ITEMS FROM INDIA

BHABHA ATOMIC RESEARCH CENTRE
Nuclear Agriculture and Biotechnology Division, Mumbai - 400085, India.

Improving the bread-making quality of Indian wheat varieties by incorporating HMW-glutenin subunits 5+10. [p. 62]

S.G. Bhagwat *, B.K. Das *, V.S. Rao **, and S. Marathe **.
* Nuclear Agriculture & Biotechnology Division and ** Food Technology Division.

Three, hexaploid wheat selections selected for the presence of glutenin subunits 5+10 and moderate rust resistance were grown at Trombay. The three selections, along with their parent lines Kalyansona and Sonalika, were assessed for their dough characteristics using a Brabender Farinograph. The selections showed improved dough strength as judged by the farinograph and were tested for bread-making quality. A modified bread-making test using 25-g whole meal and a straight-dough method were used. Two selections in the Kalyansona background had an 11 % increase and one in Sonalika had 25 % increase in specific loaf volume. Sensory evaluation by a taste panel showed a higher overall acceptance for the selections compared to that of the respective parents. The selections in Kalyansona background were higher yielding in the field at Trombay.

Publication.

• Bhagwat SG, Das BK, Rao VS, Marathe SA, and Jawali N. 2000. Use of molecular techniques in wheat quality improvement. In: Proc DAE-BRNS Symp, Use of Nuclear and Molecular Techniques in Crop Improvement. 6-8 Dec, 2000. BARC, Mumbai, India. pp. 289-296.

Screening of Indian wheat cultivars for the Glu-D1d allele (HMW-glutenin subunits 5+10) by PCR. [p. 62-63]

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

Bread-making quality is largely associated with the HMW-glutenin subunits (Payne et al. 1987). These proteins are coded by the Glu-A1, Glu-B1, and Glu-D1 loci. Of these three complex loci, the allelic variations at the Glu-D1 locus are particularly important in good or poor bread-making quality. The allele Glu-D1a, coding for subunits 2+12, has a Glu-1 score of 2 (poor), whereas Glu-D1d coding for subunits 5+10 has a Glu-1 score of 4 (good). These alleles are usually analyzed by SDS-PAGE. Discrepancies in relative mobility of some subunits make their identification ambiguous (D'Ovidio et al. 1994). DNA-based markers are useful for screening for the presence of subunits. The same DNA can be used to detect many genes.

D'Ovidio and Anderson (1994) have reported on two primers flanking the sequence of Glu-D1dx coding for subunit 5, which specifically amplifies a 450-bp fragment. Verghese et al. (1996) also reported on this primer during the screening of European winter wheat varieties. We screened 29 Indian wheat cultivars (plus Chinese Spring) and verified the authenticity with SDS-PAGE data. The PCR signal exactly matched the SDS-PAGE data.

A list of cultivars analyzed for SDS-PAGE and their HMW-glutenin subunit pattern is in Table 1. The HMW-glutenin subunits of the cultivars were resolved on 10 % SDS-PAGE according to Bhagwat and Bhatia (1993). The DNA was extracted from five seeds according to Krishna and Jawali (1997). The PCR reaction was in a 25 µl reaction volume containing 50 ng of genomic DNA, 125 ng of each primer, 2.5 µl of 10X buffer, 100 nM dNTP solution, and 0.75 µ of Taq-DNA Polymerase (Bangalore Genei Pvt. Ltd, India). The reaction conditions were 95 C (4 min), 94 C (1 min), 66 C (45 sec), and 73 C (30 min) for 3 cycles; 94 C (1 min), 63 C (45 sec), and 73 C (30 sec) for 30 cycles; and a final extension at 75 C for 5 min. The PCR products were resolved on 2.5 % agarose gels. The PCR signal (presence of the 450-bp band for lines with subunits 5+10 and absence for lines with 2+12) were scored (Table 1). These results exactly matched the SDS-PAGE data. Though specific amplification of the 450-bp band was desired, some nonspecific amplification was observed. We intend to use this marker in our program for wheat quality improvement.

References.

• Bhagwat SG and Bhatia CR. 1993. Selection for quality in early generation based on the HMW subunits of glutenin in bread wheat. Ind J Genet 53:208-214.
• D' Ovidio R, Porceddu E, and Lafiandra D. 1994. PCR analysis of genes encoding allelic variations of high molecular weight glutenin subunits at the Glu-D1 locus. Theor Appl Genet 88:175-180.
• D'Ovodio R and Anderson OD. 1994. PCR analysis to distinguish between alleles of a member of multigene family correlated with bread making quality. Theor Appl Genet 88:759-763.
• Krishna TG and Jawali N. 1997. DNA isolation from single or half seeds suitable for random amplified polymorphic DNA analysis. Anal Biochem 250:125-127.
• Payne PI and Lawrence GJ. 1983. Catalogue of alleles for the complex gene loci, Glu-A1, Glu-B1 and Glu-D1 which code for high molecular weigh subunits of glutenin in hexaploid wheat. Cereal Res Commun 11:29-35.
• Payne PI, Nightingale MA, Krattiger AF, and Holt LM. 1987. The relationship between glutenin subunit composition and the bread making quality of British-grown wheat varieties. J Sci Food Agric 40:51-65.
• Verghese JP, Struss D, and Kazman ME. 1996. Rapid screening of selected European winter wheat varieties and segregating populations for the Glu-D1d allele using PCR. Plant Breed 115:451-454.
  
  

Grain shape analysis in Indian wheat cultivars. [p. 63]

S.P. Shouche *, R. Rastogi *, S.G. Bhagwat **, and J.K. Sainis ***.
* Computer Division, ** Nuclear Agriculture & Biotechnology Division, and *** Molecular Biology & Agriculture Division.

Image analysis can be used to measure shape and size related parameters of wheat grains. At the Bhabha Atomic Research Centre, a software package has been developed which was used for study of grain shape and size variation in fifteen Indian wheat varieties. Grain images were captured using HP Scan Jet II CX/T scanner in transparency mode. Area, perimeter, compactness, major and minor axis length, slenderness, and spread were computed. Shape analysis was done using standard, central, normalized central, and invariant moments. Using geometric parameters and moments analysis, it was possible to distinguish the 15 cultivars. The study indicates that it may be possible to use image analysis for varietal identification in wheat.

