Agronomic characterization, Rht genotyping and high molecular weight glutenin subunit profiling of oligo derived lines in warm environment

Suman Sud and Suresh G. Bhagwat

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, India

Corresponding author: Suman Sud

E-mail: sumansud@barc.gov.in

Abstract

Poor biomass and lower spikelet number often contribute to the reduction in yield in high temperature environment.  Fourteen Oligo derivatives derived from a cross of (Oligoculm wheat x Kundan) x Selection 212 were grown in warm environment and were scored for 13 agronomic traits. Significant differences were observed among the genotypes for all the traits. Oligo derivatives were found to be superior to check cultivars for spikelet number per spike, flag leaf blade area, biomass per plant and per meter row length. However, for harvest index the oligo derivatives were inferior. The oligo derivatives were responsive to externally applied gibberellin and lacked amplification with perfect markers indicating absence of major dwarfing genes. There was variation for HMW subunits of glutenin profile. The study showed that the high biomass and spikelet number under warm environment conditions could be utilized for genetic improvement through incorporation of RhtB1b and RhtD1b.

Key Words: Allele specific primers, Dwarfing genes, Flag leaf blade, Gibberellin insensitivity, HMW glutenin, Oligo derivatives

Introduction

Ideally wheat is cultivated in cool climate, however, increasing demand for the grain has spread the crop in warmer areas. Also, cropping season in cooler areas is interrupted by short periods of higher temperature. Heat stress during early phase of growth affects tillering, number of spikelets per spike and biomass production. During grain filling period higher temperature affects grain development and results in lower grain weight and consequently grain yield (Ruwali and Bhawasar 1998). Atmson and Jacob (1977) reported a new form of wheat, which showed “Gigas” features with limited tillering. These wheats have large spikes with a maximum of 30 spikelets, robust and vigorous vegetative parts and thick large parts of spike. Tillering in these wheats is under thermoperiodic control (Batten 1985). The long spike, large grain and high vigour of the oligoculm wheat makes it an attractive resource for enhancing yield components. Oligoculm derivatives need to be evaluated for their performance in different environments. It is necessary to check if the higher yield components are expressed in warmer environments so that these can be used for yield improvement. It is also necessary to understand the status of certain important genes related to growth habit and quality for proper utilization. The objective of present investigation was to evaluate field performance of Oligo derivatives from a cross (Oligoculm wheat x Kundan) x Selection 212 under high temperature conditions, and analysis for Rht gene(s) and high molecular weight subunits of glutenin.

Materials and Methods

Plant materials

Fourteen Oligo derivatives obtained from a cross of (Oligoculm wheat x Kundan) x Selection 212 were used in the study.

Field Experiment

Oligoculm wheat derivatives and checks Kalyan Sona and HD2189 were evaluated under field conditions at Trombay location using randomized block design with five replications. Seed rate was 100 kg per hectare and 120 kg N per hectare was applied. Standard agronomic practices were followed. The diurnal temperature profile indicated that the crop was continuously exposed to temperature more than 25^oC for large part of the day (data not shown).

Rht genotyping

DNA was isolated from bulk of five plants according to method of Saghai-maroof et al. (1986). The alleles/genes RhtB1b, RhtD1b, RhtB1a and RhtD1a were amplified using allele specific primers reported by Ellis et al. (2002). The reaction was performed in 25μl containing 10pmoles of each primer, 100μM of dNTPs, and 2mM of MgCl₂, 1unit of Taq DNA polymerase and 100ng of template DNA. PCR amplification was carried out in an Eppendorf Mastercycler as per Ellis et al. (2002). Polymorphism survey with primer Xgwm261 for genotyping GA₃sensitive gene Rht8 was carried out according to Roder et al. (1999). The PCR products were separated on a 2% agarose gel prepared in 1xTBE buffer, visualized under UV light after ethidium bromide staining and photographed (Sambrook et al.1989).

Gibberellin sensitivity test

Twenty seedlings of each Oligo derivative were grown in 10^-4M gibberellic acid (GA3). Control seedlings were grown in water.  Seedling height was recorded on 12th day. Student’s t-test was performed to evaluate whether the differences in height of seedlings grown in GA3 and water were significant. Cultivars C-306 and Ajantha both lacking RhtB1b and RhtD1b were used as checks.

HMW-Glutenin subunit profiling

The high molecular weight glutenin subunit profile of Oligo derivatives was resolved on 10% SDS-PAGE according to Bhagwat and Bhatia (1993). The nomenclature of subunits was done according to Payne and Lawrence (1983).

