Quality Protein MAIZE

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QPM

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Geetika Breeding For Quality Protein Maize

InTRODUCTION and history:

InTRODUCTION and history Nutritionally maize is a relatively poor cereal when it comes to the quality of its protein (drawback of being deficient in two essential amino acids viz. , lysine and tryptophan) This problem was addressed through research breakthroughs at CIMMYT in the late 1990’s that lead to the development of quality protein maize (QPM) having twice the amount of lysine and tryptophan ( Krivanek et al ., 2007) In 1963, Lynn Bates, discovered much higher levels of lysine and tryptophan than normal in two maize landraces due to the presence of a gene called opaque-2 QPM

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Maize breeders have developed quality protein maize (QPM) by incorporating opaque-2 mutant gene. o2o2 (recessive) mutation ≥ heterozygous (O2o2) or homozygous dominant (O2O2) (higher lysine and tryptophan content than maize) (Crow and Kermicle , 2002) Breeding efforts using o2 mutants led to the development of high lysine cultivars, but adverse pleiotropic effects imposed severe constraints conventional plant breeding methodologies soft endosperm kernels harder types (disease and insect resistance, and with utilization and storage qualities similar to those of superior normal maize materials)

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As against the lysine and tryptophan levels of 2 and 0.4% in normal maize, the corresponding values in QPM are 4 and 0.8% respectively

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Several natural maize mutants conferring higher lysine and tryptophan were identified in the 1960s and 1970s High lysine mutants of maize Gene Allele Researchers Year of discovery Opaque-2 o2 Mertz, Bates and Nelson 1964 Floury-2 fl2 Nelson, Mertz and Bates 1965 Opaque-6 o6 McWhirter 1971 Opaque-7 o7 Ma and Nelson 1975 Floury-3 fl3 Ma and Nelson 1975

Utility of High Lysine and Tryptophan Maize:

Utility of High Lysine and Tryptophan Maize For food and nutritional security : superior food for human being Providing nutritious feed : for poultry, pork livestock, swine, fish, etc Promoting maize based entrepreneurship : nutritious products developed from QPM can replace fancied and highly priced industrial foods Figure 1. Pig fed high lysine/tryptophan maize (larger animal labeled QPM or Q4) compared with its sibling fed normal maize (labeled normal or N4).

History of QPM in India:

History of QPM in India Continuous breeding efforts done by International Centre for Maize and Wheat Improvement (CIMMYT) In India, these germplasm accessions were tested at different centres of AICRPM The DMR, New Delhi developed first QPM Composite variety, Shakti-1 with 0.63% tryptophan in the year 1997, and released for general cultivation in 1998 National Agricultural Technology Project (NATP) on QPM was launched by Indian Council of Agricultural Research (ICAR) wherein QPM germplasm received from CIMMYT And was acclimatized to suit the local agroclimatic conditions and via repeated selection, hard endosperm modified opaque-2 maize inbred lines and their crosses have been identified in India by multidisciplinary team of multi-institutes

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Name of hybrid Type of hybrid Institute Year Protein content in grain (%) Tryptophan content in protein (%) Shaktiman-1 three-way cross RAU, Pusa 2001 9.60 1.01 Shaktiman-2 Single cross RAU, Pusa 2004 9.30 1.04 HQPM-1 Single cross (1 st yellow grain ) CCSHAU, Karnal 2005 9.36 0.94 Shaktiman-3 Single cross RAU, Pusa 2006 9.63 0.73 Shaktiman-4 Single cross RAU, Pusa 2006 9.98 0.93 HQPM-5 Single cross CCSHAU, Karnal 2007 9.80 0.76 HQPM-7 Single cross CCSHAU, Karnal 2008 9.42 0.72 Vivek QPM-9 Single cross VPKAS, Almora 2008 8.46 0.83

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HQPM-1 HQPM-5 HQPM-7 VIVEK QPM-9 Shakti-1

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Shaktiman-1 Shaktiman-2 Shaktiman-3 Shaktiman-4

Nutritive value of QPM:

Nutritive value of QPM Figure: 1 Structure of maize kernel (Source: www.fao.org ) Hull- high fibre, germ- rich in oil and starchy endosperm (71%) largest concentration- prolamine (<50%) QPM- lowering of zeins by 30%

Storage proteins in maize:

Storage proteins in maize Zeins Rich in proline and glutamine consist of albumins, globulins, glutelins and prolamins (constitute about 50-60%) Four types α , β , γ , and δ Contains higher proportion of Leucine (18.7%), Phenylalanine (5.2%) Isoleucine (3.8%), Valine (3.6%) Tyrosine (3.5%) Non- Zeins Other proteins such as globulins (3%), glutelins (34%) and albumins (3%)

Cont………:

Cont……… Smaller amounts of: Threonine (3%), Histidine and Cysteine (1%), Methionine (0.9%) Lysine (0.1%) Devoid of tryptophan as it is absent from the major prolamin fraction ( α -zeins) of maize kernel. non-zein protein fraction is balanced and rich in lysine and tryptophan (Vasal, 2000).

