Hossain, S., Panozzo, J. F., Pittock, C. and Ford, R. 2011. Quantitative trait loci analysis of seed coat color components for selective breeding in chickpea (Cicer arietinumL.). Can. J. Plant Sci. 91: 49–55. Chickpea (Cicer arietinum L.) is an annual grain legume, grown worldwide for human consumption with the potential to attract premium prices in markets such as India, Bangladesh and southern Asia. The ability to accurately select for seed coat color, an important export quality trait, would greatly benefit chickpea breeding programs. In order to determine the major genomic loci governing the color trait, the color components of CIE L* (luminance), CIE a* (red/green color) and CIE b* (blue/yellow color), C* (chroma or saturation of the color) and h° (hue or purity of the color) were mapped, and associated molecular markers were identified. A linkage map was constructed with 80 SSR markers distributed over 10 linkage groups at an average marker density of 2.8 cM. Two major quantitative trait loci (QTL), which accounted for up to 36 and 49% of the genetic variance and several smaller genetic effects were determined to govern the color components. These were consistent across two differing environments. Once validated, the markers that are close to and flanking these QTL and significantly associated with the minor gene effects will be useful in future color selective breeding programs.

Chickpea (Cicer arietinum L.) is an annual diploid (2n= 2x= 16) grain legume, grown worldwide for human consumption. Selection for variation in the physical seed characters of chickpea will enable future strategic breeding of varieties with the potential to attract premium prices in overseas markets. Seed shape is therefore a major current breeding objective, for which an understanding of the genetics of inheritance is required. For this, two recombinant inbred line (RIL) populations derived from intraspecific crosses of a kabuli-type (S95362; light cream colour) crossed to two desi-types (Howzat and ICC3996; medium tan and dark tan colour, respectively) were studied. In order to discretely characterize seed shape, a new Roundness Index (RI) tool was developed, calculated from the ratio of two seed size indices (SSI). The genetic parameters estimated were genotypic and total phenotypic variance for RI, genotype vs environment interaction and broad sense heritability. The low genotype x environment interaction (<9% of total variation for both populations) and high magnitude of heritability suggested the environmental stability of the seed shape trait. Segregation ratios for different seed shape among the RIL populations indicated control of seed shape under two genes. Subsequently, one putative quantitative trait loci (QTL) was identified on linkage group LG 2 between markers TA110-TA27 and accounted for 9% of the phenotypic variance. One QTL for stem colour and flowering time also detected on LG 2 between markers TR58-TR19 (explained 16% phenotypic variance) and LG 3 between markers TS19-TR56 (explained 23% phenotypic variance) respectively.

Chickpea is valued for its nutritive seed composition which is high in protein content and used increasingly as a substitute for animal protein. High quality seed has the potential to attract premium prices. Hence, the breeding of desirable quality traits, including seed size for desi and kabuli-types, is of major importance. For this, two RIL populations derived from intraspecific crosses of a kabuli-type (S95362; light cream colour) crossed to two desi-types (Howzat and ICC3996; medium tan and dark tan colour, respectively) were assessed across two environments. Fitting of seed size group ratios to inheritance models indicated that seed size is governed by two major complementary genes, where small size is dominant. The low genotype x environment interaction (<6.0% of the total variation for either population) suggests limited environmental influence on this trait. Subsequently, two major quantitative trait loci (QTL) were identified, one on LG 4 (QTL(1)) and one on LG 1 (QTL(2)), that together accounted for 20% of the seed size trait and may be targeted for future fine mapping and associated selectable marker development. These same loci also accounted for 37% of the phenotypic variance for 100-seed weight across the two environments, indicating the close genetic relationship between seed size and weight.

Six hundred accessions of chickpea (Cicer arietinum L.) landraces and its wild relatives from 28 different countries, available at Australian Temperate and Field Crops Collection (ATFCC) were screened for tolerance to salt under greenhouse conditions using three sampling strategies; (1) random sampling of 200 accessions from different countries, (2) restricted random sampling of 200 accessions from geographical regions with salinity problems and high diversity (Middle East and West & South Asia) and (3) as for strategy 1 but with a reduced representation of accessions from the geographical regions used in strategy 2. Degree of salt tolerance was based on necrosis scores and shoot biomass reduction relative to unstressed controls at harvest after subjecting stressed plants to salt treatment from 21 to 42 days after sowing. There was a wide variation in salinity tolerance determined by both measures. For sampling strategies 1, 2 and 3 respectively; 24, 28 and 14% of accessions were salt tolerant. Accessions from the middle east and south Asian (regions with salinity problem, a long history of chickpea cultivation and high diversity) gave a higher probability (P < 0.01) of getting salt tolerant accessions.

Trifolium repens (white clover) and Trifolium subterraneum (subterranean clover) have proved to be good model systems for the investigation of Rhizobium symbiosis and plant defence systems. Considerable research has been done on the interaction of these clovers with Rhizobium leguminosarum bv. trifolii at the practical agricultural level. This work has now been supplemented with the application of many recent molecular techniques. Our research has investigated the interactions involved in development, defence and symbiosis of clovers. We have taken the dual approach of constructing transgenic plant tools and Proteome analysis to examine the activity of phytohormones and flavonoids as well as the roles of pathogenesis-related (PR) protein genes in these biological processes.

