BHABHA ATOMIC RESEARCH CENTRE
Nuclear Agriculture and Biotechnology Division, Mumbai-400085, India.
B.K. Das and S.G. Bhagwat (Nuclear Agriculture and Biotechnology Division), and N. Eswaran, A. Saini, and N. Jawali (Molecular Biology and Agriculture Division).
For combining desirable HMW-glutenin subunits and durable rust-resistance genes, intervarietal crosses involving recent bread wheat cultivars and some of our experimental lines were made. The cultivars and experimental lines used in the crosses were PBW-343, HUW-206, WH-542, HD-2385, HD-2285, Vidisha, KS-1, and SK-1. The F2 populations were scored for morphology, flowering time, and rust reaction after artificial inoculation. A study of genetic diversity of Indian cultivars using PCR-based markers is being investigated. The association of molecular markers with quality traits, agronomically important traits, and rust resistance genes also are being studied.
B.K. Das and S.G. Bhagwat.
In breeding for improvement of bread-making quality in wheat, small-scale tests for assessing gluten strength or bread-making quality is required in order to select in early generations where sample size is the limiting factor. An SDS-sedimentation volume test using 6 g of whole meal (Axford et al. 1979 ) is a small-scale test that is used routinely to assess bread-making quality. A sedimentation test using 1 g of whole meal was reported for durum wheat (Dick and Quick 1983). We used the 1-g test on 19 experimental lines of bread wheat. The protein percentage in these lines ranged from 12.9 to 15.4. A sedimentation test was performed using both 6-g and 1-g samples. Results from the 6-g test volume was recorded in milliliters and 1-g test the sedimentation height in millimeters. A significant positive correlation ( r = +0.753**) exists between the sedimentation values obtained by both the methods. We therefore conclude that the 1-g sedimentation test may be useful for selecting early generation bread wheat lines.
References.
S.G. Bhagwat.
The sphaerococcum trait previously was transferred to an agronomically suitable background by backcrossing. Among the BC5F4 segregants, crosses were made between sphaerococcum trait carriers and noncarriers. The parents and F1 plants were raised in pots. At anthesis, culm length of the tallest tiller, spike length, spikelet number, flag leaf blade length, and maximum width were measured. Spikelet number/cm of spike length was calculated. Stomatal impressions were made from the middle of the upper surface of the flag-leaf blade and stomatal frequency/mm2 was estimated.
Among the parents, sphaerococcum morphology was associated with a 29 % reduction in the culm length and a 23 % reduction in the spike length. The number of spikelets remained unchanged. Number of spikelets/cm spike length increased by about 30 %. Flag-leaf blades were 11 % wider and 3.6 % longer.
The F1s were intermediate in culm and spike length, but the flag-leaf blades were larger. Flag-leaf blade area estimates for the sphaerococcum-type parent, nonsphaerococcum-type parent and the F1 were 26.0 ± 0.95, 21.4 ± 1.32, and 29.6 ± 0.61, respectively, and stomata/mm2 were 70.5 ± 2.52, 67.4 ± 1.12, and 59.9 ± 2.71, respectively. These results indicate that the sphaerococcum morphology is associated with higher stomatal frequency for the same leaf area. The shortening of the culm in the sphaerococcum type appears to be related to the shortening of parenchymatous cells in the stem.
S.G. Bhagwat, J.K. Sainis, and S.P. Shouche; and R. Rastogi (Computer Division).
Three selections derived from three backcrosses and the recurrent parent were used to study grain morphometry. Grains were imaged in a downward position using a scanner in the transparency mode. Image analysis was made using the Comprehensive Image Processing Software, which was developed at the Bhabha Atomic Research Centre. Significant differences were detected in geometric parameters and moments. Using 45 parameters, Euclidean distances were calculated when it was possible to differentiate between the partners in four out of the six pair combinations. Previously, we had observed that the system was able to distinguish between wheat varieties, and our recent results show that this system can distinguish between closely resembling wheat samples.
BHARATHIAR UNIVERSITY
Cytogenetics Laboratory, Department of Botany, Coimbatore-641 046, India.
V.R.K. Reddy, K.M. Gothandam, and G. Kalaiselvi.
Specific genes for resistance to leaf, stem and stripe rusts present singly or in combination were transferred from hexaploid wheat stocks to seven Indian hexaploid wheat cultivars, HD 2687, PBW 343, MACS 2496, HW 2034, HW 4001, RAJ 1555, and K 8962. The genes include five leaf rust-resistance genes (Lr19, Lr24, Lr28, Lr32, and Lr37), three stem rust-resistance genes (Sr24, Sr25, and Sr38), and one stripe rust-resistance gene (Yr17). A simple backcross method was used to transfer these genes; NILs of each in BC2F5 and BC5F5 were made from all the 35 cross combinations. The lines were screened for resistance to individual rust races as seedlings in the glasshouse and with a mixture of races at adult plat stage in natural/artificial epiphytic conditions in the field. Immune to moderately resistant reactions at seedling stage and highly resistant reactions at adult plant stage provided by these genes strongly advocate the use of specific rust resistance gene (gene complex) for durable resistance. The lines with Sr24 and Sr25 showed a variable reaction pattern, probably due to interactive effects with genes already present in the recurrent parent. Lines with the leaf rust-resistance genes Lr28 showed resistance also to stem and stripe rust indicating the presence in the donor parents of additional resistance genes.
Thirty-two cultivars of T. aestivum were analyzed for their allelic variations of HMW-glutenin subunits by SDS-PAGE. A numeration system was followed for designating the HMW-glutenin subunits. The Glu-1 quality score was calculated according to the composition of HMW-glutenin subunits. A total of 10 alleles were identified, three (a, b, and c) at the Glu-A1 locus, four (a, b, c, and d) at the Glu-B1 locus, and three (a, b, and d) at the Glu-D1 locus. The most frequent HMW-glutenin subunits were 2* at Glu-A1, 7 at Glu-B1, and 5+10 at Glu-D1. The most frequent protein combinations are 2*, 7+8, 2+12 and 2*, 7, 5+10. The Glu-1 quality score ranged from 5-10. The Glu-1 quality score 8 is present in a large number of cultivars.
