303, PBW-373, Blue Boy, PBW-343, RSP-81, WW-19, UP-2425, and PBW-452 were the most promising parents, exhibiting high standard heterosis for grain yield/plant and yield components in some specific cross combinations. The results suggest that these varieties could be utilized effectively in a wheat improvement program with heterosis breeding.

Acknowledgments. Our thanks to G.S. Sethi, HPKVV, Palampur (HP) and S.K. Nayar, IARI, Regional Station, Tuti Kundi, Simla for supplying seed of the winter wheats.

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