Plant breeding has played a vital role in the advancement of human civilization (Arnel, 2006). The domestication of plants implied that man graduated from being a hunter-gatherer and could now settle down and focus his creativity on improving other aspects of civilization. The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Recent advances in biotechnology, genomics and molecular marker applications have laid the foundation for molecular plant breeding, a science that is revolutionizing 21st century crop improvement (Moose and Mumm, 2008).
This report proposes a crop improvement plan for a 'generic' crop species termed; Crop A. The characteristics of the crop, limitations and areas of improvement are described in the first part of the report. This is followed by a Breeding program, incorporating the application of molecular markers assisted selection. QTL and GRC analysis techniques are used to identify molecular marker sequences.
Features of 'Crop A'
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Crop A is a diploid perennial tree crop. It has a generation and selection time of five years. It produces 1000 perfect flowers every year mainly during one month. Reproduction is mostly vegetative. The species is essentially allogamous and self-incompatible. The self-incompatible system plays a role in preserving the genetic heterozygosity and prevents in breeding depression (Ozaki, 2007). It is generally propagated via cuttings; breeding material is largely 'hybrid'. The main product is extracted from the leaves; water availability is the main limitation on yield.
Breeding tree crops is a long term operation; this is especially true for perennial tree crops which are characterized by long generation and selection time. This has several consequences namely; breeding intensity is limited and the number of generations achieved between breeding stages is reduced to a minimum (Baudouin et al., 1997).
Other limitations on the tree crop 'A' are mentioned below:
Major limitation on yield is water availability
Lack of formal crossing/breeding undertaken
10 year period between planting and maximum yield
Although land intensive, the perennial nature of the crop simplifies germplasm management, in addition the small number of generation units reduces the risk of losing variation (Baudouin et al., 1997). The main aim of the breeding program proposed is to increase water use efficiency in the crop in order to maintain if not increase yield.
Breeding for Water use efficiency (WUE): Underlying Principles
Water availability is a limiting factor for yield production in Crop A. The relationship between yield and water availability can be described as
Yield = T - WUE - HI (Acquaah, 2007)
T= total seasonal crop transpiration
WUE= water use efficiency
HI = crop harvest index
This relationship clearly indicates that focus for breeding drought resistance should be on water use efficiency.
Water use efficiency (Wi) is defined as the net CO2 assimilation (A) rate to stomatal conductance for water vapour (gw), it is a determinant of transpiration efficiency and the ratio of biomass accumulation to water used by transpiration. Wi is known to be estimated using instantaneous gas exchange (A/gw) or integrated over diverse time scales using carbon isotopic composition (δ13C) of plant material. Differences of δ13C under specific environmental conditions could be measured by studying differences in stomatal openness. QTL analysis can be used to identify the genes responsible for Wi. However the perennial nature of the tree crop makes construction of segregating population difficult, in addition Wi is a complex trait that relates to a large number of physiological processes, thus it is important to study the genetic architecture in parallel of δ13C along with related traits at the leaf level such stomatal responses. A few studies have been carried out in trees species regarding Wi and δ13C, mostly in connection with traits such a growth, phenology and leaf morphology.
Crop improvement plan
The main objectives for initiating the breeding program
Water use efficiency ↔ High yield
4.2 BREEDING SCHEME
The scheme applied is half sib-progeny selection; individuals in half sib selection are evaluated on their half sib progeny. The advantages of this procedure include 1) the procedure is rapid to conduct 2) progeny testing increases the success of selection especially if quantitative gene action occurs or heritability is low.
4.2.1 Ecotype Selection
Always on Time
Marked to Standard
Germplasm present for selection is largely 'hybrid' mainly produced via vegetative propagation; cuttings. Trees are usually replanted only if with significantly higher yields (>50%). Selecting a relevant ideotype for a drought target environment is essential.
Testers are selected from existing populations known to produce higher yield. It is selected based on phenotypic characteristics that are related to a more efficient water use system such as deep rooting systems, leaf anatomy; waxy, thickness. Selection of testers from existing population reduces the time of the breeding cycle. Success of the selection process depends on the heritability of the trait observed; the heritability of the trait must be high (Cilas, 2003). Enzymatic markers as well as molecular markers are used to measure genetic diversity. Clone performance in terms of their specific value and combining ability are also assessed (Baudouin et al., 1997).