Transfer of sphaerococcum character in agronomically suitable background. [p. 64]

S.G. Bhagwat.

Triticum sphaerococcum has characteristics such as short stature, erect leaves, and round grains that may be advantageous in a wheat cultivar. A T. sphaerococcum accession from our collection was crossed to cultivar Kalyansona. The later was used as a recurrent parent and selection was for T. sphaerococcum plant type and grain shape. After four backcrosses and subsequent selfings, a morphologically uniform-looking population was obtained. The recurrent parent and the selection were grown in the field at Trombay. The spacing between lines was 1 ft and each line was 5-ft long. The plot had three replications and culm length and spikelet number were determined on 15 plants from each replication. Culm length of the main tiller, spikelet number/main tiller, 100-kernel weight, and harvest index based on air-dry weight of above ground plant parts were estimated (Table 2). The selection showed 92 % biological yield and 89 % grain yield compared to the recurrent parent. The harvest index was marginally lower. Spikelet number/spike was comparable (Table 2). A reduction in the culm length and more rounded grain were characteristic of the selection. The results indicate that the sphaerococcum locus is able to reduce the culm length effectively in the presence of the semidwarfing gene Rht-1. No reduction in the spikelet number was observed, although the spikes were more condensed in the selection.

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

Effectiveness of rust-resistant wheat NILs with various rust-resistance genes. [p. 64-67]

V.R.K. Reddy, S. Arumugam, and K.M. Gothandam.

A total of 27 rust resistance genes (Lr, Sr, and Yr), present singly or in combination with other gene(s) and obtained from 33 wheat donor parents, were transferred into 14 Indian hexaploid wheat cultivars through a backcross breeding program. The genes were incorporated into the Indian wheats and selected in either BC5F5 or BC6F5. The list of recurrent and donor parents are given in Table 1.

Indian hexaploid bread wheat cultivars with specific rust-resistance genes for three different types of rusts were evaluated for leaf, stem, and stripe rust resistance. Studies evaluated the effectiveness of a specific rust-resistance gene in different wheat genetic backgrounds and obtained from different donor parents. The original wheat cultivars and their rust-resistant NILs were grown in the field in two rows of 1.5 m. All the lines were artificially inoculated with a mixture of rust races of individual (leaf, stem, and stripe) rusts. Four leaf rust-resistance genes (Lr9, Lr19, Lr32, and Lr37), four stem rust-resistance genes (Sr26, Sr27, Sr36, and Sr38), and eight stripe rust resistance genes (Yr9, Yr11, Yr12, Yr13, Yr14, Yr15, Yr16, and Yr17) were found highly effective in all 14 Indian wheat genetic backgrounds (Table 1, Table 2, and Table 3). Leaf rust-resistance gene Lr25, stem rust-resistance gene Sr28, and stripe rust-resistance gene Yr8 were less effective in the Indian wheats under study. Other effective rust-resistance genes include Lr26, Lr28, Lr29, and Lr satu were effective in 11 Indian wheats and Sr24 and Sr31 were effective in 13 and 11 Indian wheats, respectively. The source of donor parent also was important for effectiveness of a particular rust-resistance gene. For example, Lr9, Lr19, Sr26, Sr27, and Yr9, which are obtained from more than one donor parent, were equally effective in all the fourteen Indian wheat genetic backgrounds. On the other hand, Lr24 was effective in all 14 Indian wheats only when it is obtained from the donor parent 'Darf/3Ag/Kite'. Similarly, Sr25 was effective in all the Indian wheat genetic backgrounds only when it was obtained from the donor parent 'Cook*6/C 80-1'. The original sources of Lr26, Sr24, Sr25, and Sr31 are not completely resistant to rust. The resistance provided by these genes in several Indian wheats may be either cumulative or due to the interaction with rust-resistance genes already present in the recurrent Indian wheats.

Publications. [p. 67-68]

• Reddy VRK. 2000. Induced mutations in wheat and triticale - mutagenic parameters. J Cytol Genet 1(NS)(2):In press.
• Reddy VRK. 2000. Differential rust reaction in Indian hexaploid wheats. J Cytol Genet. 1(NS)(2):In press.
• Reddy VRK. 2000. Incorporation of leaf and stem rust resistance genes into Indian wheat cultivars. In: Proc Natl Conf Conservation of Biodiversity, DAV College, Muzaffarnagar. (In press).
• Reddy VRK. 2001. Polygenic variability for various quantitative characters in some cereal crops. Adv Pl Sci 14(II):In press.
• Reddy VRK. 2001. Heterochromatic connectives in triticales. Adv Pl Sci 14(II):In press.
• Reddy VRK. 2001. Mutation breeding in hexaploid triticales - desirable mutants. Adv Pl Sci 14(I):In press.
• Reddy VRK and Binumol M. 2000. Radiosensitivity in wheat and triticale - effect of ploidy level. Adv Pl Sci 13(I):107-118.
• Reddy VRK and Dhamayanthi KPM. 2001. Induction of haploidy in Capsicum annuum L. In: Proc Diamond Jubilee Symp "Hundred Years of Post-Mendelian Genetics and Plant Breeding - Retrospects and Prospects", 20-23 Feb, 2001. IARI., New Delhi (abstract).
• Reddy VRK, Gothandam KM, and Kalaiselvi G. 2001. Transfer of specific genes for rust resistance into Indian hexaploid wheat cultivars. In: Proc Diamond Jubilee Symp "Hundred Years of Post-Mendelian Genetics and Plant Breeding - Retrospects and Prospects", 20-23 Feb, 2001. IARI., New Delhi (abstract).
• Reddy VRK and Manju S. 2000. Hybrid weakness in Indian hexaploid and tetraploid wheats. Adv Plant Sci 13(I):51-54.
• Reddy VRK and Viswanathan PV. 2001. Effect of R/D substitutions and/or modified rye chromosomes on kernel characters in triticales. Ind J Genet 61(1):In press.
• Viswanathan P and Reddy VRK. 2000. Effect of ploidy level on chlorophyll mutations in wheat and triticale. J Ind Bot Soc 79(1):In press.
  

CH. CHARAN SINGH UNIVERSITY
Department of Agricultural Botany, Meerut - 250 004, India.