Root morphology is both genetically programmed and environmentally determined. We have begun an analysis into the components of root development by: (a) constructing a range of transgenic clover plants to assess some of the genetic programs involved as both roots and nodules are initiated and develop. These transgenic plants report on auxin activity, flavonoid synthesis and chitinase expression and suggest a role for flavonoids as regulators of auxin levels; and (b) determining in cereals the effect of both added auxin and specific microorganisms on the initiation and development of modified root outgrowths and lateral roots. Appropriate combinations of auxin, the nitrogen fixing Acetobacter diazotrophicus, and rice variety did give rise to some plants which grew slowly for over 12 months in a nitrogen-free medium.

One of the chief predators of subterranean clover (Trifolium subterraneum) pastures is redlegged earth mite (Halotydeus destructor; RLEM). Subterranean clover pathogenesis-related (PR) proteins induced by RLEM attack and ethephon treatment were surveyed, and PR proteins with peroxidase, β-1,3-glucanase and chitinase activities were detected. A protein co-migrating with a chitinase activity, induced by RLEM predation and treatment with ethephon, was isolated. It was purified and the N-terminal amino acid sequence determined. Using a degenerate oligonucleotide primer designed from this sequence, a corresponding cDNA fragment was amplified by reverse transcriptase-PCR, then cloned, and used as a probe to screen a subterranean clover cv. Karridale genomic library. The cDNA and a 97% homologous genomic clone were sequenced and the deduced amino acid sequence revealed an open reading frame of 157 amino acids capable of encoding a peptide of 16 478 Da. Significant homology (80%) was found between this protein and an abscisic acid (ABA)-responsive protein from Pisum sativum of unknown function which is an intracellular pathogenesis-related (IPR) protein. The gene encoding this protein also has homology to pea ‘disease response resistance genes’ and to proteins from other plant species in the PR-10 family. The induced protein was designated TsPR-10a due to its homology to other PR-10 proteins. Genomic Southern analysis indicates that the gene encoding this protein, designated Ypr10a, is a member of a multigene family with at least three members. Northern blot analysis indicates that the subterranean clover Ypr10a mRNA, or homologous transcript, level is strongly induced by ethephon treatment in both root and aerial tissues of 3 week old plants. The rapid induction kinetics of Ypr10a mRNA under ethephon treatment, its correlation with a putative chitinase activity, and homology to other PR-protein genes, suggests a pathogenesis-related role for TsPR-10a protein in subterranean clover.

White clover was transformed with a tobacco basic chitinase promoter:GUS fusion. Basic chitinase promoter activity was detected by histochemical staining. Comparison of the spatial and temporal expression of the chitinase promoter-driven GUS gene in tobacco to that in white clover indicates that transcription from the promoter is induced by similar developmental and environmental response programs in each species. Wound-responsiveness of the white clover transgene was rapid and localised following mechanical and aphid (Family Aphididae) wounding. Developmental expression of the transgene during root morphogenesis reveals strong expression in tap and lateral root meristems but expression in lateral root meristems was observed only after the emergence through the tap root epidermis. No expression of the transgene was detected in the pericycle or the dividing cells of the developing lateral root. The expression of the tobacco basic chitinase promoter:GUS transgene in white clover was then used as a marker to examine the differences between the early developmental pathways leading to lateral root formation and those involoved in nodule formation in response to Rhizobium inoculation. Inoculation of the zone of emerging root hairs with a nodulation-competent Rhizobium strain ANU845(pRI4003), triggered transient transgene expression 2 to 4 h post-inoculation. No transgene expression was detectable after inoculation with purified Nod factor from strain ANU843. Our results suggest that lateral roots and nodules differ both in some of the mechanisms required to initiate cell division, and in their ongoing development after the emergence from the root epidermis.

Trifolium subterraneum (subterranean clover) is of considerable economic importance to the Australian rural industries as a pasture legume. In addition to its commercial value, it has a number of specific attributes—such as small seed size, diploidy, self-fertilization, the ability to be transformed and small genome—which make it a prime target for the modern techniques of molecular genetics. We report genetic and physiological factors that control the production and excretion of the lipooligosaccharide molecules formed by Rhizobium leguminosarum bv. trifolii in the formation of the symbiosis with subterranean clover. These molecules, synthesized by the products of the nodulation (nod) genes, are a major determinant of nodule occupancy and the strain selection imposed by the host plant. In addition, we have investigated which plant genes and proteins are activated in subterranean clover when they are either physically wounded, infected with Rhizobium, or attacked by red-legged earth mites. To analyse these interactions more precisely, we have cloned plant genes involved in the phenylpropanoid pathway and used their promoters to construct transgenic subterranean clover plants. Our studies provide an insight into the nature and consequences of the chemical exchange between plants and invading microbes.