A brief description, about methods, techniques, source of germplasm and promising tools about breeding for NUE in Maize.

1. Breeding for Nitrogen Use Efficiency (NUE) in Maize Ignacio A. Ciampitti Purdue University iciampit@purdue.edu

2. Introduction Nitrogen (N) fertilizer, most important input for maize production N uptake and N remobilization is subjected to genetic variability (in general, >50% of grain originates from the stover) NUE, defined as the grain yield per unit of N available from the soil, including N fertilizer Two main components, N uptake efficiency (N uptake/N from soil) and N utilization efficiency (yield/N uptake)

3. Source of Germplasm or Initial Material

4. Breeding Techniques Full-sib recurrent selection (e.g. Omoigui et al. 2007) Development of Low N availability pool maize populations under controlled stress condition, selection using an index (yield, stay green, ASI, etc.) Half-sib family method (e.g. Muruli and Paulsen, 1981) Intercrossed with a synthetic population to form a N-efficient and N-inefficient synthetic (tested under four N supply conditions) Hybrid production (e.g. Uribelarrea et al., 2007) Divergent selection for grain protein affects NUE in maize hybrids, crossing inbreds from IHP and ILP Synthetic cultivars (e.g. Uribelarrea et al., 2007) Four inbreds crossed in all directions and synthetic were formed of the reciprocal crosses (hybrids used as testers) Another methods: Mass selection, backcross, reciprocal recurrent selection, etc.

5. Genetic basis in NUE Indirect outcome of breeding for higher yields. The NUE is a highly complex, polygenically controlled quantitative trait Why? Moose and Below, 2009

6. Quantitative trait loci (QTL) Previous studies have identified QTL controlling NUE and some of their component traits (Agrama et al. 1999; Bertin and Gallais 2001; Hirel et al. 2001; Gallais and Hirel 2004 QTLs for N-uptake and N utilization efficiency (at high N input five QTLs explained 39% of phenotypic variance) QTLs for leaf nitrate content QTLs for glutamine synthetase (GS) activity QTLs for glutamate dehydrogenase (GDH) QTLs will have value in combining transgenes with genetic backgrounds that maximize trait expression and stability

7. QTLs Location in chromosomes (Gallais and Hirel, 2004)

8. Candidate Genes for NUE Genes for which allelic variation could be responsible for a part of the observed variation Large number of genes from C and N metabolism have been mapped (complex network) Some possible genes associated with NUE: genes coding for GS, Nr, sh2(affect starch and protein content), ADPGppase, Invertase, Sucrose-Phosphate-Synthase (SPS) and Sucrose-Synthetase (SuS), QTLs affecting grain protein and NUE components

9. Possible function of the GS isoenzymes within the maize plant (Martin et al., 2006)

10. RNA Expression Profiling Another source to identify different candidate genes Maize reproductive tissues during grain filling period Chiou et al. (2007) reported that only two miRNAs, miR395 and miR399, have been identified to be important in nutrient stresses responses Complexity of the trait, in other experiment 122 genes were upregulated by N and 204 were downregulated by N

11. Transgenes for improving NUE Root worm resistance, larger and healthier root system, possible lead to greater N uptake Projected release of transgenic maize hybrids with drought tolerance could indirectly increase the NUE trait Direct effect, through over expression of GS enzyme, increased of 30% in maize yield (Martin et al., 2006) Delayed of senescence, high citokinin levels, “stay green” phenotype, more photosynthesis extent the N uptake period (affected NUE) Future possible transgenes: overexpression NADH-GOGAT (more grain weight) , asparagine synthetase (more N per seed unit), reduced activity of citokinin oxidase (more kernels per ear)

12. Selection for “secondary traits” Correlated trait should have higher heritability (less environmental influence), to be easier and economic to measure and present a high correlation with the trait of interest. Geiger, 2009

14. Lowering the amount of N required (Nreq) to obtain a target grain yield without N applied (GY0)

15. N uptake per plant remained constant over the time, other strategies are required (biotechnology ???)Moose and Below, 2009

17. Selection based on an index composed of grain yield and different phenological traits was shown to improve breeding progress considerably. Analyzed a second-cycle DH-line population Observed a significant linear regression of grain yield under LN on the number of LN-specific yield QTL The yield increase per QTL amounted to about 1% on average. Geiger et al., 2006

18. Conclusions For increase the sink and the source, the progress understanding the genetics behind of the physiological pathways. A large germplasm source and breeding techniques are available for breeders with a broad range of genetic variability. A large number of structural genes encoding enzymes of the N and C metabolism have been mapped to the maize. Easily identified by RNA profiling experiments and other techniques available for improving NUE are the transgenes, gene shuffling and RNAi. In terms of transgenes, any transgene improvement on grain yield can impact indirectly on maize NUE trait. The projected release of transgenic maize hybrids with drought tolerance could indirectly increase the NUE trait. More research is needed in the genetic bases of NUE, quantitative genetic approach using molecular markers, genomics, and combining both agronomic and physiological studies.

19. Below, 2002