Distribution of grain hardness in Indian wheat varieties and landraces

Rajiv Sharma¹*, Anita Rawat², B.K. Misra and S. Nagarajan

Division of Genetics, Indian Agricultural Research Institute, New Delhi- 110012 India

¹ Present address: International Maize and Wheat Improvement Center (CIMMYT), Afghanistan, Kabul, Afghanistan

² Present address: Department of Biotechnology, Meerut Institute of Engineering and Technology (MIET), Meerut, India.

*Corresponding author: Rajiv Sharma (E-mail: rajivksharma@rediffmail.com)

First two authors have contributed equally.

Abstract

Global trade requires wheat to be classified into hard or soft. The trait is understood to be under the control of ‘ha’ locus on chromosome 5D. Puroindolines a and b explain the biochemical basis of grain hardness. Several methods to measure grain hardness in wheat have been suggested. Single Kernel Characterization System (SKCS) has now come to be globally accepted as the most appropriate method to screen wheat genotypes for hardness. The paper reports the SKCS hardness score of 111 Indian bread wheat varieties, 174 aestivum genetic stocks and a collection of 35 durum germplasm lines and varieties. Classified into five categories ranging from very soft to very hard, 37.5% of all material screened was categorised as very hard with an SKCS score of over 90. Whereas only five per cent of the material screened was grouped as soft or very soft scoring less than 54 SKCS score.

Key words: Indian wheat, SKCS- single kernel characterization system, Triticum aestivum, grain hardness

Introduction

Grain hardness is an important determinant of the end-product quality in wheat. It is the main classification parameter for global wheat trade. Grain hardness or kernel hardness (KH) affects parameters such as milling yield, starch damage and baking properties. Soft wheat has soft endosperm texture, requires less energy for milling and yield smaller particles with less starch damage upon milling than do hard wheat. Soft wheat is suitable for making cakes, cookies, pastries and some types of noodles, whereas, hard wheats are used for making breads and other yeast leavened foods (Morris and Rose, 1996).

Kernel hardness is reported to be under the control of a single locus Ha on short arm of 5D chromosome (Law et al., 1978, Morris, 2002). In addition, loci other than the Ha may also be involved in modifying grain hardness. For example, chromosomes 1A and 6D are also reported to affect grain hardness (Perretant et al.2000). Although Ha locus is referred to as hard but the trait conferred is soft. Biochemical basis of grain hardness suggests that a 15kDa protein, friabilin is present on the surface of water washed soft wheat starch, scarce on the surface of hard wheat starch, and absent on durum (Greenwell and Schofield, 1986). Friabilin was reported to be inherited additively in accordance with Hardness allele dosage (soft kernel texture with abundant friabilin, HaHaHa, hard kernel texture with scarce friabilin, hahaha, along with two intermediate levels of friabilin and hardness, HaHaha and Hahaha) Bettge et al., 1995). Friabilin was later reported to comprise of two polypeptides, puroindoline-a and puroindoline-b (Blochet et al., 1991, 1993). Puroindolines contain a tryptophan rich region that appears to be involved in lipid binding. The genes pin-a, pin-b and Gsp-1 (Grain softness protein-1) have been shown to be tightly linked to the Ha locus on chromosome 5D (Greenwell and Schofield 1989; Giroux and Morris 1997). It is now established that mutations in pin sequences lead to reduced or no friabilin content on water washed wheat starch and the loss of softness (Morris, 2002). The first reported mutation was a single nucleotide base change in Pinb in the codon of Gly-46, converting it into serine (the allele having been named as Pinb-D1b) (Giroux and Morris, 1997). Another mutation, first in Pina was a null allele causing no protein or mRNA transcript (Pina-D1b) (Giroux and Morris, 1998). Presently, over nine mutations have been reported in Pinb and two in Pina. The pin-a gene however, is reported to have a predominant role in controlling wheat kernel hardness as a normal (i.e. wild type) puroindoline a and mutant puroindoline b together soften the grain texture slightly more than a normal puroindoline b in the absence of puroindoline a (Morris 2002).

Good amount of information is available for this trait among the world collections of wheat varieties and genetic resources, however, this information on Indian released cultivars and germplasm lines was lacking mainly because of the lack of a proven and acceptable means of measuring hardness. This study was undertaken to assess the grain hardness level of different released wheat varieties, and a collection of bread and durum germplasm lines.

Materials and Methods

The single-kernel characterization system (SKCS) model 4100 (Perten Instruments North America, Inc., Reno, NV) was designed to score hardness of wheat grains as numerical value based on kernel texture (hardness or softness) characteristics. Hardness score is based on the mean and distribution of various expressions of texture, size, and moisture data generated from crushing a predetermined sample of seeds. In the present study, 300 wheat kernels were taken as sample size. The SKCS is designed to singulate the kernels, weigh them, and then crush them between a toothed rotor and progressively narrowing crescent-shaped gap. The force deformation profile during the crushing of the kernel and the conductivity between the rotor and electrically isolated crescent are measured against time. That information is algorithmically processed to provide the weight, size, moisture, and hardness of the kernel. Processing a 300-kernel sample takes approximately three minutes. Generated reports utilize the mean values and standard deviations of individual kernel data obtained from the 300-kernel sample. Final hardness score is based on the distribution of the data for the individual kernel measurements. Other reported data are kernel size (diameter), moisture content, and weight. A collection of 111 wheat varieties spanning different agroclimatic and growing conditions, 174 germplasm lines and 35 durum varieties and genetic resources were screened (the wheat genetic material was available in Cereal lab, Division of Genetics, IARI).  The wheat material was sown in a one-meter row at IARI research farm at New Delhi and a random sample of 300 grains was used for the experiment under report. Although the SKCS data are not directly amenable to testing the significance of differences among cultivars because of lack of true replications, the very high level of expression of the hardness gene as measured by the SKCS qualitatively separates the soft and hard classes.