 

P.K. Gupta, H.S. Balyan, M. Prasad (Present address IPK, Corrensstrasse 3, D-06466, Gatersleben, Germany), J.K. Roy, N. Kumar, S. Sharma, and P.L. Kulwal.

Marker assisted selection for some quality traits in bread wheat. [p. 68]

This project, funded by the National Agriculture Technology Project (NATP) of Indian Council of Agricultural Research in April 2000, is a continuation of earlier work done between 1995-00 at the Wheat Biotechnology Network Programme of the Department of Biotechnology, Government of India. The major objective of this new project is to develop molecular markers for MAS for some quality traits in bread wheat. These activities will include (i) developing framework maps using SSR, SAMPL, and AFLP markers with three sets of RILs for three traits (grain protein content (GPC), preharvest sprouting tolerance (PHST), and grain weight (GW)); (ii) QTL interval mapping for identifying molecular markers or chromosome segments associated with each trait and for determining the precise position and effect of individual QTLs or chromosome segments; (iii) determining stability of identified QTLs through a study of RILs over environments; (iv) validating studies involving DNA markers identified to be associated with QTLs; (v) testing the efficacy of MAS for grain-quality improvement; and (vi) training and developing linkages with breeders for the effective use of MAS by practicing wheat breeders.

Another independent study on developing molecular markers was initiated during 2000­01 and involved analyses of 14 different morphological traits and 519 polymorphic molecular markers (including SSR, APLP, and SAMPL markers). The data for these traits and markers were recorded in 55 exotic accessions of bread wheat, and the regression of each of the morphological traits on each molecular marker was examined. This study was initiated to estimate genetic diversity using molecular markers and was extended for developing molecular markers using association analysis.

Under a CSIR-Emeritus Scientist Project sanctioned to PKG, we also studied ribosomal DNA (rDNA) polymorphism in wild barley and bread wheat through a study of intraspecific length variation in the intergenic spacer (IGS) and the sequence variation in the ITS (Internal Transcribed Spacer) regions. This study demonstrated a relationship between rDNA polymorphism with ecogeographical distribution of wild barley in Israel and Jordan.

QTL interval mapping for grain quality and other traits in bread wheat. [p. 68-70]

Genotyping of RILs for QTL interval mapping. We currently are studying QTL interval mapping for grain-quality traits including GPC, PHST, and GW and other economic and physiomorphological traits in bread wheat using SSR, SAMPL, and AFLP markers. Three RIL populations (100 RILs in each set) derived from crosses between parents differing for the above traits are being used (Table 1). These RIL populations also are being evaluated in multilocation trials. The data already has been recorded on early growth habit (prostrate vs. erect), days-to-heading and days-to-maturity. Data on the grain quality traits will be recorded at maturity/harvest (in April/May, 2001). Of the ~400 wmc SSR primer pairs that were available, a total of 140 primer pairs were polymorphic between the parents of the three RIL populations (Table 1). Genotyping of the RILs of the GPC population with 47 primers, RILs of the PHST population with 24 primers, and RILs of the GW population with 15 primers has been completed and more SSR primers are being used for genotyping. As a collaborative activity, genotyping of the above three sets of RILs using additional STMS primer pairs also is being done by Manoj Prasad (currently visiting Germany on an Alexander von Humboldt Fellowship) at IPK, Gatersleben in the laboratory of Marion Röder. The segregation data for SSRs, collected both at Meerut and Gatersleben, will be used to prepare separate framework linkage maps for each of the three different RIL populations. Using the linkage maps and the data from the field trials, QTL interval mapping will be done. We also will analyze for epistasis and 'genotype x environment' interaction.

BSA using SAMPL and AFLP. Using the SAMPL approach, a total of 1,185 bands were amplified in the six parent genotypes of the three RIL populations. Five hundred and sixty-eight of these bands were found to be polymorphic. A BSA was done using these polymorphic SAMPL markers. Associations of six bands with GPC, seven bands with PHST, and four bands with GW were detected (Table 2). SAMPL markers also are being used to genotype the RILs, framework maps also will be prepared from this data, and QTL interval mapping will be done as with SSRs. This approach also will be used with AFLPs.

Table 2. Details of the SAMPL primers, number of bands amplified, and putative linked markers with three grain quality traits. The figures in parentheses are the percentage of polymorphic bands. Putative markers are designated on the basis of the nomenclature for AFLPs proposed at 9th International Wheat Genetics Symposium. X is symbol for a DNA marker, ccu stands for Ch. Charan Singh University; E indicates use of the rare-cutter enzyme EcoRI; S6 is the SAMPL primer and the three digits at the end represent the molecular weight in base pairs.

SAMPL Primers		Grain protein content	Putative markers	Preharvest sprouting tolerance	Putative markers	Grain weight	Putative markers
SAMPL-6 x M-CAG		74 (43.24)	XccuES6400	74 (37.83)	XccuES6430	60 (46.67)	---
			XccuES6390		XccuES6370		XccuES6300
SAMPL-6 x M-CTT		112 (51.78)	XccuES6350	83 (49.39)	XccuES6410	80 (52.50)	XccuES6355
					XccuES6345		XccuES6340
					XccuES6280		XccuES6330
SAMPL-6 x M-CTC		129 (43.41)	XccuES6365	92 (39.13)	---	80 (51.25)	---
			XccuES6325
SAMPL-6 x M-CAT		94 (51.06)	---	87 (43.67)	XccuES6150	73 (54.79)	XccuES6320
			XccuES6110
SAMPL-6 x M-CAA		57 (49.12)	---	40 (62.50)	---	50 (54.00)	---
Total		466 (47.63)	6	376 (44.68)	7	343 (51.89)	4

Analyses of association of molecular markers with different traits in bread wheat. Association analyses involving 14 morphological traits and a large number of molecular markers (AFLP, SAMPL, and STMS) were made using 55 exotic wheat genotypes originating in 29 countries on six continents. We wanted to identify molecular markers showing association with each of the 14 different traits. The associated markers will be subsequently mapped to determine the location of QTL for different traits and will be used for MAS in wheat breeding.