One hundred twenty cultivars of bread wheat were evaluated for 16 yield and associated traits. All the genotypes were grouped into 11 clusters. Cluster I was the largest and included 62 cultivars followed by cluster IV with 20, cluster VI with 13, and cluster XI with 11. The remaining cultivars were distributed in seven clusters with two cultivars in each cluster. Analysis of variance for each individual character showed highly significant differences among the cultivars for all the sixteen characters. The mean performance of each cluster showed appreciable differences for all the characters. The characters of harvest index, protein content, flag leaf area, and straw strength contributed maximum to genetic divergence. Characters such as days-to-heading, days-to-maturity, plant height, and threshability contributed a minimum to the genetic divergence. The phenotypic, genotypic, and environmental correlation coefficients studies revealed that days-to-heading, days-to-maturity, spikelets/ear, straw strength, and protein content were positively correlated with grain yield. Harvest index exhibited a positive correlation with 1,000-kernel weight, texture, and biological yield; and a negative correlation with the other characters. Protein content exhibited positive correlation with spike length, 1,000-kernel weight, and grain yield. Based on genetic divergence and the mean performance of yield and other traits, six diverse and superior genotypes were selected. These genotypes may be involved in a multiple-crossing program to recover transgressive segregates with high genetic yield potential.
Eighty-five bread , 11 emmer , and 23 durum wheats were crossed to three hexaploid testers to determine their genotypic status with reference to necrosis and chlorosis genes. The testers are T. aestivum subsp. macha var. subletschchumicum (Ne1ne2 Ch1ch2) and T. aestivum cultivars C 306 (Ne1ne2 ch1Ch2) and Sonalika (ne1Ne2 ch1Ch2).
In aestivum wheats, 52 out of 85 varieties have Ne2
and Ch2 (ne1Ne2 ch1Ch2) and 12 have Ne1 and
Ch2 (Ne1ne2 ch1Ch2). The remaining 21 varieties
are noncarriers (ne1ne2 ch1ch2) for both genes. In dicoccum
wheat, six of the 11 varieties have Ne1 and Ch1
(Ne1ne2 Ch1ch2), two have Ne1 (Ne1ne2 ch1ch2),
and the remaining three are noncarriers. In the durum wheats,
19 of 23 varieties have Ne1 and Ch1 (Ne1ne2 Ch1ch2)
and the remaining four are noncarriers for both genes. Allelic
variation at the Ch (Chs, Chm, and Chw)
and Ne (Nes, Nem, and New) loci were
observed.
CH. CHARAN SINGH UNIVERSITY
Department of Agricultural Botany, Meerut 250 004, India.
P.K. Gupta, H.S. Balyan, S. Kumar, M. Prasad (Present address IPK, Corrensstrasse 3, D- 06466), J. K. Roy, N. Kumar, S. Sharma, P.L. Kulwal, S. Rustgi, and R. Singh.
Construction of molecular genetic map using SSRs with International Wheat Microsatellite Mapping Network (IWMMN). Ten centers (including Meerut, India) of the IWMMN collaborated in extending the SSR genetic map. As part of the Wheat Microsatellite Consortium (WMC), large numbers of SSR primer pairs (~ 400) were developed. Fifty-eight of 176 primer pairs tested were polymorphic between the parents of ITMI mapping population 'W7984/Opata 85' that was used earlier for the construction of RFLP maps in bread wheat. Using this population and the framework map, a total of 66 microsatellite loci were mapped, which were distributed on 20 of the 21 chromosomes (except on chromosome 6D). These 66 SSR loci are added to the existing 384 SSR loci earlier mapped in bread wheat. This important study with 20 authors has been accepted for publication and will appear in the forthcoming issue of the journal Theoretical and Applied Genetics.
QTL mapping for different economic traits in bread wheat - QTL interval-mapping using the ITMI mapping population and the ITMI map for QTL analysis for yield and yield-contributing traits. Using QTL Cartographer software, QTL interval mapping for seven yield and yield-contributing traits (tillers/plant, spike length, spikelets/spike, grains/spike, biological yield, harvest index, and grain yield) was undertaken in bread wheat using a set of 110 RILs of the ITMI mapping population. For each trait, values were averaged from 15 plants/genotype (five from each of the three replications). Data on a set of 358 mapped molecular markers (both genotype data and genetic distances) were provided by M.S. Röder, IPK, Gatersleben, Germany, and were used for QTL analysis. CIM resolved 29 QTLs (LOD = 2.1 to 5.5) distributed on 16 chromosomes (six from the A genome and five each from B and D genomes). Seven molecular markers associated with QTLs also showed significant marker-trait association, both in regression and t-tests and can be used for MAS. QTL effects (R2) ranged from 5.3 to 50.6 %, and, when measured irrespective of LOD score, gave a characteristic L-shaped distribution suggesting that there are many minor QTLs that should also be taken into account during MAS. Multitrait, composite-interval mapping (MCIM) for two groups of correlated traits (tillers/plant, biological yield, harvest index, and grain yield; and spike length, spikelets/spike, and grains/spike) detected 41 QTLs for seven individual traits. CIM also may give both false positives and false negatives and several QTL may be pleiotropic, influencing more than one trait. In some cases, for the same QTL, LOD scores in MCIM were generally higher than those in CIM, thus placing higher level of confidence and reducing the possibility of false positives in these QTL identified both in CIM and MCIM.
QTL analysis for growth and leaf characters.