Improved protein quality of opaque-2 mutants:

Improved protein quality of opaque-2 mutants Reduction in levels of zeins by various mutant allele is accomplished either by : reduction in levels of various zein sub-units, rate of accumulation of zeins, increase in methionine content, Effect on timing and pattern of storage protein accumulation.

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Mutant gene o-2 defective basic-domain- leucine-zipper transcription factor Decrease in leucine From (18.7%) more tryptophan for niacin biosynthesis Dominant form 02 expression of 22 KDa α -zeins LKR - gene mutant allele 02 increase in non-zein fraction differentially regulates and reduces zein gene transcription floury-2 mutants encode 22KD α -zein gene with a defective signal polypeptide reduction in 4 types of zeins Mucronate mutants frame shift mutation in 16 KDa γ -zein

Problems associated with high lysine mutants:

Problems associated with high lysine mutants Consequence Effect Reported By Reduced yield than normal maize due to less density per unit volume as starch is loosely packed with lot of air spaces Singh and Venkatesh , 2006 Low grain consistence soft, chalky endosperm that dried slowly making it prone to damage Toro et al., (2003) Farinaceous endosperm that retains water a thick pericarp, more susceptibility to diseases and pests, higher storage losses and also affects harvest ability Toro et al., (2003)

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Consequence Effect Reported By Changes in texture of grains o2 mutants (reduced levels of a- zeins ) resulting in small unexpanded protein bodies Geetha et al., 1991 o15 mutants (reduces γ - zeins ) leads to smaller number of protein bodies. floury-2 (fl-2), Mucronate (Mc), and defective endosperm (De B30) result in irregularly shaped protein bodies.

Genetics of High Lysine and Tryptophan Maize:

Genetics of High Lysine and Tryptophan Maize The development of high lysine/tryptophan maize involves manipulating three distinct genetic systems: The simple recessive allele of the opaque-2 gene, Modifiers/enhancers of the o2o2-containing endosperm to confer higher lysine and tryptophan, Genes that modify the opaque-2-induced soft endosperm to hard endosperm.

Simple recessive allele of the opaque-2 gene:

Simple recessive allele of the opaque-2 gene The presence of o2 in the homozygous recessive (o2o2) state is a pre-requisite for the entire process of obtaining high lysine/tryptophan maize Simple recessive inheritance of o2 gene O2O2 Χ o2o2 O2o2 1/4 O2O2; 1/2 O2o2; 1/4 o2o2 Figure: 2 . Simple recessive inheritance of the o2 gene.

Modifiers/enhancers of the o2o2 containing endosperm :

Modifiers/enhancers of the o2o2 containing endosperm Protein fraction Percentage of total protein (g/100 g protein) Number Name Normal endosperm Soft endosperm I Ablumins , globulins and soluble nitrogen 6.6 17.0 II Zeins (alpha, beta, delta, gamma) 48.7 9.7 III Zein like 14.0 13.4 IV Glutelin like 9.2 17.2 V Glutelin 17.0 34.5 Residue 4.5 8.1 Table 1. Protein fraction distribution of endosperm samples of normal and soft endosperm (o2). Increase in the proportion of non-zein proteins (Fractions I, IV, and V) (Gibbon and Larkins, 2005) Source: Cited by Bjarnason and Vasal (1992)

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It consists of minor modifying loci that affect lysine and tryptophan levels in the endosperm due to decrease in zein fraction from 47.2% in normal maize to 22.8% in opaque-2 mutants Table:2 Lysine and tryptophan levels as % of total protein in whole grain flour of normal and o2o2 maize Normal maize o2o2 maize Lysine ( a) 1.6-2.6 (mean 2.0) 2.7-4.5 (mean 4.0) Tryptophan (b) 0.2-0.6 (mean 0.4) 0.5-1.1 (mean 0.8) a Moro et al. (1996). b CIMMYT tropical lowland sub program Genes governing the levels of lysine have been mapped to the chromosome2, 4 and 7 (Wang et al., 2001; Wu et al., 2002) and include eEF1A (7L), eEF1A (4S), eEF1A (2S), FAA (1L), FAA (2S), FAA (2L), FAA (3S), FAA (4L), FAA (5L), FAA (7L), FAA (8S) and FAA (9S). Lysine or tryptophan levels must be monitored while developing new cultivars