Results

Hardness among released bread wheat varieties ranged from very soft to hard types (Table 1). As per the manufacturer’s specifications, genotypes scoring higher than 75 were categorized as hard whereas those scoring less than 35 were grouped as soft type wheat. But earlier wheat with SKCS score of 36 and 39 has been classified as soft (Morris et al. 2001). The SKCS produces a four-class frequency distribution of hardness data for each sample, with class limits of <33, 34–46, 47–59, and >60 (Morris et al. 2001). However, different countries have formulated and adopted their own classification system. Since most of our genotypes fell under hard category, we made three categories for the hard wheat and only two categories for soft type wheat (Table 2). Out of the 111 wheat varieties screened for hardness, an overwhelming majority of over 93% was categorized as hard. A total of 52.3 % scored between 75 and 89 and 18.9% scored over 90, latter being categorized as very hard. The situation was no different in case of aestivum germplasm in which 85.7% scored over 75. In fact over 43% germplasm lines were categorized as very hard, thereby implying that genetic stocks are predominantly of hard type as is indicated by the fact that around 95% of all germplasm lines screened were grouped into hard type. In case of durum wheat, all the lines screened scored over 75 and over 68% scored over 90. This in fact was expected since durums lack D genome that houses the softness conferring ha locus. Over all, 95% of the material screened was grouped into hard type and over 82% of the material scored over 75.

Association analysis (Table 3) revealed that grain weight and grain diameter was negatively associated with grain hardness in all the agroclimatic zones except for grain diameter in PZ. Grain moisture was positively associated with grain hardness in all the zones except in NHZ.

Discussion

The results revealed that most Indian varieties and the genetic stocks were of hard type. Wheat breeders in India, in accordance with the consumer preference have for ages selected for hard wheat. The shine and sound of hard lustrous wheat has been a preference of Indian consumers for making atta, the whole meal flour used in India for making the largely consumed chapati, the Indian flat leavened bread.

For the purpose of wheat cultivation and for official recommendation of commercial varieties, India has been divided into several agroclimatic zones. The major ones are North Western Plains Zone (NWPZ), North Eastern Plains Zone (NEPZ), Central Zone (CZ), Northern Hills Zone (NHZ), and Peninsular Zone (PZ). NWPZ offers longest wheat growth duration of up to 145 days which gets reduced by up to 20 days in NEPZ, remains around 100 days in CZ and is reduced further and hovers around 85-90 days in PZ. NHZ is unique because of being a region of low temperatures and high humidity even at the time of crop maturity.

The association of hardness with the other three parameters was also analyzed. Hardness was found to be negatively correlated with the kernel weight.  This indicated that higher kernel weight correlated with a lower SKCS index (Nadolska-Orczyk 2009). SKCS grain hardness was negatively influenced by average grain weight and grain diameter (Paynter and Portmann, 2005). A negative association of grain hardness with weight and diameter was observed (Table 3) in the present study too. The association of hardness with the other three parameters remained almost unchanged among the varieties suitable for different agroclimatic conditions except for the two unique climate zones of Northern Hills Zone and Peninsular Zone (Table 3). Hardness and grain moisture showed positive association in all the zones except NHZ in which it was negative. This is perhaps because of low temperatures and high humidity prevailing even at maturity stage so that only those varieties were hard which retained minimum of moisture, whereas in other zones the minimum of moisture that grains retained did not interfere with the hardness and thus even showed positive association with hardness. This association was maximum (0.77) in the most productive NWPZ. The other interesting deviation was observed among the varieties of PZ in which hardness showed a positive association with grain diameter as opposed to being negative in all other zones. Perhaps in other zones, increase in diameter brings along some degree of softness whereas in PZ, the zone with shortest growing duration of about 85-90 days only and relatively higher temperature regimes throughout the growing period, grain size is already a limiting factor and under such conditions genetical potential to have increased diameter goes well and retain hardness as well. On the contrary, a smaller diameter under such conditions would become too small to retain hardness.

The frequency distribution of the SKCS hardness data of all wheats suggested that all were pure and none of them was a mixture of soft and hard types. The distribution of hardness among zones was on expected lines (Table 4). Except for NHZ, 80-100% of the released varieties fell under top two hard categories. In the NHZ over 60% of varieties scored less than 75 whereas in all other zones, over 80% varieties scored more than 75. The reduced grain hardness among the varieties of NHZ is because of the climatic requirements of the zone that favours only such varieties to be successful high yielder in the zone that mature under low temperature high humidity regimes. The only varieties showing an SKCS score less than 34 and falling under the category of very soft were Lerma rojo (34) and Safed Lerma (27) released during 1965 in the early phase of green revolution in the country. The trend among Indian wheat cultivars is clearly of hard type (Table 4). Soft varieties can be developed by increasing the functional copy number of the puroindolines which can impact grain softness in bread wheat.

The same has perhaps been achieved by wheat breeders who themselves as consumers knew that hard type yielded better atta that gave preferred chapati quality and higher flour yield.

Acknowledgements

The authors would like to thank Department of Biotechnology (DBT), India for providing financial assistance.

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