QTL interval mapping for four growth characters (early growth
habit, days-to-heading, days-to-maturity, and plant height), and
association studies for two leaf characters (leaf color, scored
as dark and pale green; and leaf waxiness scored as waxy and nonwaxy)
also were conducted utilizing the ITMI mapping population. Using
QTL Cartographer software, the CIM for all the four growth characters
and MCIM for three correlated traits (excluding plant height),
were made. For the growth characters, CIM suggested the presence
of 14 QTLs (LOD = 2.0-12.7), of which only six were common with
those among the 18 QTLs identified by MCIM. Some false positives
among QTLs identified by CIM is possible. The 14 molecular markers
that were closest, one for each of the 14 QTLs identified by CIM,
also were tested for marker-trait association using regression
and t-tests. Five markers showed significant association and,
therefore, are recommended for MAS. Incidentally, the QTLs associated
with these five markers were identified by both CIM and MCIM,
thus placing a higher level of confidence in these markers. Some
of the QTLs identified by CIM and joint MCIM also affected more
than one trait. During CIM for individual traits, the phenotypic
variation explained by all QTLs that were identified at LOD score
> 2.0 or above together accounted for approximately 1791
% of the phenotypic variation. However, QTL effects, when measured
irrespective of LOD score, exhibited a characteristic L-shaped
distribution. The two leaf characters had a 100 % correlation.
Tests of independence of attributes involving each of the 358
molecular markers with the two leaf traits identified 14 molecular
markers spread over seven different chromosomes, each showing
significant association with both leaf color and leaf waxiness.
Some of these markers are the same and also exhibited association
with some growth and yield traits studied earlier, thus adding
to their utility in wheat breeding through MAS.
QTL analysis for preharvest sprouting tolerance.
QTL interval mapping for preharvest-sprouting tolerance (PHST)
was also conducted using the ITMI mapping population. At crop
maturity, data for PHST were recorded on each of 110 RILs belonging
to the population. At the time of physiological maturity, 15 spikes
from each of the 110 RILs (five each per genotype per replication)
were harvested separately and scored for tolerance to preharvest
sprouting following Baier (Ann Wheat Newslet 1987; 33:40). Observations
on sprouting were recorded after 10 days. The data on PHST were
scored on the scale of 19. Although there was a narrow range
of variability between parents of ITMI population for PHST, a
wide range of variability was available among the RILs derived
from the cross. This encouraged us to conduct composite-interval
mapping leading to identification of five QTLs on four chromosomes
(1D, 2D, 4B, and 6A). Individual QTL effects (R2) ranged from
5.9121.07 %. The total R2 value calculated after summing
individual QTL effects was 55.02 %. Individual QTL effects exhibited
an L-shaped distribution, again explaining presence of large number
of QTLs with moderate to small effects.
QTL interval mapping using trait-specific mapping populations - preparation of framework map and QTL analysis for grain protein content. In a continuation of earlier studies, QTL interval mapping for grain protein content (GPC) using a set of 100 RILs derived from a cross between the parents WL711 (low GPC) and PH132 (high GPC) was conducted. From the GPC data on RILs evaluated in five different environments at three different locations, QTLs were mapping by single marker analysis (SMA), interval mapping (IM), and composite interval mapping (CIM) using QTL-Cartographer V. 1.21. For this purpose a framework genetic map was prepared using the above population. The map had 173 loci involving 171 SSR primer pairs. As many as 13 QTLs with a LOD score > 2.5 were detected on eight different chromosomes. The maximum number of QTLs (10) were detected by CIM, followed by SMA and IM (five each). Of the 13 QTLs, seven were identified by CIM at a LOD score higher than the threshold LOD calculated for each environment after 1,000 permutations. Two of these seven QTLs also were identified by all the three methods. The present map is being saturated using SAMPL and AFLP markers. Genotyping of RILs with 13 primer combinations using SAMPL primers 6 and 7 with one AFLP primer pair has already been completed.
Preparation of framework maps for preharvest sprouting tolerance
and grain weight and their proposed use in QTL interval mapping.
The ongoing project Marker assisted selection for some quality
traits in bread wheat, funded by National Agricultural Technology
Project (NATP) of Indian Council of Agricultural Research (ICAR),
also involves preparation of the framework linkage maps for other
two grain quality traits of PHST and grain weight (GW). We have
a mapping population of 100 RILs for each of these traits. The
data on these traits at three different locations were recorded
in the year 2001 and also will be recorded in 2002. Data also
are being collected on other traits including early growth habit,
days-to-heading, days-to-maturity, leaf color, leaf waxiness,
plant height, tillers/plant, spike length, spikelets/spike, grains/spike,
grain yield, harvest index, preharvest sprouting tolerance, and
grain weight. This data collected over environments will be used
for finding 'genotype x environment' interactions. At present,
RILs of PHST are being genotyped using SAMPL markers; genotyping
has already been completed using four primer combinations with
SAMPL primer 6.
After completing the genotyping of PHST RILs with SAMPL primers
6 and 7, and AFLP primers, genotyping of GW RILs will be done.
Using the molecular genotyping data, framework maps will be prepared
that will be used for QTL interval mapping.
Association analysis identifies molecular markers for different traits in bread wheat. Molecular markers linked with QTLs/major genes for traits of interest are being routinely developed in several crops using material derived from planned crosses such as F2, RILs, and DH populations. However, the unavailability of mapping populations and the substantial time needed to develop such populations are sometimes major limitations in the development of molecular markers for specific traits. To overcome this difficulty, association studies involving the use of germ plasm collections for the development of molecular markers has been conducted. During the present study, binary data for SSR, AFLP, and SAMPL markers used earlier for diversity studies were utilized for a study of marker-trait associations involving data scored on 14 phenotypic traits in 55 elite and exotic wheat genotypes. Using both simple linear regression and multiple regression methods, a total of 351 molecular markers (131 SSR, 166 AFLP, and 54 SAMPL) were identified, each of which showed significant association with at least one of the 14 traits. Out of the above 351 markers, 47 were common in both the regression methods (23 SSR, 21 AFLP, and 3 SAMPL). The 47 markers that were common in both analyses are important and can be used for MAS in wheat breeding after conducting the necessary validation studies.