Association analysis using SAMPL markers. Two SAMPL primers, each in combination with one AFLP primer, amplified a total of 87 bands in 55 genotypes. Forty-three of these SAMPL bands were polymorphic. Data for each of the 14 different individual traits scored on 55 genotypes was separately regressed on these 43 SAMPL markers using backward multiple regression approach. Significant regression of each of the 13 of the 14 traits (excluding 1,000-kernel weight) on a variable number of markers (a total of 38 markers) out of the 43 different polymorphic markers was detected. Of these 13 traits, harvest index showed significant regression on only one marker, whereas grain yield has a significant regression on maximum number (18) of markers. The remaining 11 traits showed significant regression on four to 16 different SAMPL markers. These associated markers explained 5 % (harvest index) to 66 % (spikelets/spike) of the variation in the different traits studied.

Association analysis using AFLP markers. Eight AFLP primer pairs amplified a total of 615 bands in 55 genotypes studied. Two hundred and fifty-five of these AFLP bands were polymorphic. Data for each of the 14 different individual traits scored on 55 genotypes were separately regressed on these 255 AFLP markers using backward multiple regression approach. Significant regression of each of the 14 traits on variable number of polymorphic markers was detected. Grains/spike showed regression on 45 AFLP markers, whereas plant height, days-to-maturity, days-to-flowering, tiller number, spike length, biological yield, grain yield, and harvest index each showed significant regression on 53 different AFLP markers. The remaining traits showed regression on sets of 46-51 different markers. These associated markers together explained 98-100 % variation in each of the different traits examined.

Association analysis using STMS markers. Twenty STMS primers amplified a total of 221 polymorphic bands in 55 genotypes. Data of 14 different individual traits scored on 55 genotypes was separately regressed on these 221 markers using backward multiple regression approach. Significant regression of all the 14 traits on a variable number of different STMS markers was detected. Harvest index showed significant regression on 45 STMS markers, whereas peduncle length showed significant regression on 52 STMS markers. The remaining 12 traits showed significant regression on sets of 47-51 different STMS markers. The associated markers were able to explain 97­100 % variation in each of the different traits studied.

Ribosomal DNA polymorphism in collections of exotic bread wheat and wild barley, Hordeum spontaneum (length variation in IGS and sequence variation in ITS region). Ribosomal DNA polymorphism in wheat and wild barley was studied through the study of variation in the length of intergenic spacer (IGS) regions of 50 exotic wheat genotypes and 327 wild barley genotypes. The bread wheats were the same as those used for the association studies. Wild barley was collected from 27 different populations in Jordan and four microniches in Israel. Sequence variation in the internal transcribed spacer (ITS) regions of rDNA also was studied in six genotypes of wheat and in 10 genotypes of wild barley.

IGS length variation in wheat. In order to study the variation in IGS length in wheat, genomic DNA was digested with the SacI enzyme and after DNA hybridization the digested DNA was probed with rDNA probe pTA71. In 50 wheat genotypes, 2-7 bands were detected. One of these bands , ~3,880 bp, was common to all the genotypes and presumably represents major part of transcribed region of rDNA repeats as was earlier shown in barley. The remaining 1-6 bands of variable size perhaps represented the IGS region of the rDNA repeat units. This variation in size of the above bands is attributed to the variation in the number of subrepeats in the IGS region.

Of the 50 genotypes studied, the maximum number of genotypes (34 %) showed one band, 30 % had two bands, 20 % had three bands, 8 % had four bands, 6 % had five bands, and the remaining (2 %) genotype (E3086) had six bands. Currently, we are making efforts to assign these six fragments following aneuploid analysis to the eight different rDNA loci reported in hexaploid wheat.

Length and sequence variation in the ITS region of wild barley. The length and sequence variation of ITS region of rDNA repeat units of 10 wild barley genotypes was examined. These accessions were from different locations in Jordan. The ITS region, including the 5.8S rRNA gene, was PCR amplified using ITS-4 and ITS-5 primers, and the amplified products were cloned and sequenced using an automated sequencer. Both insertions/deletions (indels) and substitutions (SNPs) were detected in both the ITS-1 and ITS-2 regions. The length of ITS-1 ranged from 214-217 bp and that of ITS-2 ranged from 215-217 bp. Sequence divergence, based on nucleotide substitutions, ranged from 0.16-1.70 %. Five potentially informative sites were found.

Publications. [p. 71-72]

