Joint research project

Lotus interspecific hybrids: a new material to breed new varieties and clone genes involved in  proanthocyanidin biosynthesis and salt tolerance in legumes.

Project leaders
Francesco Paolocci, Oscar Adolfo Ruiz
Agreement
ARGENTINA - CONICET - Consejo Nacional de Investigaciones Científicas y Técnicas
Call
CNR/CONICET 2011-2012
Department
Agriculture and Food
Thematic area
Biology, agriculture and food sciences
Status of the project
Extended
Report for renewal
e-argentina-x-rinnovo-accordi-bilaterale-23-09-2010-final-report-cnr-conicet.pdf

Research proposal

 
Within the frame of the 2009-2010 bilateral project, the two Research Units have produced and characterized hybrids obtained through controlled pollination between plants belonging to a diploid, wild accession of Lotus corniculatus recovered from saline area in Spain, and plants from a commercial, highly productive Lotus tenuis (Lt) variety largely grown in South America. The ultimate goal of this research was in fact to output superior Lotus genotypes to breed new forage varieties, and to deep our understanding on the genetic control of proanthocyanidin (PA) biosynthesis and salt tolerance in legumes.
PAs, also known as condensed tannins, are oligomers or polymers of flavan-3-ol units that are present in the fruits, bark, leaves and seeds of many plants, where they provide protection against predation (Dixon et al., 2005). The presence of PAs is a major quality factor for forage crops as legume species with moderate concentrations of leaf PAs (3-6% dry matter) prevents ruminal bloating in sheep and cattle. Leaf PAs in fact slow down the degradation of dietary protein to ammonia by rumen microorganisms and increase protein outflow from the rumen, when ruminants are fed with fresh legume pastures. Overall, PAs increase the absorption of amino acids in the small intestine of the animal and the nutritive value of forage legumes while decreasing the excretion of urea and methane in the environment by ruminants. It is noteworthy that methane produced by ruminants is one of the major pollutant responsible for the greenhouse effect. However, high dietary PA concentrations (7–12% dry matter) depress voluntary feed intake, digestive efficiency and animal productivity (Tanner, 2004). Moreover, some particular PAs have been found to confer pathogen resistance in ruminants (Barry & McNabb, 1999). Species of the genus Lotus, many of which are native from the Mediterranean Sea region, have been naturalized in South America and are important forage crops in marginal environments. PA concentration shows a significant degree of variation among different Lotus species: cultivated tetraploid L. corniculatus contains PA concentrations adequate for animal nutrition. On the contrary, L. tenuis, is as diploid forage species well adapted to the restrictive conditions of the main areas devoted to cattle breeding in Argentina, but it contains PA levels well below those considered to be optimal, and exhibits an extremely low degree of intra-specific variation regarding with trait (Escaray et al., 2008). Thus, the modification of PA concentration in cultivated L. tenuis is a desirable goal for plant breeders.
Until recently, Lotus corniculatus has been regarded as a polyploidy species. The discovery of a natural, diploid and PA-rich accession of Lotus corniculatus (hereinafter refereed to as LcPD) that spontaneously grows in sandy and saline soil in Spain has disclosed the possibility to intercross these plants with those from a well adapted and highly productive Lt variety largely grown in south America to obtain hybrid plants with a balance presence of leaf PAs, resistant to salt stress as LcPD and as productive as Lt. Indeed four hybrids lines (LH1-LH4) have been produced and characterized, both morphologically and molecularly, during the years 2009-2010. These hybrid lines show a total amount of leaf PAs of about 6% DW (i.e just in the optimal range to confer bloat safety) which is basically intermediate between the two parents. This evidence suggests that the inheritance of this trait could be additive (Passeri et al. 2010). Molecular studies have allowed  the two RUs to identify among the structural and regulatory genes involved into PA synthesis those that are differently expressed in leaves of hybrid lines and their parents, such as the structural genes DFR, ANR, ANR and LAR1 and the regulatory R2R3MYB gene LcTT2 (Paolocci et al. 2007; Escaray et al. 2010). Likewise, preliminary tests have shown that hybrids show a tolerance to salt stress as much as the LcPD parent, whereas their forage production is not dissimilar from that of the best parent (Lt) (Escaray et al. 2009 and 2010).
Overall, we have showed that it is possible to transfer the leaf PA and salt resistance traits across Lotus spp. The resulting interspecific hybrids represent a genetic material of outstanding relevance to breed superior Lotus varieties and for dissecting the genetic control and genes involved in a) the biosynthesis of PAs in leaves and b) salt stress resistance in forage legumes. To reach these goals, the hybrids will be used: 1) as donor material of useful traits in backcrosses with the Lt parental line; 2) to produce a segregating population for the leaf PA and salt tolerance traits.
References:
Barry & McNabb (1999). British Journal of Nutrition 81: 263–272.
Dixon et al. (2005). New phytologist 165:9-28.
Escaray et al (2008). Lotus Newsletter 38(2): 101-104
Escary et al (2009) BioCell 33:pp 35
Escaray et al.(2010) European COST Action Meeting 2010 (COST Action: FA0605-INPAS) Valencia, Spain.
Paolocci et al. (2007) Plant Physiology 143: 504-516.
Passeri et al. (2010). Italian Society of Plant Genetics Annual 54° Annual Congress Matera 27-30 September 2010
Tanner GJ (2004) Condensed tannins. In KM Davies, ed, Plant Pigments and Their Manipulation. Annual Plant Reviews, Vol 12. Blackwell Publishing-CRC Press, Boca Raton, FL, pp 150–184.

Research goals

 

Improving Lt varieties using interspecific hybrids as donor of useful traits. Hybrid lines will be backcrossed to Lt, the most productive parent, and the progeny (BC1) will be evaluated for forage production, PA accumulation and salt resistance. The Argentinean seed company GENTOS will perform the crosses and test forage production, whereas the two RUs will perform molecular and physiological analyses of BC1 progeny.
Production and evaluation of a segregating population.  To get insight into the genetic control of salt resistance and leaf PA traits in Lotus, the hybrids will be intercrossed to produce a segregating population (F2). The progeny lines will be evaluated for the content of leaf PAs and for their ability to growth under salt stress conditions. The F2 lines will be sorted according to the most divergent characters (i.e PA-rich vs PA-depleted lines, and salt tolerant vs salt susceptible lines) and nuclei acids isolated from lines with contrasting phenotypes for molecular analyses.
Functional evaluation of a candidate regulatory gene of PA in legumes . The full length cDNA of the R2R3MYB LcTT2 gene, whose expression correlates with the content of leaf PAs in Lotus spp. and with the expression of the key PA structural genes, will be isolated from LcPD and ectopically expressed in different Lotus and Medicago backgrounds. The resulting transgenic tissues and/or plants will be evaluated for the accumulation of PAs and the expression of PA structural gene.

Last update: 29/03/2024