Development and use of EST-derived SSR and SNP markers in bread wheat. SSRs and SNPs are the second and third generation markers of choice and will be used extensively in the future for a variety of purposes. These markers are preferred because of their ubiquity and uniform distribution in nuclear and organellar genomes of plants and animals. Because their development is labor-intensive and expensive, their large-scale use in plant genotyping and MAS may take time. To reduce the development cost of these markers in bread wheat, we used the ever-expanding EST database. EST-derived SSRs/cSSRs and SNPs/cSNPs have many intrinsic advantages over genomic SSRs and SNPs. They are free by-products of the EST database and can be quickly obtained by electronic search of the database. Furthermore, because they are present in expressed regions of genome, which are generally conserved, these markers also have high transferability, and therefore also are useful in comparative genomics. Their presence in the expressed regions of genome also make them particularly useful for studies involving marker-trait association. We developed two, user-friendly tools, MSL and SNPL, which can be easily used by the beginners to mine SNPs and SSRs, respectively. With the help of these tools, we detected 1,083 SSRs (17 motif length) and 19 SNPs, using EST databases (dbEST and EMEST located at NCBI and EBI sites, respectively). We have designed primers for 36 cSSRs. Four of these primer pairs also were synthesized and used successfully for PCR amplification of SSRs. These primers also were tested on two accessions of barley to elucidate their functionality beyond genera (i.e., to test their transferability). All the four primer-pairs tested are functional; they amplified the products of accepted sizes both in wheat and barley. Generally these primer-pairs are intraspecifically monomorphic, but one of the primer-pairs was polymorphic between the two genotypes of bread wheat that represent parents of GPC mapping population. All the four primer-pairs tested showed polymorphism between barley and wheat.
Mapping of SAMPL markers and their conversion into SCAR markers for MAS. SAMPL markers can be used for mapping. Those markers associated with a trait of interest can be converted into PCR-based markers, SCARs, which can be easily used for MAS. Cloned SAMPL fragments also can be used as probes in RFLP and FISH to study their genomic distribution. We used SAMPL markers in bread wheat for the first time to study genetic diversity and possible marker-trait associations (Theor Appl Genet 104:465-472. 2002). The polymorphic SAMPL bands are now being used for preparation of framework maps using three mapping populations developed for individual traits (grain protein content, preharvest sprouting tolerance, and seed weight), and the SAMPL bands identified to be associated with these traits are being converted into SCARs through sequencing of the corresponding amplified products.
Study of ribosomal DNA in wild barley (Hordeum spontaneum).
Intergenic spacer (IGS) length polymorphism in wild barley.
A total of 112 accessions of wild barley belonging to three different
microsites (Evolution Canyon, Tabigha, and Neve Yaar) from Israel
having contrasting ecogeographical conditions were analyzed for
IGS length polymorphism. Genomic DNA was digested with SacI and,
after DNA hybridization, was probed with rDNA probe pTA71. Repeat-unit
lengths varied from 9.2-11.0 kb. We observed 12 lines constituting
34 different rDNA phenotypes, which were largely correlated with
different ecogeographic conditions prevailing at the microsites.
In the accessions from Neve Yaar, homogenization of IGS length
at the two loci located on two different chromosomes were observed.
FISH analysis ruled out the possibility of loss of one of the
two NOR loci.
Methylation status of rDNA.
For analyzing methylation status at rDNA repeat units, we used
a pair of isochizomers, HpaII (CG methylation-sensitive) and MspI
(methylation insensitive), and also the methylation-sensitive
restriction enzyme BamHI. HpaII cleaved the ribosomal-repeat unit
at a solitary site giving a single band of the size of repeat
unit, whereas MspI cleaved it at several sites giving multiple
bands and suggesting that rDNA repeat units in barley are heavily
methylated.
BamHI also gave multiple bands of variable densities, suggesting that the site for this enzyme is not uniformly methylated in all ribosomal-repeat units. A combination of BamHI bands in a genotype was designated as the phenotype of that accession, and seven such phenotypes were available. Out of a total of 11 BamHI bands, four (1.8, 3.8, 7.2, and 8.9 kb) were present in all the genotypes and the remaining seven (2.4, 3.0, 3.6, 4.1, 4.6, 5.0, and 5.9 kb) were polymorphic by presence or absence in different genotypes giving rise to seven different phenotypes. Out of seven phenotypes, six are microsite specific. Thus, the present study resolves microsite-specific methylation of different sites within the intergenic spacer, suggesting that natural selection also plays an important role in methylation of these different sites that may perhaps have a role in regulation of gene expression.
Identification of a T1BL·1RS translocation in some wheat genotypes using three different approaches. The short arm of chromosome 1 of rye is known to have genes for resistance to important fungal diseases and insect pests and high yield. Chromosome arm 1RS also is one of the most widely utilized sources of alien chromatin in wheat improvement. Therefore, knowledge of wheat varieties containing the T1BL·1RS translocation will allow judicious selection of material to be used in a crossing program by the plant breeders for wheat improvement. In view of the above, a total of 19 genotypes/important Indian wheat variety were studied for the identification of the T1BL·1RS translocation using three different approaches including study of satellite chromosomes in mitosis, GISH, and STMS-marker analysis. The results suggested that eight wheat varieties (PBW373, PBW343, PBW175, UP2338, UP2425, UP2418, UP2382, and CPAN3004) of the 19 examined have the T1BL·1RS translocation. These varieties may be considered as a useful source of germ plasm in wheat breeding.
INDIAN AGRICULTURAL RESEARCH INSTITUTE REGIONAL STATION
Wellington - 643 231, the Nilgiris, Tamilnadu, India.
M. Sivasamy, A.J. Prabakaran, K.A. Nayeem, and R.N. Brahma.
The damage caused to tea plantations and hilly vegetables due to winter frost at high altitudes of the Nilgiri hills in Tamilnadu, India, is a common phenomenon. However, there was no such report on wheat until now. Winter or ground frost usually occurs every year in the high-altitude, open lands of the Nilgiri Hills during the winter months (from 15 November-15 February). On a frosty night, room temperature can be 0-2°C and soil temperature below 0°C. The dew on the leaf surface of grass, crops, bushes, and tea plants growing in the open lands at high altitudes will crystallize to form ice. Temperatures the following day can reach 18-22°C quickly in the bright sunshine. Because of the high diurnal variation, extreme low temperatures result in the drying and scorching of the leaves. Plants of the grasslands and tea plantations and other crops may wither totally. These cold conditions usually occur for 3-10 consecutive days, followed by a warm period, and again by frosty nights. In a particular year, 1-3 cold periods can occur. The frosts do not affect the vegetative stages of wheat, barley, rye, oats, and Brassica.
