Seed quality and the effect of introducing Cicer echinospermum to improve disease and pest resistance in desi chickpea

The utilisation and consumption of food crops for human nutrition demand acceptable seed quality traits to enable efficient processing into food products. Desi chickpea (Cicer arietinum L.) is a staple food of the Indian subcontinent, and domestication has led to cultivars with seeds that are an acceptable size, colour, and shape, easy to mill, and quick to hydrate and cook. Domestication has also severely restricted the gene pool, so breeders are looking to “wild” chickpea relatives as sources of novel genes that may provide agronomic benefits such as disease and pest resistance. Cicer echinospermum (a“wild” relative of cultivated chickpea with black, echinate seeds) was used in the breeding programme to introgress phytophthora root rot and root‐lesion nematode resistance genes into adapted C. arietinum backgrounds. The resulting C. echinospermum derivative lines were compared with commercial desi cultivars to examine any effects on seed quality attributes. The C. echinospermum derivatives had similar visual seed characteristics to the C. arietinum cultivars with, on average, lower milling performance and quicker cooking times; however, a few individual derivative lines met or exceeded the average cultivar milling performance. This paper shows the quality variation within the C. echinospermum derivatives, compares them with commercial desi cultivars, and confirms their potential to improve disease resistance whilst retaining the basic seed quality traits important for commercialisation and exporting new cultivars.

gene pool, so breeders are looking to "wild" chickpea relatives as sources of novel genes that may provide agronomic benefits such as disease and pest resistance. Cicer echinospermum (a"wild" relative of cultivated chickpea with black, echinate seeds) was used in the breeding programme to introgress phytophthora root rot and rootlesion nematode resistance genes into adapted C. arietinum backgrounds. The resulting C. echinospermum derivative lines were compared with commercial desi cultivars to examine any effects on seed quality attributes. The C. echinospermum derivatives had similar visual seed characteristics to the C. arietinum cultivars with, on average, lower milling performance and quicker cooking times; however, a few individual derivative lines met or exceeded the average cultivar milling performance. This paper shows the quality variation within the C. echinospermum derivatives, compares them with commercial desi cultivars, and confirms their potential to improve disease resistance whilst retaining the basic seed quality traits important for commercialisation and exporting new cultivars.