Genes modifying opaque-2-induced soft endosperm to hard endosperm. :

Genes modifying opaque-2-induced soft endosperm to hard endosperm . Breeding requires selection based on a third, distinct genetic system, also comprised of minor modifying loci, that convert the mutant endosperm of the soft/opaque/floury phenotype to a hard/vitreous phenotype similar to normal maize. Role of gamma zeins to recover hard endosperm phenotype, given that the o2-modified (hard endosperm) grains have approximately double the amount of gamma zein in the endosperm as the o2-only mutants. (Wallace et al., 1990) The beneficial alleles of the modifying loci (controlling gamma zein production) can be selected using a rapid, low-cost, light-table method

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Figure3 . Soft endosperm o2 ears showing splitting of pericarp Figure 4. Ears of Pool 25, cycle 0, soft endosperm o2 maize (left) and ears of its improved version (cycle 18) (right). Both c0 and c18 have high lysine and tryptophan levels. However, c18 (having desirable kernel characteristics) is considered QPM Thus, QPM looks like common maize and can be differentiated only through laboratory biochemical tests having o2 gene , high lys and try and hard endosperm

Breeding Quality Protein Maize:

Breeding Quality Protein Maize elite non-QPM inbred lines and open-pollinated varieties (OPVs) backcrossing, pedigree crosses elite non QPM germplasm Χ elite QPM donors QPM Different methods for maize inbred production can be used in a breeding program (include the recurrent, the pedigree, and single seed descent selection methods, within family full-sib mating) ‘Modified back crossing-cum-recurrent selection’, (innovative breeding procedure) was designed, owning to nature and complexity of kernel modification trait Two unique and essential steps in the development of QPM germplasm : Identification of segregants in a poulation having the o2 allele in the homozygous recessive (o2o2) condition with a hard endosperm Identification and confirmation of QPM quality (percentage of tryptophan and protein in sample) through laboratory analyses

Early efforts and experiences in using o2 cultivars:

Early efforts and experiences in using o2 cultivars conversion normal genotypes o2 mutant versions Elite inbred lines making good hybrid combinations were converted through standard back cross approach The search then continued for new mutants and this led to the recognition of ‘o2 endosperm modifier genes’ that alter the phenotype of o2 mutants Two interesting alternatives were considered: (i) exploiting double-mutant combinations; and (ii) simultaneous use of o2 gene and the genetic modifiers of the o2 locus.

From opaque-2 to QPM: Endosperm modifiers:

From opaque-2 to QPM: Endosperm modifiers Various endosperm modifier genes interact to improve grain characteristics improve the kernel hardiness and appearance and increase kernel weight and density endosperm modifier genes could be used along with o-2 gene either singly or in combination with other mutants such as sugary-2 (Su-2) acceptable characteristics in the final product Such double mutant combination resulted in maize lines high lysine, lesser ear rot, vitreous grains and better protein digestibility even though yield was affected and were not always vitreous Most successful and rewarding option a combined use of o2 and the genetic modifiers of the opaque phenotype

QPM breeding approaches, methods, components, steps, and tools.:

QPM breeding approaches, methods, components, steps, and tools.

Light table for identification of o2o2 :

Light table for identification of o2o2 Figure:5 Side view of a type (b) light table with the top open . Figure:6 Screening maize kernels on a light table. Custom-made box differentiate hard endosperm maize types from soft o2o2 genotypes made of wood on all sides, except for the top surface (semi-transparent glass or plastic) Inside the box, one or more fluorescent (or other type) bulbs are placed To view kernel characteristics PRINCIPAL o2o2- softness- seen opaque O2o2 or O2O2 – translucent Less opaque- implies more modifiers

modification scores and Selection of scale :

modification scores and Selection of scale Gradation in the opaqueness is scored on a 1-to-5 scale for easy descriptions of the various classes and to enable statistical analysis. Percentage of opaqueness is visually assessed Type (Modification score) 1: Not opaque Type (Modification score) 2: 25% opaque advanced stages Type (Modification score) 3: 50% opaque F2 generation Type (Modification score) 4: 75% opaque Type (Modification score) 5: 100% opaque Figure: 7 Kernels on a light table sorted into modification classes. Types 1 to 3 would be considered QPM, provided their protein quality is verified . F3 and F4

Biochemical laboratory quantitative determination :