At the IARI Regional Station, wheat is sown year round, generation after generation, to reduce the time for varietal improvement by taking advantage of favorable weather conditions. However, farmers follow the normal sowing times, kharif (June-July) and Rabi (November-December). From our observations, it is now evident that both bread and dicoccum wheat are damaged by frost at flowering. We confirmed that flowering during frosty conditions resulted in the deformation of the lower spikelets and, subsequently, no spikelets were found on the lower portion of the spike. The remaining spikelets on the upper portion were sterile or the fertilized embryos aborted. The spikes of the lower surface were male sterile and entirely open. The spikes that emerged after a frost period had perfect seed set. This type of crop damage was noticed mainly on wheat grown in open fields rather than on slopes or near tree plantations. The crop loss varied between 50-100 %.
M. Sivasamy, M.K. Menon, R.N. Brahma, S.M.S. Tomar (Division of Genetics), A.J. Prabakaran, and K.A. Nayeem.
An early maturing, high-yielding wheat variety coupled with resistance to rusts and blight is needed for the northeastern Plains Zone of India (comprising the states of Bihar, Jarkand, parts of Uttar Pradesh, West Bengal, and Assam) as more area is used for late-sown crops in the predominantly ricewheat cropping system. The early maturing variety HW 2045 was bred at the IARI-Regional Station, Wellington, through a backcross method using HD 2402, which has the Th. ponticum-derived, linked genes Lr19 and Sr25 and the mutant line 'Sunstar* 6/C 80-1' developed by Knott and McIntosh, in a white-seeded background with highly reduced yellow pigmentation in the endosperm. HW 2045 was released for cultivation in the northeast India during 2001-02. This variety possesses remarkable resistance to all existing pathotypes of stem, leaf, and stripe rusts and moderate resistance against the foliar blight. This variety also exhibited slow senescence contributing to increased grain yield under the late-sown conditions. In the All-India Coordinated Yield Trial, the variety HW 2045 has an average yield of 41.2 q/ha when compared to the best check NW 1014, 39.7 q/ha. This is the first time a variety with Lr19 and Sr25 has been released for commercial cultivation in Gangetic Plains of India. HW 2045 will act as genetic barrier against any fresh epidemic of rusts in the plains of North Eastern India.
INDIAN AGRICULTURAL RESEARCH INSTITUTE
Division of Genetics, New Delhi - 110012, India.
S.S. Singh, G.P. Singh, J.B. Sharma, Nanak Chand, D.N. Sharma, J.B. Singh (Division of Genetics); P.C. Pandey and M.R.S. Kaim (Nuclear Research Laboratory); and T. Mohapatra and K.P. Singh (National Research Centre on Plant Biotechnology).
India has achieved spectacular progress in wheat production due to conceptual wheat breeding and now ranks as the second largest wheat-producing nation after China. The replacement of tall wheats susceptible to lodging by semidwarf, high-yielding, input-responsive and rust resistant wheats in the early 1960s ushered in the green revolution. The national average for wheat yields has increased from about one t/ha in the 1960s to nearly 2.7 t/ha in the later half of 1990s. Wheat production rose from 11 million tons in 1960-61 to 75.4 million tons in 1999-2000 with a buffer stock of 26.5 million tons. World wheat production is 570 million tons, and trade in 2001-02 is estimated to be 107.3 million tons, which is 4.3 million tons more than last year despite a decline in wheat production. India's share of exports is expected to be 3 million tons in 2001-02 because of several limitations, including grain quality. The world price of high-grade wheat is $124 USD/t, whereas Indian wheat is worth around $105 USD/t. Therefore, the cost of wheat production must be reduced and grain quality improved to make the Indian wheat internationally competitive (Nagarajan 2001).
The 1 % annual genetic gain in productivity gain during the last 25 years was due to newer generations of high-yielding varieties, but the yield potential has slowed considerably in recent decades. In addition, the cost of cultivation has increased; fertilizer contributed nearly 26 % followed by labor (18.67 %), harvesting (13.91 %), threshing (12.19 %), and land preparation (10.02 %) (Nagarajan 2001). Therefore, the competitiveness of wheat farming will depend on opportunities for dramatically reducing unit costs of production, which can be achieved by shifting the yield frontiers and/or increasing the efficient use of inputs (Pingali 1999).
For achieving the breakthrough in yield potential breeding at CIMMYT Mexico have developed a new wheat type called Buitre through 20 years of prebreeding, genetic manipulation and countless recombinations. This unique ideotype has robust stem, a long spike (> 30 cm), multiple spikelets and florets, large leaf area and broad leaves. This change should increase yields by improving the harvest index and input use efficiency. However, due to some unknown physiological imbalance or disorder the spikes remains largely sterile and resulting grains are mostly shriveled in addition, the plants are generally highly susceptible to rust, specially leaf rust and stripe rust (Rajaram and van Ginkel 1996).
The Indian Agricultural Research Institute, New Delhi, initiated strategic research in 1994 to further enhance wheat productivity by designing an NPT utilizing some local types characterized by very long spikes but with shriveled grains. These local types have low tillering and are highly susceptible to rusts. The NPT has combined the three yield components (grain weight, grain number/spike, and tillers/plant) along with dark-green, thick and broad leaves, thick stem, higher biomass and resistance to leaf and stem rusts. The NPT is the first of its kind in the country and in the world. Several NPT lines exhibiting increased yield potential at low levels of fertilizer application due to improved physiological efficiency. These lines also are resistant to leaf and stem rusts and a better grain quality because of high nitrate-reductase activity (high NR type) and grain protein content near 12.5 %.