K E Y W O R D S
chickpea, Cicer echinospermum, cooking, domestication, milling, seed quality traits, wild species 1 | INTRODUCTION Chickpea (Cicer arietinum L.) is the second largest pulse crop in the world and is particularly important in the Indian subcontinent, Central and West Asia, and North Africa as a source of protein, carbohydrate, vitamins, and minerals. The species can be divided into two main groups on the basis of seed type. Kabuli chickpeas will not be discussed in this paper. Desi chickpeas generally have a small, angular shaped seed with a thick, darker coloured testa (seed coat). Some desi chickpeas are cooked whole or roasted, but most are decorticated and split to produce dhal, and the dhal can be further processed into flour (besan).
Australia is the largest exporter of chickpea, producing and exporting 960,000 tonnes annually, averaged over 5 years: (FAO, 2019. The industry is concentrated in the north-east region of Australia (northern New South Wales and Queensland) where production is almost exclusively of the desi type. The main biotic production constraints for chickpea in northeastern Australia are ascochyta blight (Ascochyta rabiei) and phytophthora root rot (Phytophthora medicaginis). Both diseases are capable of up to 100% mortality under conducive conditions (Du, Zhao, Raju, Davies, & Trethowan, 2012;Knights, Southwell, Schwinghamer, & Harden, 2008). Moderate levels of resistance to ascochyta blight has now been incorporated in C. arietinum cultivars and breeding lines, although there is some evidence that the pathogen may be adapting to become a threat to this resistance (Mehmood et al., 2017). Only moderate resistance to phytophthora root rot (P. medicaginis) had been identified in the cultigen (Brinsmead, Rettke, Irwin, Ryley, & Langdon, 1985). The presence of root-lesion nematodes (Pratylenchus thornei and Pratylenchus neglectus) can also constrain the production of chickpea, and they are widely distributed across the Australian grain-growing regions (Hollaway, Vanstone, Nobbs, Smith, & Brown, 2008;Reen, Mumford, & Thompson, 2019;Riley & Kelly, 2002;Riley & Wouts, 2001;Thompson et al., 2010).
The seeds of these "wild" relatives are, however, very different to domesticated chickpeas visually, and their quality attributes are largely unknown. The seeds of C. echinospermum, for example, are dark brown to black with a thick and echinate testa that is resistant to water absorption (Robertson, Ocampo, & Singh, 1997). In comparison, domesticated C. arietinum chickpea cultivars have smooth, yellow-brown testa and are generally easily hydrated in water. Backcrossing of the "wild" relatives into C. arietinum backgrounds that have acceptable seed characteristics and are adapted to local conditions is therefore necessary to achieve cultivars that can be successfully grown and marketed.
The Australian chickpea breeding programme targeted several "wild" relatives of C. arietinum as alternative sources of improved phytophthora root rot and root-lesion nematode resistance. The C. echinospermum accessions generated showed superior resistance to phytophthora root rot  and root-lesion nematodes (Thompson et al., 2011); hence, a backcrossing programme was developed to introgress disease resistance genes into adapted Australian C. arietinum backgrounds. The resulting C. echinospermum derivative lines examined in this study have a theoretical 25% C. echinospermum in their genome. They have similar yields to C. arietinum cultivars and no agronomic penalties (Knights etal., 2008).
This paper reports an investigation into the quality characteristics of these C. echinospermum derivatives compared with Australian C. arietinum desi cultivars.
The weight of 100 unsized seeds was measured and recorded.
Seed was then sieved into sizes according to APQ-103 Australian Pulse Quality Laboratory Manual (Burridge, Hensing, & Petterson, 2001). Seed weight is indicative of seed size. The size in majority (either 6 -7 mm or 7 -8 mm) was subsequently used for all seed quality analyses.
Seed coat content was determined by peeling the testa off seeds soaked overnight, drying both the testa and cotyledons (50 C oven until no weight change is recorded), and the result expressed as a percentage of the total dry seed weight.

| Water absorption measurements
Hydration properties were determined according to Wood and Harden (2006). Briefly, the weight and volume of seeds were recorded 0,2, 4.5, 7, and 24hr after immersion in distilled water. Unhydratable (hard) seeds are those seeds that do not imbibe water and were recorded as a percentage (by weight of the initial dry sample weight).
Plots of seed weight versus soaking time were used with a threeparameter model y =I(1+ α (1− β x )), where y = weight during soaking; I = initial weight, α = maximum weight increase; 1− β = rate of imbibition; and x = hours soaking. The asymptote of the curve, I + α, is a reliable estimator of maximum hydration, and α is designated as H max . The shape of the curve, β, determines how quickly the curve nears the asymptote (rate of imbibition), with 1 − β being the proportion of the total weight gain achieved after 1 hr (designated as H rate ).

| Milling quality and dhal colour
Unconditioned seed from the 6-7 mm and 7-8 mm of size classes was separately milled using the Sheller component of an SK Engineering Mill. The sheller gap was set to the size of seed to ensure optimum splitting results (Wood & Malcolmson, 2011). Dehulling efficiency (DE x ) and splitting yield (SY x ) for seed of x-mm size class were calculated according to Wood, Knights, & Harden (2008) The resulting dhal colour was measured as described for whole seeds (above).

| Cooking
Cooking time of whole seeds, soaked overnight in distilled water, was measured by the texture analysis method APQ-102.2 Australian Pulse Quality Laboratory Manual (Burridge, Hensing, & Petterson, 2001), modified slightly by squashing nine desi seeds rather than an approximate 5 g of seed to enhance direct comparisons between samples.
Cooking time of dhal was also measured by this method; however, the dhal was not soaked before cooking, and 25 g of cooked dhal was placed in a custom-built Perspex dish of 50 mm inner diameter and 10 mm high side walls for the test. For both methods, the texture analyser (TA-XT , Perten) cross-head was fitted with a 40 mm diameter 2 Perspex disc, descending at a test speed of 2.0 mms −1 with a load cell of 25 kg. The force (in Newtons) required to squash the seed or dhal sample to 75% deformation was recorded and is an indication of the time the sample would require, with quicker cooking samples having a lower force.