Culm length and leaf area

The Oligo derivatives were grown in field and the culm length of tallest tiller from the ground to the base of spike was recorded. Data on ten plants from each derivative were taken. Flag leaf blade area was estimated on four fully developed flag leaves randomly chosen from the plot. Leaf blade length and maximum leaf width were measured and area was estimated according to Quarry and Jones (1977).

Measurements of yield and yield components

Each genotype was grown in one meter row in completely randomized design. The data on each trait was average of observations recorded on five plants of each genotype. Data for yield and yield components including plant height (cm), leaf area (cm²), spike length (cm), spikelet number per spike, grain number per spike, grain yield per spike (g), hundred kernel weight (g), tillers per plant, biomass per meter row length (g) were recorded at maturity.

Statistical analysis

Analysis of variance for testing genotypic differences for all the characters was performed using Plant Breeding and Genetics package of Windostat Advanced Level Statistical Software, version 7.10 (Indostat Services, Hyderabad, India).

Results

Yield components and yield

The mean values for yield components measured from Oligo derivatives and checks are presented in Table 1. All Oligo derivatives were significantly higher in plant height than semi dwarf checks HD2189 (RhtB1b) and Kalyan Sona (RhtB1b). Flag leaf area recorded 10 days after flag leaf emergence (Table 1) showed that all Oligo derivatives had larger flag leaf blade than both the checks. All Oligo derivatives had higher number of spikelets than checks HD2189 and Kalyan Sona. Eight Oligo derivatives: Oligo 2 (50.0), Oligo 3 (50.2), Oligo 6 (51.2), Oligo 7 (55.2), Oligo 8 (53.0), Oligo 10 (51.0), Oligo 13 (52.3) and Oligo 14 (53.0) had grain number per spike significantly higher than HD2189 (43.1) (Table 1).  Twelve oligo derivatives had grain yield per spike significantly higher than Kalyan Sona and six had significantly higher than both the checks Kalyan Sona (1.42g) and HD2189 (1.78g). Six of the derivatives viz; Oligo 1 (4.05g), Oligo 2 (4.99g), Oligo 3 (4.39g), Oligo 7 (4.06g), Oligo 13 (4.44g), and Oligo 14 (4.05g) had hundred kernel weight higher than checks HD2189 (3.8g) and Kalyan Sona (3.2g). All Oligo derivatives had tiller number per plant significantly lower than both the check varieties.

Eight derivatives Oligo 1 (24.5g),  Oligo 3 (25.8g), Oligo 5 (28.0g), Oligo 6 (24.2g), Oligo 8 (29.4g), Oligo 10 (28.5g), Oligo 11  (25.2g), Oligo 13  (26.3g) had biomass significantly higher than checks HD2189 (21.0g) and Kalyan Sona (17.0g). Eleven Oligo derivatives Oligo 1 (376.7g), Oligo 2 (305.4g), Oligo 3 (303.6g), Oligo 4 (287.2g), Oligo 5 (325.4g), Oligo 6 (306.6g), Oligo 7 (284.8g), Oligo 8 (285.0g), Oligo 9 (339.4g), and Oligo 10 (289.7g), Oligo 12 (308.0g) had biomass per metre significantly higher than check HD2189 (232.9g) and all derivatives had biomass per metre significantly more than variety Kalyan Sona. Oligo derivatives yielded poorly on area basis. All Oligo derivatives had yield lower than check variety HD2189.

Response to gibberellin

The response of seedlings to externally applied GA3 showed that there was about 14-27% increase in seedling height for the Oligo derivatives over the seedling grown in water (Table 2). The check varieties C-306 and Ajantha showed 24.7 and 23.5% increase in height in response to GA3. 

Rht status

The Oligo derivatives showed amplification of a 237-bp and 264-bp band with primer pair BF-WR1 and DF2-WR2 corresponding to wild type allele RhtB1a and RhtD1a (Fig. 1). The check variety HD2189 which carries RhtB1b and RhtD1a showed amplification of 237-bp and 264-bp band with the corresponding perfect marker (BF-MR1, DF2-WR2) while the variety Sonalika carries RhtB1a and RhtD1b showed presence of 237-bp and 254-bp band with primer pair BF-WR1 and DF-MR2 (Table 2). Two out of fourteen Oligo derivatives carried 192-bp allele at the Xgwm261 locus and the rest showed presence of 165-bp allele (Fig. 2). 

HMW subunit profile

The Oligo derivatives were found to vary in their HMW subunit composition (Fig. 3). All the Oligo derivatives showed the presence of subunits GluD1d coded subunits 2+12. Oligo 2 and 3 showed the presence of subunit 1 (GluA1a) and the rest showed the presence of null allele (GluA1c) of the GluA1 locus. At the GluB1 locus Oligo 1 and 4 to12 showed the presence of subunits 13+16 (Glu B1f), Oligo 2, 13, 14 showed 7+8 (Glu B1b) and Oligo 3 showed the presence of subunit 7+9 (Glu B1c).