4.2.2 Breeding Program Scheme
Marker assisted selection
Test for Variation using Molecular Markers
Test for Variation using Molecular Markers
Test for Variation using Molecular Markers
Development of linkage maps using pseudo test cross mapping strategy.
Source Population F0
QTL analysis or GCR marker assisted analysis for identification of molecular markers for desired traits.
The source population (F0) is selected on the basis of phenotypic traits; they are then crossed with the common tester. The half sib progeny (F1) is then evaluated and two superior varieties assessed on the basis of phenotypic characteristics are selected and crossed for the following generation. The progeny (F2) obtained from these crosses are then selected for superior varieties on the basis of phenotypic traits. The varieties selected are crossed with each other (F3).
Test for Variation using Molecular Markers
4.2.3 QTL analysis to identify traits
Allogamous species such as forest trees are often in linkage equilibrium, thus it is likely that the alleles at QTLs and at maker loci are randomly associated in different individuals, consequently it is harder to establish marker-QTL associations at the population level in large mating populations (Polmion, 1996). Progeny issued from the cross between two heterozygous parents (F1 cross) are used to study QTLs (Lespinasse, 2000). A pseudo-test cross could be established for each parent using RAPDs and SSRs markers, MAP MAKER 3.0. Then a consensus map between the two parental maps could be established using homologous markers segregating in both parents, JOINMAP software version 1.4 (Stam 1993) is used to generate the map. Parental maps are constructed prior to the synthetic map as they allow for examination of colinearity between parental genomes and prevents the merging of homologous chromosomes which would otherwise have been rearranged
QTLs for the following traits related too drought resistance can be identified; deep rooting system, leaf anatomy such as a waxy coat and thickness, cellular traits: osmotic adjustment, this trait has been considered valuable when searching for improved yield under water limiting conditions (Pita et al., 2005). Stomatal conductance may also be used, this has been identified as a useful tool in breeding for improved yield under water stressed conditions mainly because 1) water loss by transpiration and carbon gain thus promoting growth 2) water saving by stomatal closure and increased heat stress (Pita et al., 2005). Moreover stomatal conductance has been reported to be under genetic control in Pima cotton (Gossypium barbadense L.), and prove to be a useful tool to predict higher yields in hot environments (Lu et al., 2000). Infra-red thermometer can be used to indicate stomatal openness.
At least 100 progeny plants should be screened and grown under different environmental conditions this is essential for verification of stability of the QTLs identified (Lanaud et al., 2000).
4.2.4 Marker Assisted Selection
Post the assembly of markers into genetic maps and the identification of QTLs for the desired traits, the markers along with the QTLs and phenotypic characteristics can be used to track the presence of valuable characteristic is large segregating populations as part of the crop breeding program (Murphy, 2007). This allows for accurate screening of seedlings several years before the characters can be evaluated in the field, this makes it possible for the accumulation of different factors in a genotype of interest and shortens the number of generations needed in for the breeding program (Dirlewanger, 2004)
4.2.5 Genetic-regression-combined marker trait association (GRC)
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This technique bypasses the use of planned populations, molecular marker-trait associations have been identified by the combination between germplasm and regression technique. A comparison between QTL analysis and GRC analysis is presented in Figure 2. Multiple regression analysis can be used to select molecular markers associated with morpho-biochemical traits, these methods are increasingly being used in many crops (Vijayan et al., 2006). This technique has been applied for the identification of number of traits; drought tolerance in tea (Camellia) (Mishra and Mandi, 2004), 6 agronomic traits in mulberry (Morus india L.) (Vijayan et al., 2006).
Figure 2: Comparison in marker development between linkage-based (Source: Collard and Mackill, 2008) and germplasm-regression combined marker -trait association identification (Ruan, 2009)
Optimal accession from germplasm (collected from water stressed areas and trees that display 50% higher yield) is evaluated for phenotypic traits of interest; Leaf yield attributing leaf anatomy; waxy, thickness deep rooting systems. DNA is then extracted from the chosen accessions and amplified using PCR analysis with optimal markers. Sequence-tagged site (STS), sequence characterized amplified region (SCAR) and single nucleotide polymorphism (SNPs) markers can be used due to their high polymorphic information content (Ruan, 2010). These markers are currently the most appropriate maker class for MAS (Collard and Mackill, 2008).