| Comparison between the conventional and C. echinospermum derivative groups
The C. echinospermum parent had black seeds with a thick and echinate testa, yet it was possible to recover seed of derivative lines that had testa similar in visual smoothness, colour, and thickness to the C. arietinum cultivars and did not show any of the "primitive" features of their C. echinospermum parent (Figure 1). From a visual perspective, the seeds of these derivative lines would be acceptable for export and human consumption markets.
The testa of the C. echinospermum parent is also known to be resistant to water absorption (i.e., hard seeded; strong seed dormancy). Testing for the significance of the random term genotype showed that genotype was significant for all the quality parameters indicating genotypic variation within the two groups. As a whole, the C. echinospermum derivatives did not differ significantly (5% probability level) from the C. arietinum cultivars for many of the quality parameters (Tables 1 and 2). There was, on average, no significant difference in the groups predicted means for seed size, testa content, or colour, dhal colour, or parameters related to water imbibition (H max or H rate ).
However, the C. echinospermum derivative group did produce seed that was, on average, significantly more difficult to dehull (DE) and split (SY) than the C. arietinum cultivars (a disadvantage), and the resulting dhal had a significantly shorter cooking time requirement (an a dvantage). Table 3 shows the range in genotype predicted means within the C. echinospermum derivative group and C. arietinum cultivar group for each quality parameter. There was little difference in the ranges between the groups for rate of imbibition (H rate ), seed colour (brightness L* and yellowness b*), dhal colour (brightness L* and redness a*), and dhal cooking time. The derivative group showed a tightening of the range compared with the cultivar group for 100 seed weight and dehulling efficiency, suggesting that the C. echinospermum parentage is reducing the variability of these traits.
In comparison, the derivative group showed an expansion in predicted value ranges for testa content, seed colour (yellowness b*), splitting yield (for the 6-mm size class), maximum hydration (H max ), and the cooking time of whole seeds, suggesting that the C. echinospermum parentage is contributing to a broadening of variability in these traits.
Both groups showed a lower splitting yield for the 7 -8 mm size class compared with the smaller 6 -7 mm size class (Table 1), in agreement with previous reports that, of the two most abundant seed size classes of a genotype, the smaller seeds are generally easier to split (Agrawal & Singh, 2003;Wood, Knights, & Harden, 2008). Although the derivative group, on average, had poorer milling performance than the C. arietinum cultivars, there was a significant amount of variation, and a few of the individual C. echinospermum derivatives met or exceeded the average C. arietinum cultivar dehulling efficiency and/or splitting yield (Figure 2). An easy-to-mill C. echinospermum derivative was identified and studied in relation to this trait (Wood et al., 2008;Wood, Knights, Campbell, & Choct, 2014a, 2014b, 2014c; however, this is the first time a wider group of C. echinospermum derivative lines has been examined more broadly over numerous years and sites. The C. echinospermum derivative group predicted means show reduced milling yields (both DE and SY) compared with the desi cultivar group (Figure 2). There was also a large amount of variation in softening of cooked seeds and dhal from individual C. echinospermum derivative lines. Dhal from all the individual C. echinospermum derivatives, except one, had predicted means that were softer after cooking (i.e., quicker cooking) than the respective C. arietinum cultivar group mean ( Figure 3). Figure 4 shows both the raw data and the predicted means for each genotype within each group for the three quality parameters where the C. echinospermum derivative group and the C. arietinum cultivar group means were significantly different. Predicted means for each genotype, averaged over environment, clearly reduce the variability compared with the raw data. However, it is apparent that both the actual data and the predicted means of genotypes within each group show significant differences between the groups for milling performance (DE and SY) and dhal cooking.
It is unclear why the derivative group was unable to be dehulled and split as easily as the cultivar group. The lower milling quality of the derivatives could not be attributed to a thicker testa, inherited from the C. echinospermum parent (Murray, 1984), because there was no significant difference between the average testa content of the C. echinospermum derivatives and the C. arietinum cultivars (Table 1). It may be due to differences in the testa (composition or morphology) because the testa of the parent C. echinospermum is very different, at least in appearance, to cultivated desi chickpeas. Wood et al. (2014a) examined the broad chemical composition of the testa of three desi genotypes, one being a C. echinospermum derivative, and although this line had some significant differences in composition, it was often within the values of the other two desi genotypes. However, its testa did contain significantly less insoluble nonstarch polysaccharides (NSPs) and more soluble NSPs (Wood et al., 2014a), as well as some differences in the testa NSP composition (Wood etal., 2014c). Assuming that this derivative line is representative of the C. echinospermum derivative group, a reduced level of insoluble NSPs may indicate that the testa is less brittle and more pliable; these characteristics would reduce the cracking and brittleness of the testa and therefore make its removal more difficult. Deeper investigation of the minerals in the testa also revealed a very different composition for the derivative line (Wood et al., 2014b). It was significantly higher in total ash, iron (Fe), potassium (K), and manganese (Mn)and significantly lower in boron (B), calcium (Ca), magnesium (Mg), sodium (Na), and zinc (Zn)than the other two desi genotypes examined. It remains unclear, however, F I G U R E 2 The Cicer echinospermum derivatives were generally not as efficiently dehulled or split during milling as the Cicer arietinum cultivar group.
Horizontal lines indicate the predicted means of the C. arietinum cultivar group for dehulling efficiency (dashed) and splitting yield (solid). Bars show predicted means for each C. echinospermum derivative for dehulling efficiency (white) and splitting yield (blue) F I G U R E 3 The Cicer echinospermum derivative dhal and seeds were softer (lower force required to squash the sample after 25 min of cooking) than the Cicer arietinum cultivar group.
Horizontal lines indicate the predicted mean of the C. arietinum cultivar group for whole seed cooking (dashed) and dhal cooking (solid). Bars show predicted means for each C. echinospermum derivative for whole seed cooking (white) and dhal cooking (blue) whether or how these potential differences in testa composition may contribute to a reduced milling performance.
Another possible reason for the differences in milling performance, particularly SY, is tighter adhesion between the two cotyledons in C. echinospermum derivatives so that splitting them cleanly becomes more difficult. A C. echinospermum derivative line was previously found to contain significantly more soluble arabinose in the intermediate fractions abraded from the surface of dhal that appears to be associated with more difficult-to-mill samples via cell wall adherence and more boron potentially acting as bridging agents for cell wall stabilisation between dhal (Wood et al., 2014b(Wood et al., , 2014c. In this case, preconditioning might improve the milling performance of the C. echinospermum derivatives in a commercial milling plant and the dhal quality by reducing abrasion. If not, the lower dhal extraction of C. echinospermum derivatives would have adverse economic implications for millers, although the observed differences in milling quality were small relative to the genotypic variation existing within the cultigen. Conversely, the anticipated reduction in seed "shatter" (splitting) caused by weather damage and/or low moisture contents would be advantageous to farmers yield and profitability.
Although there was no difference between the groups for whole seed cooking, the shorter cooking times required for C. echinospermum derivative dhal would be an advantage over current C. arietinum cultivars. Poorer milling performance leads to an increase in abrasion of cotyledons during the milling process, which might facilitate more rapid water absorption and speeding up the cooking process. An alternative explanation for the quicker cooking is potential differences in the cotyledon carbohydrate and/or protein chemistry of the C. echinospermum derivatives. Wood et al. (2014aWood et al. ( , 2014bWood et al. ( , 2014c examined the composition of one C. echinospermum derivative and two desi chickpea genotypes and found that cotyledons of the derivative line contained similar protein content (although higher amounts of several of the most abundant amino acids: glutamic acid, leucine, and lysine), similar starch content, and significantly higher amounts of total NSP content, particularly arabinose (in both soluble and insoluble fractions). Arabinose (2,3,5-Ara residues, likely derived from arabinan) were found to be present in higher levels in dhal of a fast-cooking desi cultivar compared with a slow-cooking cultivar (Wood et al., 2018). More research is required to confirm whether these isolated associations are representative of a wider relationship (i.e., whether C. echinospermum derivatives have higher dhal arabinose content leading to quicker cooking).