• Gupta PK. 1999. Intracellular protein transport and signal sequences: The 1999 Nobel Prize in Medicine. Proc Natl Acad Sci India, Sec B. 69 (Part III & IV):187-203.
• Gupta PK. 1999. Plant genomics: A new discipline in Botany. J Ind Bot Soc 78:181-189.
• Gupta PK. 2000. Use of molecular markers in wheat breeding. In: Natl Symp Biotechnology for Sustainability in agriculture. G B Pant Uni of Agri & Tech, Pantnagar, India, 27-29 April, 2000. pp. 124-125.
• Gupta PK. 2000. Signal transduction in the nervous system: The 2000 Noble Prize for physiology or medicine. Curr Sci 79:1145-1152.
• Gupta PK. 2000. Transgenic crops, molecular marker technology and genomics for crop improvement. In: Proc of the DAE-BRNS Symp 'The Use of Nuclear and Molecular Techniques in Crop Improvement'. BARC, Mumbai, India. 6-8 December, 2000. pp. 9-20.
• Gupta PK and Varshney RK. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113:163-185.
• Gupta PK. 2001. Molecular maps, isochors and DNA sequences. In: Plant Biology in the Era of Genomics and Proteomics Workshop, Bose Institute, Kolkata, India, 23-25 March, 2001.
• Gupta PK. 2001. A memoir of Professor Ram Parikshan Roy. In: Bibliographical Memoirs of INSA Fellows. Indian National Science Academy, New Delhi, India. 22:in press.
• Gupta PK. 2001. Applied cytogenetics: classical and molecular. In: Natl Symp 'Meeting the Future Needs of Higher Education in Genetics and Related Disciplines: Courses and Curriculum'. Indian Soc Genet & Pl Breed, IARI, New Delhi. 21-23 April, 1998. (in press).
• Gupta PK. 2001. Chromosome and genome research in plants: some recent developments. The Nucleus (invited paper, in press).
• Gupta PK, Dhaliwal HS, Balyan HS, Harjit-Singh, Prasad M, Varshney RK, and Roy JK. 2000. Molecular markers for some quality traits for marker assisted selection in bread wheat. In: Proc 6th Internat Cong of Plant Mol Biol, Quebec, Canada, 18-24 June, 2000.
• Gupta PK, Roy JK, and Prasad M. 2001. Single nucleotide polymorphisms (SNPs): a new paradigm in molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Sci 80:524-535.
• Gupta PK, Varshney RK, and Roy JK. 2001. Plant genomics for crop improvement. In: Perspectives in Cytology and Genetics (Manna GK and Roy SC eds). 10:23-40.
• Gupta PK, Varshney RK, and Prasad M. 2001. Molecular markers: Principles and methodology. In: Molecular Techniques in Crop Improvement (Jain, SM, Ahloowalia, BS, and Brar DS eds). Kluwer Academic Publishers, Dordrecht, Netherlands. (invited paper, in press).
• Kumar N, Kulwal PL, Roy JK, Balyan HS, Dhaliwal HS, and Gupta PK. 2001. Towards QTL interval mapping for some economic traits in bread wheat using molecular markers. In: Proc Natl Symp Perspectives in Genome Analysis, CCMB, Hyderabad, India, 23-24 February, 2001. p. 45.
• Roy JK, Prasad M, Balyan HS, and Gupta PK. 2001. Use of SAMPL for gene tagging and genetic diversity in bread wheat. In: Proc Natl Symp Perspectives in Genome Analysis, CCMB, Hyderabad, India, 23-24 February, 2001. pp. 44.
• Singh H, Prasad M, Varshney RK, Roy JK, Balyan HS, Dhaliwal HS, and Gupta PK. 2001. STMS markers for grain protein content and their validation using near-isogenic lines in bread wheat. Plant Breed (In press).
• Sharma S, Roy JK, Balyan HS, and Gupta PK. 2001. Study of intraspecific sequence variation in the internal transcribed spacer (ITS) region of ribosomal DNA in barley and wheat genotypes. In: Proc Natl Symp Perspectives in Genome Analysis, CCMB, Hyderabad, India, 23-24 February, 2001. p. 43.
• Varshney RK, Kumar A, Balyan HS, Roy JK, Prasad M, and Gupta PK. 2000. Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat. Plant Mol Biol Rep 18:5-16.
• Varshney RK, Prasad M, Roy JK, Balyan HS, and Gupta PK. 2000. Development and use of molecular markers for gene tagging and diversity studies in bread wheat. In: Proc 3rd Internat Crop Sci Cong, Hamburg, Germany, 17-22 August, 2000.
• Varshney RK, Prasad M, Roy JK, Kumar N, Harjit-Singh, Dhaliwal HS, Balyan HS, and Gupta PK. 2000. Identification of eight chromosomes and a microsatellite marker on 1AS associated with QTL for grain weight in bread wheat. Theor Appl Genet 100:1290-1294.
• Varshney RK, Prasad M, Roy JK, Röder MS, Balyan HS, and Gupta PK. 2001. Integrated physical maps of 2DL, 6BS and 7DL, carrying loci for grain protein content and pre-harvest sprouting tolerance in bread wheat. Cereal Res Commun (In press).
  

INDIAN AGRICULTURAL RESEARCH INSTITUTE REGIONAL STATION
Wellington - 643 231, the Nilgiris, Tamilnadu, India

 

Developing a dwarf dicoccum wheat with resistance to stripe rust and powdery mildew. [p. 72]

M. Sivasamy, R.N. Brahea, M.K. Menon, Aloka Saikia, and R. Asir.

Next to bread and durum wheats, dicoccum or Khapli wheat is widely cultivated in southern India. The taller cultivar NP 200 was the most popular Khapli wheat until the dwarf dicoccum variety DDK 1001 was released for commercial cultivation in 1996. All the presently grown cultivars, DDK1001, DDK 1009, and NP200, are highly susceptible to yellow rust at Wellington, Nilgiris Hills, which is a hot spot for wheat foliar diseases. Thus, we are making efforts to develop a dwarf dicoccum with resistance to yellow rust and powdery miIdew.

The Mexican dwarf dicoccum (MD), which has a high level of resistance to the prevalent yellow rust races (P and I) at Wellington, was chosen as a parent for crosses. However, this line is highly susceptible to powdery mildew and has very poor tillering. Lines from the crosses 'MACS 2912/NP 200//MD', 'MACS 2931/NP200//MD', 'MACS 2938/JHW 2000//MD', and 'DDK l00l/HW 2000//MD' had very good resistance to yellow rust and powdery mildew. Resistance to yellow rust in MD is controlled by a recessive gene. Thus, all the F1 plants in all crosses were susceptible to yellow rust. Resistant lines were selected in the F2 and advanced to the F5 generation. The resistance to powdery mildew was most likely derived from the lines MACS 2912, MACS 2931, MACS 2938, and DDK 1001, which are resistant to powdery mildew. All of these cultivars have a dwarfing gene from T. durum. The constituted resistant lines have profuse tillering coupled with improved grain quality. These lines will be evaluated soon in the All-India coordinated yield trials.

Incidence of wheat diseases in the Nilgiri Hills, southern India. [p. 72-73]

R.N. Brahma, M. Sivasamy, and A. Saikia.

Bread wheat and dicoccum wheat are the important cereal crops of the Nilgiri Hills. These crops are cultivated both during the winter and summer seasons. Nearly all the economically important diseases of wheat occur in this region, because of the cool climatic condition prevalent here even during the summer. We report here the present disease situation of these crops in this area.

Stem, leaf, and stripe rusts. All three wheat rusts occur throughout the year in the Nilgiri Hills where the summer crops play an important role in the survival and dissemination of rust pathogens. Interestingly, the wheat crop also is sown at different time intervals during the summer in this region. Thus, crops in different stages of growth provide good conditions for the rust pathogen to continuously infect the crop. The crop sown in the later part of the summer plays an important role in supplying rust inoculum to the winter crop. This infection pattern continues from season to season.

The Southern Hills, which comprise the Nilgiri and Palney Hills, are an important source of rust inoculum, particularly for stem and leaf rust and play a crucial role in causing new epidemics of rusts in the plains of India. When cyclonic turbulence occurs in the Bay of Bengal between October and November, air currents carry and deposit the rust uredospores in the plains of India.

