The main objective of the research project was to develop strategies to reduce RFO concentration in lentils and chickpeas. This was accomplished by:
1. Characterising the amounts of RFO in lentil and chickpea germplasm;
2. Characterising the synthetic pathway by isolating the principal genes involved in the RFO synthetic pathway;
3. Studying the expression profile of the key genes;
4. Developing genetic maps of selected chickpea and lentil cultivars; and
5. Generating DNA-based molecular markers, to screen germplasm and use in marker-assisted selection to develop genotypes with reduced RFOs in seed.
Compared to grains, legume seeds have higher concentration of soluble carbohydrates belonging to the raffinose family oligosaccharides (RFO), including stachyose and verbascose. The RFO and other non-starch polysaccharides (dietary fibers), contribute up to one-eighth of the seed dry weight in chickpeas. The RFO in human diet and animal feed have both beneficial and undesirable effects. Humans lack the RFO-digesting enzyme alpha-galactosidase, and as a result RFOs pass undigested into the lower gut, where they are fermented by microbes. This produces gas and flatulence, and in extreme cases diarrhea. This is a major constraint in human pulse consumption in North America and Europe. Seeds with reduced RFO content could lead to increased pulse consumption in the human diet. Knowledge of the RFO content of different genotypes can guide breeders’ selections and lead to new cultivars tailored to specific markets.
We hypothesize that:
1. Genotypes of chickpeas and lentils vary in their RFO pathway expression and final concentration, and
2. Markers connected to the pathway can be used to identify lower RFO genotypes for development of new genotypes.
1. The RFO content of mature seed was determined using an HPLC method with pulsed amperometric detection.
2. RNA was prepared from developing seed tissue, and cDNA libraries made. A new RNA method was developed for developing lentil seeds, as existing methods were not efficient for this tissue. Existing databases were used to guide isolation of the lentil forms of galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (SS) genes.
3. In chickpeas, the accumulation of different sugars during seed development was analyzed; while in lentils, the expression of the responsible genes was analyzed. Association mapping was used to try to associate markers with low and high RFO accumulation. For lentils a biparental set of lines was available for part of the project. High-throughput marker systems, like Diversity Array Technology (DArT®), were applied to statistically analyze association between relatively low and high RFO accumulation and DNA markers.
4. Identification of targeted DNA-based molecular markers to screen germplasm for developing lentil and chickpea cultivars with reduced RFO was assayed. For chickpeas, activity of the pathway enzymes in low/high RFO pairs was assessed with the intent of identifying the limiting enzyme.
The RFO content of mature seed of 171 chickpea lines was determined. Stachyose was generally highest, with raffinose and small amounts of verbascose. There was substantial variation; for example, stachyose varied from 0.18 to 2.38 g/100g. However, the major Canadian-grown cultivars showed a limited range, possibly due to the limited diversity in their parents. In chickpea seeds, raffinose, stachyose and verbascose content showed a moderate broad sense heritability (0.25-0.56) when lines were grown at two locations (India field and Saskatoon greenhouse). Lentils of 20 lines showed less than two-fold variation in content of each RFO, probably also due to limited diversity in their parents. When 168 additional genotypes from ICARDA were examined, however, greater variability was found. To examine heritability, eleven lentil cultivars (seven green cotyledons and four red cotyledons) grown in ten environments were analyzed by the content and composition of soluble carbohydrates in seeds. The broad sense heritability for RFO was high (0.85).
Lentils have two major isoforms of GS gene, one of RS and one of SS.
GS was expressed until late in seed development, while RS and SS were mostly expressed during the period of rapid seed maturation and desiccation, when the products of these enzymes also accumulated most rapidly. Association mapping showed some connection between particular genome regions/markers and RFO traits. DArT markers were used to analyze population structure and genetic relationships within a specifically designed chickpea germplasm collection including wild, cultivated, and some advanced breeding lines, for seed quality traits. Association mapping identified genomic regions associated with variation in total RFO, raffinose, stachyose, and verbascose concentrations, although associations were not very strong. These potential genomic regions need to be dissected further to develop diagnostic markers for use in chickpea breeding programs. The DArT markers provided significant information about population structure, and association mapping is a useful approach to identify important genomic regions associated with RFO related traits in chickpea, although this is affected by the narrow genetic base in the domestic forms of both species, particularly the Canadian cultivars. This technique suggests marker-trait associations which then need to be verified independently. Further work is in progress.
The analysis of chickpea enzyme activity during development showed an association of RFO enzyme activity with the period of seed desiccation, for which it may be necessary. High RFO genotypes expressed about 2- to 3-fold higher activity of all RFO biosynthetic enzymes compared with low RFO concentration genotypes; GS is suggested as the most likely limiting factor. In lentils, two forms of GS peaked in expression at different times, and two SS forms also showed different expression profiles, with one barely detectable. Sequencing of multiple genotypes showed some variants in sequence, but they could not be associated with differences in RFO concentration.
This project has identified natural variation in RFO in chickpea and lentil germplasm, improved our understanding of RFO biosynthesis during seed development, identified and characterized major RFO biosynthetic genes and their expression during seed development. We also identified some key genomic regions associated with RFO concentration in chickpeas and identified allelic variation in two RFO biosynthetic genes.
This project has identified lentil and chickpea genotypes that are being used to develop varieties with reduced RFO concentration. The identified DNA markers, when completely characterized, will accelerate development of lentil and chickpea varieties with reduced RFO, thus causing less stomach discomfort. This will promote consumption of lentils and chickpeas or their products. The DNA-based markers identified in this project can also be used for other pulse crops such as peas and beans and reduce the time needed to respond to changing market demands.