Materials and Methods. Parental lines. The material involved as parental lines for the development of NPT genotypes with increased yield potential were local germ plasm Sirsa Farm Wheat (SFW) and two released wheats (Vaishali and Vidisha) with bold, lusturous grains and the tightly linked resistance genes (Lr24/Sr24) for leaf and stem rusts derived from Th. elongatum.
Breeding method. A variant of pedigree method of selection was used to handle segregating populations of the F2F5 for developing the NPT combining desirable yield components and resistance to rusts. The breeding scheme was as follows:
| 1st year | P1/P2 | Parents include SFW (a local type) and Vaishali and Vidisha (released varieties). |
| 1st year (off-season nursery) | F1 | 25-35 seeds, space-planted, harvested in bulk. |
| 2nd year | F2 | 1. Space-plant more than 2,000 plants. |
| 2. Screen for leaf rust resistance in artificial epidemics. | ||
| 3. Select and harvest superior plants showing NPT characteristics along with resistance to rusts. | ||
| 4. Select and carry forward plants with plump grains. | ||
| 3rd year | F3 | 1. Plant row progenies at commercial seeding rate |
| 2. Screen for leaf rust resistance in artificial epidemics. | ||
| 3. Select 100 spikes expressing NPT characteristics and resistance to rusts from the families. | ||
| 4. Reselect spikes with plump grains and bulk seed. | ||
| 4th year | F4 | 1. Plant bulk progenies at commercial seeding rate. |
| 2. Screen for leaf rust resistance in artificial epidemics. | ||
| 3. Select 100 spikes expressing NPT characteristics and resistance to rusts from the families. | ||
| 4. Thresh ears individually and screen for plump grain. | ||
| 5th year | F5 | 1. Plant head-row progenies; screen for leaf rust resistance in artificial epidemics. |
| 2. Bulk superior rows. | ||
| 3. Select grain. | ||
| 6th year | F6 | 1. Plant yield trials. |
| 2. Grow off-season, summer nursery. | ||
| 3. Screen for stem rust resistance in artificial epidemics. | ||
| 4. Quality test. | ||
| F7 | 1. Agronomic trials include seeding rate/variety, different nutrient levels, date of sowing/variety, and growing in a furrow-irrigated, raised-bed system. | |
| 2. Quality test samples from different nutrient levels. | ||
| 3. Fingerprint of selected lines. |
Methodology for physiological studies. The high-yielding NPTs, DL 1266-5 and DL 1266-2, along with best standard check PBW 343 were grown in the field. Phosphorus in the form of P2O5 and potassium in the form of Muriate of potash were added at the rate of 60 and 40 kg/hectare at the time of sowing. Nitrogen at 100, 150, and 200 kg/hectare in the form of urea was added in three equal splits. The first split was applied as a basal dose, and the 2nd and 3rd splits were applied at crown-root initiation and booting stage, respectively. Each fertilizer treatment was replicated three times, and all the genotypes were used for each of the treatment.
A large number of main shoots were tagged and data was collected on yield attributes including grain weight/spike, grain number/spike, and 1,000-kernel weight. In the present study, assay of nitrate-reductase activity was restricted to the laminae of the main shoot of the tagged plants only. Previous studies indicate that the laminae reduce the nitrate taken up by the plant by more than 75 % (Nair and Chatterjee 1990). Nitrate-reductase activity was assayed in vivo as suggested by Hagman and Hucklesby (1971) and Nair and Abrol (1977). The reduced N content in the grain of the final harvest was determined by the alkaline-phenol-sodium hypochloride method using a Techanicon Autoanalyser (Anonymous 1971). Biomass, grain yield, and number of ears/m2 were sampled randomly.
An analysis of variance and the least significant difference was made for all the varieties, however, our results and discussion shall be confined to the comparison between the check PBW 343 and the sister lines DL 1266-2 and DL 1266-5.
DNA fingerprinting. Seven lines, DL 1266-1, DL 1266-2, DL 1266-5, DL 1266-6, DL 1266-10, DL 1266-16, and DL 1266-17, derived from the cross 'SFW/Vaishali'; two other unrelated breeding lines, DL 1337-1 and DL 1396-11; the two parent genotypes, SFW and Vaishali; and two commercial varieties, PBW 343 and HD 2329 were used. Approximately 100 seeds/sample were germinated under aseptic conditions. Seedlings were harvested, bulked, frozen in liquid nitrogen, and used for DNA isolation. DNA was isolated by the standard CTAB method of Doyle and Doyle (1990), purified by RNAse treatment followed by phenol chloroform extraction, dissolved in 10 mm Tris-Cl buffer, quantified by analyzing gels using uncut lambda DNA as standard, diluted to 5 ng/ml, and used in PCR.
Thirty-three STMS markers already mapped on the hexaploid wheat genome, one per chromosome arm, were selected. Custom-synthesized primers were used in the PCR amplification. The reaction mixture contained 10 ng of template DNA, 20 ng of each primer, 250 µm of each dNTP, 1x PCR assay buffer, and 0.2 unit of Taq DNA polymerase. Amplification was in a thermal cycler (Perkin-Elmer model 9600) with the following specifications: 94°C for 5 min, 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and at 72°C for 7 min. The amplified products were separated on a 3 % metaphor agarose gel (FMC, USA). The samples were electrophoresed using 1x TBE buffer, stained using gelstar dye, and photographed using poloroid photographic system. The bands were scored and used to make a binary data matrix. Jaccard's similarity coefficient was calculated and used to establish genetic relationship based on clustering. The computer package NTSYS.PC was used for cluster analysis.