Discussion

Improvement of yield in wheat is constant requirement for which new resources are required. Yield in unfavorable environment can be improved if a donor with ability to express the superior trait in the unfavorable environment is available. The Oligo derivatives have their vigour derived from “Gigas”. In warmer environment plant vigour is often the most limiting factor (Fischer 1984). It is also observed that the tiller number and spikelet number per spike is affected by elevated temperature (Johnson and Kanemasu 1983). The Oligo derivatives lacked major dwarfing gene as seen by their responsiveness to gibberellin and by use of perfect markers, as a result these were taller than the semi-dwarf check varieties. Biomasses per plant and per meter were significantly higher in many of the Oligoculm derivatives indicating their superior ability to accumulate biomass. However, harvest index in the derivatives was generally lower. Introduction of a semi-dwarfing gene would improve the partitioning of dry matter in grain. Grain yield per spike was higher in ten derivatives while on area basis the derivatives were poorer. The ability of the derivatives could be improved by introduction of semi-dwarfing gene(s). As a plant population the performance is superior for the semi-dwarfs as compared to the tall stature varieties. The spikelet number per spike and flag leaf area were significantly higher than the checks, hence the derivatives can be used as a source of these yield components for improvement in warm environment. Gigas genotypes and Oligo derivatives were found to produce stunted phenotype in short day and low temperature conditions (Atmson and Jacob 1977). Under warm conditions in which the experiments were carried out the expression was normal and hence it can be used as a donor of the traits. The Oligo derivatives however lacked tolerance to heat stress as indicated by their rapid leaf senescence. The limited number of tillers in Oligo lines could be a useful trait in warm environment as it avoids formation of non-productive late tillers. The Glu-1 scores of the Oligo derivatives ranged between six to eight indicating there is scope to choose among the lines according to the end use requirement. 

The Oligo derivatives were developed from a cross (Oligoculm wheat x Kundan) x Selection 212 (K.P. Singh, 2002, personal communication). Kundan is a semi dwarf cultivar known to carry RhtD1b, however all the derivatives were tall. No amplification was observed either for RhtB1b and RhtD1b specific primers in Oligo derived lines; however, the wild type alleles (RhtB1a, RhtD1a) were amplified by the specific primers (Fig. 1). The culm length and GA3 sensitivity data support the conclusion that the Oligo derived lines were devoid of the major semi-dwarfing genes RhtB1b and RhtD1b. The Norin-10 genes are known to improve the partitioning of dry matter and also improve the performance as a population (Gent and Kiyomoto 1998). Introducing these genes in the oligo derivatives could improve their agronomic performance. Introgression of dwarfing genes (RhtB1b and RhtD1b) into wheat cultivars during green revolution led to high yields, lodging resistance, improved assimilates partioning into grain (Peng et al. 1999). No height reduction was found in presence 192-bp allele as compared to genotypes with 165-bp allele. This indicated that height neutral allele was present in these lines which was not associated with Rht8 gene.

Flag leaf is single most important source of photosynthetic assimilates to the grain in wheat (Lupton 1973) with about 40% contribution to final grain weight (Ibrahim and Abo Elenin 1977). The large flag leaf blades of Oligo derivatives Oligo 2 (45.7cm²), Oligo 4 (47.0 cm²), Oligo 5 (41.0cm²), Oligo 6 (40.3cm² ), Oligo 11 (40.7cm²), Oligo 12 (40.3cm²) and Oligo 14 (43.8cm ²) indicated that these can serve as donor for larger flag leaf area. Heat stress affects grain development and also leads to severe reduction in leaf area, spikelets per spike, floret fertility, grains per spike and spike dry weight resulting into poor sink capacity and future source capability of plant (Sharma and Tandon 1977). Biomass or total plant weight is an important criterion for selecting for higher yield under heat stress (Reynolds et al.1994). The data showed that some of the lines had poor Glu-1 score, which would result in poor dough strength. The lines with high Glu-1 scores would be expected to give stronger dough. The variability in Glu-1 scores offers scope to select for strong or weak dough as per requirement. On the other hand it may be necessary to alter HMW subunit composition of the best lines for appropriate dough properties.

Conclusions

The oligo derivatives were superior to check varieties in some yield components such as hundred kernel weight, spikelet number per spike, grain yield per spike, flag leaf blade area and biomass per meter row length in warmer environment and could be a useful resource. There was variation in the HMW subunit pattern. Recombining the superior yield components and semi dwarf habit with improved thermotolerance would be necessary to obtain high yielding types.

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