Biochemical laboratory quantitative determination Only single parameter tryptophan is analyzed on a routine basis. lysine (Lys) and tryptophan ( Trp ) values are highly correlated The value of lysine is four times that of tryptophan and have well established relationship Figure:8 Correlation between tryptophan and lysine content in 307 samples of tropical germplasm. Source: H. Cordova and A. Krivanek, 2006, personal communication

Components of QPM breeding:

Components of QPM breeding Elite source germplasm : using best germplasm Genetic progress determined by eliteness ensures maximum genetic gain in a pedigree breeding The parents used in a cross must complement each other so that the source hybrid must have the full complement of target traits. QPM donors by selection for modified grain texture in QPM backgrounds using various selection schemes from soft o2 materials that showed varying degrees of kernel modification due to differential accumulation of modifier genes.

Two approaches exploited:

Two approaches exploited The initial cycle followed by modified ear-to-row system Involved recombination of superior hard endosperm o2 families yellow white Controlled full-sib pollination Selection was practised for modified ears and modified kernels at all stages Intra population selection for genetic modifiers in o2 backgrounds exhibiting a higher frequency of modified o2 kernels Yellow H.E.o2 White H.E. o2 Selection of modified ears with good protein quality for 3-4 cycles Recombined separately

QPM testers :

QPM testers Inbred line, an OPV, or a single-cross hybrid. A desirable tester must facilitate discrimination among genotypes for combining ability and desirable traits, simultaneously identify useful hybrid products for direct use Choice of testers involves a mix of theoretical and practical considerations ( Bänziger et al., 2000) how genetically broad-based the tester should be; whether it should be high or low yielding; whether it should have a high or low frequency of target traits ; whether it has good or poor general combining ability ; whether testers should be related or unrelated.

Schematic diagram illustrating the conversion of a non-QPM to QPM:

Schematic diagram illustrating the conversion of a non-QPM to QPM

Constraints in QPM development:

Constraints in QPM development opaque-2 gene (recessive) through pollen contamination dilution and gradual loss of QPM’s nutritional quality Lack of appropriate seed multiplication and distribution systems From the standpoint of ensuring that the opaque-2 gene is retained in the grain hybrids would be preferable.

Genetic engineering for improving protein quality :

Genetic engineering for improving protein quality Various biochemical pathways are potential targets for manipulation using genetic engineering (Shewry, 2007) suppressing lysine catabolism (Houmard et al ., 2007) bifunctional lysine degradation enzyme (ZLKR/SDH) transgenic maize kernels RNAi down regulation 22 KDa α -_zeins (Segal et al., 2003) 19 KDa α -_zeins (Huang et al., 2006) higher lysine content (upto 16-20% more lysine) Reducing zein synthesis CordapA LKR/SDH simultaneous expression of deregulated lysine biosynthetic Enzyme by a single cassette (Frizzi et al . 2008)

Increasing the level of free amino acids:

Increasing the level of free amino acids Aspartic acid lysine, threonine and metheonine highly branched and under complex feed back inhibition (AK) and (DHPS) Normal maize Transgenic maize Expressing Feed back insensitive DHPS gene from Corynebacterium (Zhu et al., 2007) (Monsanto 2007) Normal lines Transgenic maize lines produce the milk protein α -lactalbumin in the endosperm . (Bicar et al . 2008)

New Methods : MAS:

New Methods : MAS Problem of Backcross conversion of normal lines, requires Selfing generation between each backcross to recover the recessive o2 allele before selecting for endosperm hardness and amino-acid modifying loci Seven seasons to complete QPM conversion Potential solutions - to use molecular markers to identify the o2 gene it has recently become possible to use MAS There are three publicly available (SSR) markers for this purpose, phi57, phi112, and umc 1066 , (J.M. Ribaut, personal communication) the sequences of which are in the maize database ( http://www.agron.missouri.edu/ssr.html ) Phi112 – dominant (do not contain recessive o2 allele )

Concluding remarks:

Concluding remarks Genomic techniques are currently being employed by some research groups in the developed countries, to investigate the patterns of gene expression in mutants influencing maize kernel texture Such knowledge could be of considerable value in improving the precision and efficiency of QPM breeding Hybrid development efforts in QPM have also progressed considerably by select some of the most promising hybrid combinations developed by CIMMYT for release in respective countries in the near future

Future thrust:

Future thrust an increasing application of molecular genetic tools in QPM research and development. This would essentially require more intensive studies in relation to the role of γ -zein proteins in enhancing lysine content, better characterization of the zein proteins, molecular tagging of endosperm modifier gene loci and isolation of the modifier genes themselves

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