Of the three rusts, leaf and stripe rusts occur in higher intensity (trace to l00 %) compared with stem rust (trace to 50 %) in the farmers' fields. However, severe stem rust occurs throughout the year at our research farm.

To combat wheat rust epidemics in the Southern Hills, we now have several very effective resistance genes , including Sr24, Sr25, Sr26, Sr27, and Sr31 for stem rust; Lr9, Lrl9, Lr24, Lr28, and Lr37 for leaf rust; and Yr9, Yrl5, and Yrl7 for stripe rust.

Recently, we have released two rust-resistant bread wheat varieties, HK 1085 (Bhavani) with Sr31, Lr24, and Yr9, and BW 2044 (Kurinji), with Sr25, Lrl9, and an unknown resistance gene for stripe rust, for the Southern Hills zone. These varieties are now serving as a genetic barrier against the spread of stem and leaf rusts to the plains of India.

Dicoccum wheat rarely is affected by leaf rust in this area. One race involved has been identified as race 16. Other than the leaf rust, yellow rust occurs severely on dicoccum wheats during the winter with a lower infection rate during the summer.

Leaf blight (Relminthoaperium antivua). Leaf blight is observed more or less throughout the year in this area. In the last several years, we found leaf blight to be most severe during the warmer months (February and March) in our research farm. Susceptible varieties like Sonalika and HW 3003 yielded very poorly and seeds were unfit for consumption. Leaf blight now appears to be even more serious than the wheat rusts.

Wheat scab. Scab is most severe during summer season, June to October, because of the prolonged moist conditions. Scab affects all the cultivated varieties in the area, including recently introduced wheat varieties HW 741, HW 1085 (Bhavani), and HW 2044 (Kurinji). The disease severely affects seed, renders it unfit for consumption, and causes poor germination.

At our research farm, we now observe wheat scab even during the winter season when cloudy and moist weather conditions folIowing short showers prevail for several days. In the last few years, we were unable to harvest good seed from the summer crop due to scab.

Powdery mildew. This disease generally is observed in the farmers' fields during the summer during prolonged moisture. However, we recently observed powdery mildew throughout the year at our research farm. This disease was observed to severely affect the crop at the seedling stage, resulting in a reduction of fresh weight of up to 84 % as compared to healthy seedlings. We have found a few powdery mildew-resistance genes, Pml, Pm2, Pm3, Pm3b, and Pm6, to be very effective against the powdery mildew pathotypes prevalent in the Nilgiri Hills. The systemic chemicals Tilt (Propiconazole), Contaf (Hexaconazole), and Topsin M are very effective against powdery miIdew.

Leaf blotch. In our conditions, the disease is minor and attacks only dicoccum wheats. Leaf blotch only was observed during the summer season and at certain locations. When inoculated with the pathogen, the Indian wheat cultivars Sonalika, Kalyansona, HD 2285, HE 2329, HD 2009, HVW 318, and HUW 234 were found to be highly resistant to this disease. No significant crop loss was observed.

Head blotch. Head blotch also occurs in this area throughout the year. In some years, head blotch may be devastating, particularly during the summer when prolonged moist conditions prevail. Grain from the infected crop is unfit for consumption. Nearly all cultivars are highly susceptible.

INDIAN AGRICULTURAL RESEARCH INSTITUTE
Division of Genetics, New Delhi - 110012, India.

 

Inheritance of novel partial genetic male sterility in hexaploid wheat. [p. 74-76]

Dalmir Singh.

Abstract. A male-sterile plant was isolated from a hexaploid wheat cross involving Sel. 212 and HD 2009. In the process of its stabilization for six selfed generations, it produced about five to six seeds/spike, demonstrating partial male sterility (p-mst). The sterility is caused by homeotic transformation of androceum whorl (anthers) to gynoecia. The inheritance of p-mst was studied in crosses involving the male sterile parent as a female and the variety Kundan (fully fertile) as the male parent. The parents, F1, and F2 generations were grown in the field and evaluated for the male sterile trait. The inheritance pattern suggested that male sterility is controlled by a single recessive gene and may have possible use in hybrid wheat breeding program.

In 1994, a male-sterile plant with open florets was detected among F3 progenies derived from a cross between two T. aestivum lines, Sel. 212 (a wheat­rye recombinant resistant to leaf and stem rusts) and HD 2009 (an Indian wheat susceptible to wheat rust). The plant was resistant to leaf and stem rusts. On selfing, seven seeds were obtained from 10 spikes and produced four viable plants.

The breeding behavior of the genetic male sterility was studied for six generations (1994­99). During this period, a large number of male-sterile spikes were bagged every year to control seed set. Selfed male-sterile spikes produced an average of 6.06 seeds/spike (Table 1), which was much lower than that of the parental line Sel. 212 (40.4 seeds/spike). The range of seed set in male-sterile spikes was 0 to 15 seeds/spike. In terms of sterility, male-sterile culture exhibited 70­100 % male sterility, amounting to partial male sterility. A similar kind of genetic male sterility has been reported in T. aestivum by Pugsley and Oram (1959), Lupton and Bingham (1966-67), Krupnov (1968), and Lemekh et al. (1971).

Morphology of the p-mst plants. The overall morphology of p-mst plants was like that of other cultivated wheat, except that it was late in maturity and fully resistant to leaf and stem rusts (resistance from rye). Microscopic observations on the florets showed that a large number of anthers had become ovaries. In other florets, anthers appeared as though they were part anther and part ovary. Varying degrees of pistilloidy have been reported due to genetic male sterility (Jan and Qualset 1977) and cytoplasmic male sterility (Endo 1980) in T. aestivum. The under developed anthers look small, yellow, and had pollen, In some florets that had three anthers, even the best anthers were not plump and had problems with dehiscence even when exposed to light and heat. The number of pollen grains appeared to be less than normal. The florets tended to remain open for a longer duration, exposing a feathery stigma. The seed set on the p-mst plants were dented or notched on the side.

Meiosis in the p-mst plants. Each year, some plants were tested for stability of pollen meiosis at first meiotic metaphase. We observed that the meiosis was normal. Female fertility was normal, including the modified ovaries, which when crossed produced multiembryomic seeds,

Genetics of the p-mst trait. After four generations of selfing, male sterility had become highly homozygous. A few spikes from p-mst plants were emasculated and pollinated with Kundan (an Indian hexaploid wheat). A sufficient number of cross seed was obtained. The parents and the F1, and F2 populations were planted at IARI, New Delhi.