| Quality attribute relationships
Plots of paired data for the quality attributes were also examined and confirmed several expected relationships: (a) Testa content There were also some expected correlations that did not eventuate. First, the parameters estimating cooking times for whole seeds and dhal showed little relationship to each other (R= .12 to .23; Figure 5a). This could be due to the confounding influence of seed size on the cooking time parameter for whole seeds as well as the involvement of the testa, whereas the dhal cooking time parameter involved only the cotyledon softening and without bias from size. Second, the maximum hydration was not related to the parameters estimating cooking times (R= −.05 and R= .20 for seeds and dhal, respectively; Figure 5b). Many researchers have previously found an association with hydration capacity (H max ) and whole seed cooking time in chickpea (Badshah, Ahmad, Aurangzeb, Mohammad, & Khan, 1987;Kaur, Singh, & Sodhi, 2005), yellow pea (Wang, Daun, & Malcolmson, 2003), dry bean (Ercan, Atli, Koeksel, & Dag, 1994), and mungbean (Antu, Sudesh, & Yadav, 2006 (2016) found that red cowpeas took longer to cook than black cowpeas, in agreement with our relationship for desi chickpeas; however, their red cowpeas were also significantly larger than the black, which was likely the main contributing factor to their longer cooking times. A more detailed investigation into the potential role of seed pigments in the seed coat with water imbibition and cooking times may be warranted.