Each quantitative trait is treated as dependent variable and the various marker genotypes (scored as 1 for present and 0 for absent) as independent. The analysis model is based on the model reported by Ruan 2010:
Y= a+ b1m1 + b2m2 +… b j m j +… bnmn + d + d… (Ruan, 2010)
Y= accession means for quantitative trait, m j = set of independent variables, representing the markers, b j= partial regression coefficients that specify empirical relationships between Y and m j, d= accessions residuals, e= random error, P values= 0.045- 0.099
Relates the variation in the dependent variable Y, to a linear function of the set off independent variables.
The best fit markers can be further tested using linear models or confirming the significance of β-statistics for each band identified. This method provides the most likely estimates of relationships between quantitative traits and various markers.
However the success of this method is largely dependent on choosing the most optimal germplasm, molecular markers and the test of identification efficiency of markers associated with the traits. (Ruan, 2010).
MASS CLONAL PROPAGATION
Mass clonal propagation could be used as a much faster and cheaper alternative to multiplying up genetic stock. Based on the desired traits the bet-performing trees could be used for propagation. Vegetative cuttings typically of stems or leaves are taken from the chosen tree(s) and cultivated in a mixture of nutrients until plantlets are regenerated. The plantlets can be sub-cultured on a massive scale. In this way a single elite clone could give rise to an entire population in a short period of time. However disadvantages of this method include 1) all the clones might be identical to the parent line, in the long term their genetic uniformity might make them vulnerable to disease 2) the other risk is of creating abnormalities in during the tissue culture process itself (Murphy, 2007). However despite these drawbacks this method is widely used in the propagation of perennials.
Breeding for desired traits through the use of conventional programmes is time consuming and labour - intensive. Genetic engineering offers a more promising alternative; wherein specific traits can be introduced without the loss of genetic integrity. However this would only be possible if efficient transformation protocols are developed.
Somatic hybridisation techniques could also be used provided a drough resistant variety is identified. Protoplasts from this variety could be fused with protoplasts from a variety display increased yield. Progenies could be slected for drought resistance using QTLs and markers.
RESEARH TOPIC FOR FUTURE BREEDING PROGRAMS: EARLY MATURITY USING AP1 GENES
A Technique proposed for reduction of the juvenile period so as to speed up generation and selection time. Crop A is characterised by an extended adolescence, during this juvenile period it is difficult to measure yield capacity which is seen mainly in mature leaves. Trait analyses often takes long periods of time; 3 to 5 years till flowers appear and it become fully productive (Cortines and Weiss, 2001). Arabidopsis has been exploited as a model crop in the identification of gene functions. Martinez- Zapater 2001 showed the ectopic expression of LFY/AP1 can significantly shorten the juvenile stage in the development of orange trees (Cortines and Weiss, 2001). AP1 gene can be cloned into Crop A in order to shorten the breeding cycle.
This report has attempted to draw out a breeding plan for the generic species 'Crop A' which is a perennial self-incompatible, diploid tree crop with a generation and selection time of 5 year. A number of tree crops are of immense economical value, however a lengthy juvenile phase has slowed down the development of breeding programs. Moreover breeding for complex traits such as 'water use efficiency' which are under polygenic control with considerable environmental influence creates a number of challenges. The dawn of MAS breeding technologies marked a new era in plant genetics, however in order to exploit the complete potential of MAS; extended research is required towards developing linkage maps and identifying QTLs. High thorough put systems such as 'association mapping' could also be developed as potentially shortcut methods to QTL discovery. New techniques that bypass the development of linkage maps and QTLs for marker discovery are also being researched such as genetic-regression-combined marker trait association. Development of these research programs provide potential for improving breeding programs used for perennial tree species. High cost continues to be a barrier that prevents the utilisation of this technique, It is imperative that more research be carried out in order to further our understanding of molecular plant breeding, joint collaboration between governments and agricultural institution all around the world is essential to make these technologies as feasible as possible (Collard and Mackill 2008).