Observations on dominance/recessive behavior of the p-mst trait in the F1 and its pattern of segregation in the F2 were recorded. A chi-square test was applied to test the goodness of fit for the assumed segregation ratio.

Male-sterile plants were highly sterile and produced 6.5 seeds (dented) per spike. The check Kundan was fully fertile and produced 43.3 seeds per spike (Table 2). All F1 hybrids were fully fertile (45.1 seeds per spike), indicating that fertility was dominant over sterility. In the F2 population, a segregation ratio of 3:1 (fertile:sterile) was observed (Table 2). Thus, male sterility in the hexaploid wheat stock used is controlled by one recessive gene. Pugeley and Oram (1959), Latypov (1974), Driscoll and sarlow (1976), Kleijer and Fossati (1976), and Sasakuma et al. (1978) also have reported single recessive genes for g-mst.

Table 2. Segregation for partial male sterility in the F2 population derived from a cross involving the male-sterile stock and the variety Kundan.

Fertile plants			Sterile plants
Parents	With plump seed	Seeds/spike	With dented seed	Seeds/spike	Chi-square 3:1	P-value between
Kundan	30	95	43.3	0	---	---
mst plants	0	262	0	1,200	---	---
F1 mst / Kundan	15	133*	45.1	0	---	---
F2 mst / Kundan	282	267	46.3	108	1.36	0.20--0.30

 

Compared with the CMS and CHA systems, the g-mst system under study offers the following advantages:

1. breeding procedures simplified by eliminating the need for cytoplasmic and fertility-restoration genes,
2. genotypes with poor anther extrusion can still be used as female parents,
3. the time delay in converting a promising new genotype into a CMS (female) inbred is avoided,
4. evaluating lines for general and specific combining ability is simpler,
5. no effect on female fertility (in fact, crossed seed set is enhanced because of the presence of multiple ovaries in the florets in the male-sterile plants),
6. least influenced by environment,
7. g-mst plants produce about 10-12 % seeds (selfed or left open), therefore, it is economical to maintain,
8. controlled by single recessive gene and can be maintained in homozygous condition, and
9. labor-intensive process of emasculation and pollination are eliminated, significantly reducing the cost of cross seed.

We noted that the g-mst system may have these disadvantages:

1. In the crossing block, there may be 10­12 % male-sterile seeds that will produce male-sterile plants on germination. Because of their slow growth, their development will be suppressed by the vigor of hybrid plants.
2. The genotype possessing the g-mst trait is slightly taller (about 110 cm) and can be utilized for hybrid development for limited input conditions.

Utilization of the g-mst system for developing wheat hybrids. The g-mst system was used to develop wheat hybrids involving 10 different genotypes. In one combination where pollinator also was tall, 53 hybrid plants produced 3,240 grams of grain yield (in a 3-m2 area). To test its repeatability, the same hybrid is being tested along with other hybrids including one best check in an area of 40 m2 (800 hybrid plants of promising combinations) during current Rabi season (2001).

References.

• Driscoll CJ and Barlow KK. 1976. Male sterility in plants. Induction, isolation and utilisation. In: Proc Symp Induced mutations in cross breeding, IAEA, Vienna, Austria. pp. 123-131.
• Endo TR. 1980. Genetic constancy of the cytoplasm. In: Genetic diversity of the cytoplasm in Triticum and Aegilops (Tsunewaki K ed). Jpn Soc Promotion Sci Publ pp. 13-48.
• Jan CC and Qualset CO. 1977. Genetic male sterility in wheat inheritance. Crop Sci 17:791-794.
• Kleijer G and Fossati A. 1976. Chromosomal location of a gene for male sterility in wheat (Triticum aestivum). Wheat Inf Serv 41-42:12-13.
• Krupnov VA. 1968. Genetic male sterility in common wheat. Genetica 4(10):28-35.
• Latypov AZ. 1974. Digenic nature of male sterile mutants of winter wheat. Sb Nauch Tr Belovrus Acad USSR 123:3-7.
• Lemekh IM, Pavlishin MM, and Savits'ka EV. 1971. Some biological characters of pollen sterile plants of Triticum aestivum in relation to their use for breeding. Nauk Pratsi Zemle Robstva Tvarnnitstva Zakhidn, USSR. 17:220-225.
• Lupton FGH and Bingham J. 1966-67. Winter wheat. In: Plant Breed Inst Cambridge Ann Rep, p. 67.
• Pugeley AT and Oram RN. 1959. Genetic male sterility in wheat. Aust Plant Breed Genet Newslett 14:1011.
• Sasakuma T, Mann SS, and Williams ND. 1978. EMS induced male sterile mutants in euplasmic and alloplasmic wheat. Crop Sci 18:850-853.

Monosomic analysis of some spike traits in Oligo, a hexaploid derivative of wheat. [p. 76]

K.P. Singh and Dalmir Singh.

India will need an estimated 109 million tons of wheat by 2020 AD. Because wheat yields have reached a plateau, wheat researchers throughout the world are making efforts to increase yield. To achieve this target, sufficient genetic variability is a basic requirement. Genetic variability for several traits has been generated through chromosome manipulation. A derivative with a long spike length (19.0 cm), a higher spikelet number (25), a high number of seed/spike (85), and amber seed was isolated from a cross involving a line of Pb. C-591 monosomic for chromosome 3A and the cultivar Oligo. The Oligo parent was uniculm and has a very long spike length (21.5 cm) and red seed color. We studied the genetics and location of these traits and located them on specific wheat chromosomes before using it in a breeding program.

Monosomic lines of Chinese Spring were identified cytologically at first meiotic metaphase in all the 21 monosomic lines and crossed as females with an Oligo derivative. All F, hybrids were grown in the field, and monosomic hybrids were identified cytologically for all the 21 monosomic lines. Seed obtained from these plants and disomic F, plants were grown in the field to obtain an F2 population. Observations were recorded on spike length and spikelet number and number of grains/spike on the parents and the F, and F2 plants. Data was analyzed by standard statistical procedures. The F2-monosomic analysis revealed that spike length was controlled by the genes present on chromosomes 2D and 6A. The genes controlling spikelet number were located on chromosome 2B, 2D, and 6D. Chromosomes 2B and 7B have genes for the number of grains/spike.