| Effect of the environment
The environment was also shown to have an impact on the group means for some of the quality attributes (

| CONCLUSION
Chickpea breeding programmes are increasingly seeking to reintroduce wild genetic diversity into domesticated crops to improve tolerance to pests and diseases. It is important to understand the potential ramifications of this on seed quality traits. It was possible to introgress C. echinospermum genetics into adapted C. arietinum backgrounds and retain the visual characteristics of desi chickpea seeds without unhydratable hard seeds in most trials and years, although three derivatives showed hard seeds at two sites.
In general, the seed quality attributes of the C. echinospermum derivatives investigated were comparable with C. arietinum released cultivars, although considerable variation was evident within both groups across environments for many traits. The C. echinospermum derivative group was, on average, more difficult to mill (dehulling and splitting), whereas the resulting dhal had a significantly shorter cooking time requirement than the commercial cultivar group.
Despite poorer milling performance, several of the derivative lines met or exceeded the cultivar average for DE and SY, so it is possible to select better performing derivative lines from the group.
Inclusion of C. echinospermum genetics lead to a broadening in variability for testa content and yellowness, SY, H max , and whole seed cooking and a contraction in variability for seed weight and DE.
Hydration capacity (H max ) was not correlated with whole seed cooking and should not be used as a predictor trait in breeding programmes. The potential of C. echinospermum derivative lines to introduce phytophthora root rot and root-lesion nematode resistance into domesticated chickpea whilst maintaining seed quality has now been confirmed. Individual lines of C. echinospermum possess not only a wide diversity of resistance to pests and diseases but also a wide diversity in seed quality attributes. Continued routine screening for quality traits within the breeding programme will be able to identify those derivate lines with suitable qualities for export and primary processing for progression to cultivar release.
(a) (b) F I G U R E 5 Scatter plots of raw data showing no associations between (a) cooking of whole seeds and dhal, and (b) maximum hydration and whole seed cooking Chemical composition and sensory analysis now need to be undertaken on a wider range of C. echinospermum derivative lines to confirm food processing, nutritional, and consumer acceptance.