Results and Discussion. Breeding work on changing plant architecture was initiated in 1994 at the Indian Agricultural Research Institute, New Delhi, with the hybridization of the released varieties Vaishali and Vidisha with a local wheat, SFW. In this endeavor, several strains were successfully designed of which DL 1266-1, DL 1266-2, DL 1266-5, DL 1266-6, DL 1267-2, DL 1267-3, and DL 1267-4 were selected to represent the NPT of wheat (Table 1). These NPT genotypes possess a high 1,000-kernel weight, a higher number of grains/spike, a higher biomass, dark green, broad leaves, thick stems, a plant height between 85-100 cm, and good root system. Thus, the negative correlation between yield components has been broken, leading to a positive correlation between grain weight and grain number/spike with optimum productive tillering capacity. In these genotypes, the physiological efficiency of partitioning of dry matter to economic yield has increased. Better synchronization (post-anthesis) of assimilate source, path, and the grain sink, resulting in the increased availability of assimilate for proper development and grainfilling may lead to high grain weight because of proper filling of all grains in all spikelets resulting in higher number of grains/spike.
| Cross | Biological yield (g/m^2^) | Grain yield (g/m^2^) | Tillers/m^2^ | Grains/spike | 1,000-kernel weight (g) |
|---|---|---|---|---|---|
| SFW/Viashali and SFW/Vidisha | 1,883-2,160 | 667-707 | 274-541 | 41-56 | 45-52 |
| Checks PBW 343, HD 2329, and UP 2338 |
1,920-2,036 | 553-625 | 446-514 | 36-42 | 36-39 |
Success in achieving high productivity on a sustained basis will depend upon our ability to develop new methods of feeding the plants. Research on breeding and feeding should be made concurrently by a team of breeders, physiologists, agronomists, and soil scientists. At the same time, wheat scientists, including breeders, physiologists, agronomists, and biotechnologists are working together to harness the maximum yield potential of these NPT wheats (Swaminathan 2000).
Exploitable yield potential. In order to know the exploitable yield potential of these NPT wheats, DL 1266-5, a newly designed wheat genotype (pedigree: SFW/Vaishali) was planted (6 rows/5 beds of 5.5 m) adjacent to an irrigation channel 2.5-m width at commercial seeding rate of 100 kg/hectare. Fertilizer was applied at 100 N:60 P:40 K and five irrigations were provided to these plots.
We observed that plants in the border row towards the irrigation channel of five beds (each 5.5 m) expressed the full potential of the NPT compared to inner rows where there was interplant competition. Table 2 indicates a highly significant increase in all yield components in the border rows when compared to the inner rows. The mean yield of one row is 1.656 kg (90.66 q/ha), which is an overall increase of 218.5 % of border rows over the inner rows. These findings strongly suggest that the NPT wheat DL 1266-5 has a very high yield potential, if grown with suitable technology such as FIRB, which utilizes and exploits the border effect. The exploitation of very high yield potential of NPT wheats will not only save the land but also reduce the cost of cultivation, which is presently very high.
| Trait | Row | Bed 1 (R1) | Bed 2 (R2) | Bed 3 (R3) | Bed 4 (R4) | Bed 5 (R5) | Total | Mean | Advantage to border row (%) |
|---|---|---|---|---|---|---|---|---|---|
| Mean no. of grains/spike | B | 92.0 | 93.2 | 91.2 | 92.8 | 90.8 | 460.0 | 92.0 | 69.4 |
| I | 54.8 | 55.0 | 53.8 | 54.2 | 53.6 | 271.4 | 54.3 | ||
| Mean spike weight (g) | B | 5.12 | 5.16 | 5.13 | 5.14 | 5.09 | 25.64 | 5.13 | 120.2 |
| I | 2.34 | 2.40 | 2.30 | 2.37 | 2.24 | 11.65 | 2.33 | ||
| 1,000-kernel weight (g) | B | 55.6 | 55.3 | 56.3 | 55.4 | 56.1 | 278.7 | 55.7 | 29.8 |
| I | 42.7 | 43.6 | 42.8 | 43.7 | 41.8 | 214.6 | 42.9 | ||
| Yield of 1 row of 5.5 m (kg) | B | 1.670 | 1.830 | 1.550 | 1.720 | 1.510 | 8.280 | 1.656 | 218.5 |
| I | 0.550 | 0.550 | 0.560 | 0.540 | 0.430 | 2.600 | 0.520 | ||
| Estimated number of tillers in a 5.5-m row | B | 326 | 355 | 302 | 335 | 297 | 1,616 | 323 | 44.8 |
| I | 222 | 229 | 243 | 228 | 192 | 1114 | 223 |
Further efforts will be needed to increase the yield potential of the NPT wheats along with incorporation of diverse genes for resistance to rusts for durable resistance. These genotypes have been crossed with indigenous and exotic germ plasm with the objective to increase the number of productive tillers/plant while keeping 1,000-kernel weight and grain number/spike constant. A large number of segregating and fixed materials were generated, which are in testing at various levels. Advanced material is in testing at different levels of inputs and FIRB in collaboration with wheat agronomists at our institute. These lines also are being analyzed for quality parameters.
Durable resistance to rusts. From the first generation, we have proceeded with the objective that Lr24 will provide high level of resistance not only in India, but also in whole Indian subcontinent including Pakistan, Bangladesh, Nepal, and Bhutan. After 1998, resistance to all the three rust pathogens was initiated with the aim to develop derivatives of NPT lines with enhanced resistance leading to durable resistance along with enhanced yield by increasing tillering capacity without affecting grain weight and number of grains/spike. A large number of cultivars/exotic and indigenous germ plasms (including PBW 343, PBW 373, UP 2338, UP 2425, HD 2687, HD 2733, NI 1202, HS 295, HS 365, HW series, WH 542, HD2009, Oasis, Milan, Pios, Opata, and Kauz) having genes for durable resistance to three rusts (Lr34, along with Lr13, Lr26, Lr23, Lr10; Sr2, Sr5, Sr31; and Yr9 and Yr18) were involved in generating NPT wheats with further increases in yield potential and durable resistance.