Breaking yield barriers in wheat-designing a new plant type. [p. 76-77]

S.S. Singh, J.B. Sharma, Nanak Chand, and D.N. Sharma.

The Green Revolution in India in the mid 1960s included large-scale planting of high yielding, semidwarf, wheat varieties developed at CIMMYT, Mexico. The semidwarf wheats reduced yield losses from lodging and were responsive to higher levels of input. This new plant architecture, which replaced a taller type, was responsible for increasing wheat yields from 1 t/ha in the early 1960s to nearly 2.7 t/ha in late l990s. However, to keep pace with the population growth, India will need 109 million tons of wheat by the year 2020. To achieve this target, average yield must be increased from 2.7 t/ha to 4.0 t/ha. To achieve this quantum jump in productivity, the Indian Agricultural Research Institute initiated strategic research in 1994­95 to design a new plant type. This plant type would be the first of its kind in India, and probably the world, which was achieved by exploiting local germ plasm with the characteristics of very few tillers, very long and lax spikes, spikelet numbers between 23 and 26, and shriveled seed. The objective was to design a new plant type with an optimum combination of three yield components, i.e., grain weight, grain number/spike, and tillers/plant, along with dark green, thick broad leaves and thick stems. A local germ plasm was crossed with two wheat varieties, Vaishali (DL 784-3) and Vidisha (DL 788-2), with the objective of incorporating the leaf and stem rust-resistance genes Lr24 and Sr24, derived from A. elongatum. Huge variability for yield-contributing traits such as plant height; maturity duration; and leaf, stem and grain characteristics was created. Selections were made for transgressive segregants for yield components and other plant characters of the local germ plasm in very large F2 populations. Bold ears were chosen from selected families in the following generations and grain selection was skewed towards families with well-filled amber grains and a grain weight above 45 g/1,000 grains. All the segregating materials in each generations were subjected to leaf rust pressure by creating artificial epidemics.

Beeders have successfully designed several lines, DL 1266-1, DL 1266-2, DL 1266-6, DL 1267-3, and DL 1267-4, combining very high yield and resistance to leaf and stem rusts when compared to best wheat varieties PBW 343, HD 2329, and UP 2338 (Table 3). These advanced lines are moderate in tillering but possess a higher number of grains/pike; high 1,000-kernel weight; higher biomass; dark-green, thick broad leaves; thick stems; a maturity duration between 120 to 135 days; and a plant height of 85­100 cm. These lines also exhibit a high level of resistance to leaf and stem rusts imparted from Lr24/Sr24 when tested as seedlings in glasshouse and as adult plants in the field. The presence of resistance genes Lr24/Sr24 in this new plant type is a unique feature. A large number of lines much improved over the F1 material are being evaluated in replicated yield trials. These superior wheat varieties with increased yield potential are targeted to wheat growing areas with high soil fertility and assured irrigation. These new wheat varieties have 15-20 % more yield potential than PBW 343, HD 2329, and UP 2338 However, their yield may increase by 20-30 % under improved production management, which also is being developed at our Institute, so as to harness the maximum yield potential in wheat bowl zone of the country. This step is towards the Ever Green Revolution in the country.

Table 3. Performance of wheat lines with a new plant type for yield and yield-contributing factors in comparison to the best checks during the 1998­99 and 1999­00 growing seasons.

Cross	Biological yield (g/sq m)	Grain yield (g/sq m)	Tillers/sq m	Grains/spike	1,000-kernel weight (g)
SFW/Vaishali  SFW/Vidisha	1,883-2,160	667-707	274-541	41-56	45-52
Checks PBW 343, HD 2329, UP 2338	1,820-2,036	553-625	446-514	36-42	36-39

SHER-E-KASHMIR UNIVERSITY OF AGRICULTURAL SCIENCES AND TECHNOLOGY
Division of Plant Breeding and Genetics, Faculty of Agriculture, R.S. Pura ­ 181 102 - Jammu, (J&K) India.

 

Cytogenetics of Ae. comosa/T. aestivam hybrids. [p. 77-78]

J.S. Bijral, Kuldip Singh, and T.R. Seiarma.

Aegilops comosa is a diploid species with the M genome that is probably involved in the parentage of Ae. geniculata, Ae. biuncialis, Ae. neglecta, and Ae. columnaris of the U-genome group and Ae. crassa, Ae. juvenalis, and Ae. vavilovii of the D-genome group. Aegilops comosa is highly resistant to yellow rust (Kimber and Feldman 1987) and could be a valuable germ plasm source for the transfer of rust resistance to bread wheat. Interspecific hybrids involving Ae. comosa (accession no. 520, the female parent) and T. aestivum (HD 2285) were produced under field conditions to transfer yellow rust-resistance gene(s) to bread wheat and also to assess the potential of Ae. comosa cytoplasm for developing wheat hybrids. This report is on the cytogenetics of these Ae. comosa­T. aestivum Fl hybrids.

For meiotic studies, immature Fl spikes were fixed in an acetic acid/alcohol solution for 24 h, and meiotic preparations made by squashing the anthers in 2 % acetocarmine.

Tetraploid (2n = 28, ABDM) hybrid plants were vigorous, tillered profusely, and resembled the male parent more closely in over all morphology. However, the Fl hybrid plants were completely self-sterile and their terminal spikelets had awns similar to those of Ae. comosa. Our efforts to produce BCI seeds failed.

Chromosome pairing in the amphihaploids averaged 2.69 bivalents (1.3 ring + 1.39 rod) + 22.6 univalents per meiocyte. The maximum chromosome pairing recorded was 4 bivalents (3 ring + 1 rod) + 20 univalents.

Acknowledgment. Our sincere thanks to Dr. H.S. Dhaliwal, Professor, Department of Genetics and Biotechnology, PAU, Ludhiana, India, for kindly supplying seed of the Ae. comosa accession no. 520.

Reference.

• Kimber G and Feldman M. 1987. Wild Wheat - An Introduction. College of Agriculture, University of Missouri, Columbia, Special Reports 353.