Physiological and biochemical studies of the NPT genotypes DL 1266-2 and DL 1266-5. Biomass tends to increase with increasing nitrogen levels, i.e., 100, 150, and 200 kg N/hectare in the standard check cultivar PBW 343, whereas in DL 1266-2 and DL 1266-5, the maximum biomass was achieved at 150 kg N/hectare. Thus, the new lines are more efficient N utilizers at lower N fertility levels, although the grain yield/m2 was not significantly higher at different nitrogen levels and between the three genotypes (Table 3). The over all harvest index was superior in DL 1266-2 and DL 1266-5 as compared to PBW 343 check, particularly at low fertility levels. We concluded from the harvest index data that the NPT wheats are more efficient mobilizers of the assimilates to the developing grain sink. The number of productive tillers/m2 is always lower in the NPT genotypes as is evident from number of spikes/m2. This means that DL 1266-2 and DL 1266-5 also are nutrient efficient, as they produce limited number of synchronous tillers with thick stems and compact, long spikes. The superior harvest index also supports this fact and indicates a superior assimilate reserve accumulation and its mobilization to the developing grain sink so that a very high number and improved grain weight result in significantly higher grain weight/spike are sustained. Such improved ideotypes predicted earlier also are expected to be nutrient-efficient based on a comparative study of pre and post-green revolution plant types (Pande et al. 1983). Analysis of grain weight/spike confirms the superiority of NPT genotypes over the check at all the three fertility levels. We may conclude that NPT lines have evolved a superior spike architecture that ensures improved transportation of assimilates to the grain. This proposal becomes obvious as remarkable increases in grain weight/spike, even at low fertility levels, is achieved in these NPT genotypes. Thus, the need to develop newand more efficient and improved plant type to meet the future demands particularly under low fertility conditions is met. As for the analysis of grain-yield components, the grain number/spike, grain weight/spike, and 1,000-kernel weight show remarkable superiority even at lower fertility levels in these NPT lines compared to the check cultivar.
| Character | Genotype | Nitrogen levels (kg per hectare N) | |||
|---|---|---|---|---|---|
| 100 | 150 | 200 | Mean | ||
| Biomass/m^2^ (g) | DL 1266-2 | 1,280 | 1,585 | 1,566 | 1,477 |
| DL 1266-5 | 1,541 | 1,741 | 1,632 | 1,638 | |
| PBW 343 (C) | 1,491 | 1,665 | 1,784 | 1,647 | |
| Grain yield/m2 (g) | DL 1266-2 | 589 | 691 | 653 | 644 |
| DL 1266-5 | 681 | 790 | 729 | 733 | |
| PBW 343 (C) | 650 | 759 | 700 | 703 | |
| Number of spikes/m^2^ | DL 1266-2 | 261 | 285 | 310 | 285 |
| DL 1266-5 | 308 | 335 | 331 | 325 | |
| PBW 343 (C) | 456 | 462 | 496 | 471 | |
| Grain weight/spike (g) | DL 1266-2 | 4.66 | 4.92 | 4.80 | 4.79 |
| DL 1266-5 | 5.70 | 5.79 | 5.32 | 5.60 | |
| PBW 343 (C) | 2.69 | 2.71 | 2.86 | 2.75 | |
| Number of grains/spike (g) | DL 1266-2 | 76 | 85 | 89 | 83.33 |
| DL 1266-5 | 100 | 100 | 98 | 99.33 | |
| PBW 343 (C) | 58 | 60 | 66 | 61.33 | |
| 1,000-kernel weight (g) | DL 1266-2 | 58.99 | 57.73 | 54.39 | 57.00 |
| DL 1266-5 | 56.77 | 57.84 | 54.06 | 56.22 | |
| PBW 343 (C) | 46.81 | 45.75 | 43.26 | 45.27 | |
| Protein % | DL 1266-2 | 12.06 | 13.10 | 14.41 | 13.19 |
| DL 1266-5 | 12.17 | 13.01 | 13.70 | 12.96 | |
| PBW 343 (C) | 11.27 | 12.06 | 11.76 | 11.70 | |
Grain protein is an important quality attribute in wheat that also determines its export potential. In general, when compared to the check, the grain protein in the NPT lines was more than 1 % at 100 and 150 kg N/ha, more than 2 % at 200 kg N/ha. DL 1266-5 had the highest grain protein/m2 followed by check, irrespective of fertility levels. These results indicate that DL 1266-5 was the most efficient nitrogen utilizer irrespective of nitrogen fertility levels and harvested maximum grain protein/m2. Shera (a post-green revolution variety), which also produced synchronous and limited number of tillers, also was efficient utilizer of applied nitrogen (Pande et al. 1983; Abrol and Nair 1976; Abrol et al. 1976), however it did not have the earliness, thick stem, or heavy and compact spikes that the NPT genotype DL 1266-5 possesses. Among the NPTs from the mid 1980s, Shera was considered the most efficient utilizer of applied N and showed maximum nitrate-reductase activity at different nitrogen fertility levels (Nair and Abrol 1977). The newly designed plant type, although much superior to existing cultivars, possesses tremendous scope for further improvement in both quantitative and qualitative yields.
Finger printing for protection of NPT genotypes. The markers amplified a maximum of two alleles. Of the 33 markers used, 20 detected polymorphism among the genotypes used in the study. The polymorphic markers in combination precisely identified each genotype. The lines derived from the cross 'SFW/Vaishali' were closer genetically to the parents than the other lines and the varieties used in the analysis. Maximum similarity was observed between sister lines DL 1266-2 and DL 1266-10. The lines DL 1396-11 was most divergent from others. The line DL 1266-1, DL 1266-2, DL 1266-5, DL 1266-10, DL 1266-16, and DL 1266-17 were more close to the parent SFW, whereas DL 1266-6 was closer to the Vaishali parent.
STMS-based DNA fingerprints of the NPT wheat lines can be used for protection of these lines from any unauthorized use. STMS markers already were mapped and the polymorphism was in the nonoverlapping genomic regions. Moreover, STMS is PCR based and highly reproducible between laboratories. Therefore, the STMS-based fingerprints developed for the wheat lines can be used with high degree of confidence.
Conclusion. The NPT lines with increased yield potential and better grain quality are highly suitable for export purposes and will put India on the export map of the world in addition to eliminating the problem of malnutrition in poorer section of society.
References.