Gene transformation and chromosomal translocation – A plant breeder’s vista

K.K.Vinod, Neway Mengistu, Nicholas Adam Crowley and Jeffery Ryan Sullivan

(This was the subject of a threaded discussion at University of Nebraska-Lincoln)

When a breeder is looking to incorporate “alien” genes into a line, two good choices he has are translocation and transformation (transgenic events). With a little luck, time, and effort their results can show great benefit to commercial crops. The two methods are similar because you are adding DNA segments to an existing genome without conventional breeding methods. Both of these methods are used to add a desired gene(s) to a crop which lacks the gene of interest. Chromosome translocation is caused by the interchange of parts between non-homologous chromosomes. Transgenic inserts add new, although more controllable segments, to an existing genome. The two methods require selfing and selection to be successful.

Translocation mostly gives a successful result in polyploid crops. It has been tried in wheat and rye to transfer disease resistant genes from their wild relatives. Moreover, translocation lines are more acceptable in polyploidy systems, wherein other chromosomes in the genome can compensate for a lost arm/part of chromosome eventuate in translocation events. Chromosomal translocations are random events. Translocations therefore differ from crossing over by randomness of insertion points. Any chromosomal segment can get attached to any arm of another chromosome. Classical examples are random translocations caused by transposable elements. When a chromosomal fragment carrying a desirable gene is getting translocated to a cultivated variety, the event may result in transferring of some other unknown alleles along with the gene of interest. For example, if a chromosomal segment from a wild relative carrying a disease resistance gene is translocated into a cultivated variety, it may also carry some undesirable wild traits into the cultivar. In the earlier days translocation was not much utilized in crop improvement due to this impediment. Nowadays, by use of transposable elements translocations and insertions are being utilized for site directed mutagenesis and random transfer of genetic elements.

Transformation techniques use biolistics/particle bombardment or Agrobacterium tumefaciens to insert a gene. These transformation techniques are “quick” means to introduce a gene into the plant of interest. Transformation events, try to incorporate new resistance, tolerance, or quality traits. Examples of transgene insert performed in soybean (RoundupReady® soyaben), cotton (Bt-cotton), rice (golden rice - enriched with vitamin A) etc., give a good reasoning to suggest that it can work. However, most of the traits are novel, which produce a function beneficial to humankind/cropping systems and the genes responsible are not found in that specific crop or any of its relatives. Transferring a trait using transgene insert can be manipulated by genetic engineering techniques to isolate the gene and manipulate it through cloning. In the case of a transgene, the insertion is a random event and can occur any where in the genome. This will result in hemizygosity for the transgene.

Although the advantages of these two methods are a great benefit to breeders, both have their own considerations when using them for adding desirable genes to a crop. The translocated disease resistant gene would contain surrounding chromosome segments from the wild relative. The surrounding genome would likely be undesirable since it is being donated by a distant relative to the crop. Moreover, it may take longer years to find a stable line that contain the disease resistant gene. In theory, transgenic events seem simple, and scientists/geneticists have found ways to make it as easy as it sounds, but when breeders work with translocation lines, it is hard to get everything seem simple. One case in which the breeders/scientists have used to make this technique easier is the Ph mutant in wheat. This mutation allows homoeologous pairing and crossing over between alien chromosome and its crop homoeologue, allowing transfer of chromosome segment containing the alien gene. And also if the crop is a kind of polyploid chromosomal translocation may be preferred than transgene insert - because of better tolerance of the new chromosome fragment in polyploids. On the other hand, many diseases are controlled by a single gene resistance which may not justify transfer of a chromosome fragment. A transgene insert may accomplish the job very well. However, transgene methods are still questionable by some and this must be measured when developing transgenic crops. Transgenic methods must also be isolated and sequenced for the desired gene.

Notwithstanding the fact that these methods have similarities and differences, both techniques can be used for transferring desirable traits like disease resistance. The similarity between the translocation and transgene lies in the hemizygosity it produced. Since corresponding allele(s) from the translocated fragment are not found in the homologous pair, or transgene is attached only to one chromosome, hemizygosity produces a situation where only one allele is present in excess on one chromosome, while it is totally absent on its pair. As breeders, hemizygosity is not a desirable situation as we have the threat of missing the event upto 50% among gametes. The best solution is to self the plants to generate a homozygous line for the transferred gene. This homozygous line can be used for further breeding programmes. Another similarity can be from incorporation of many novel genes from translocation and transformation. Translocation between species can provide more than one beneficial gene. This is also the case in transformation; a good example is YieldGuard® plus corn hybrids. These incorporate herbicide resistance, and various insecticide genetic events to produce a corn hybrid that is beneficial to the farmer.

One way, in which these methods differ, lie in the mode of prediction of the gene transformation event itself, i.e. marker. In the case of transformation, an antibiotic resistance, herbicide tolerant or gus gene is added for easy phenotypic identification in early stages of development. In translocation crosses, phenotypic markers that may be by chance linked to the gene being transferred need to be looked to identify the plant with the alien translocation.

The major difference between a transgene insert and the translocation event is that, we know the number and nature of the genes inserted in the transgenic event, while we are unsure of the number and nature of alleles inserted through a translocation event. Shorter the translocation insert more stable will be the translocated event, while the question of stability of the transgene is still not resolved. Depending upon the length of the translocated fragments, the number of alleles will vary which may comprise of many introns and exons. Transgene insert usually has gene of interest along with antibiotic or herbicide resistance markers and/or gus markers plus the promoter regions and plasmid fractions.

When using the translocation technique in a breeding program, a breeder must consider the difficult task of achieving fertilization from the parents and possible seed abortion and using embryo rescue. In the case of transformation this is not a problem. Transformation can cause problems when the gene of interest is placed in an existing gene for interest and produces a mutant or lethal plant.

My Professor's Dilemma

K.K.Vinod and M. Maheswaran

Yesterday my Professor, Dr M Maheswaran wrote me after reading the book “Emperor of All Maladies- A Biography of Cancer” by Siddhartha Mukherjee.

He wrote,

Genes talk to genes and pathways to pathways in perfect pitch, producing a familiar yet foreign music that rolls faster and faster into a lethal rhythm.

If the data did not fit the dogma then the dogma, not the data, needed to be changed.

After reading the book and understanding the biological causes behind the cancer genetics I felt very bad about things what we are teaching and doing in understanding the genetics of many of the traits we deal with crops and exploit the results in practical plant breeding. Many of the concepts of predictions we make based on the classical genetics, whether it is Mendelian or Galtonian, remain irrelevant considering the biological implications on a particular phenotype. For example, p53 gene, an unassuming name, has major role in the development of human cancer than any other component of the genome. The gene get its name from the product it encodes, p53, which is a polypeptide having a molecular weight of 53 kilo Daltons. p53 was thought for number of years to be a dominantly acting oncogene, but in 1990, it was recognized as the tumour suppressor gene that, when absent, is responsible for a rare inherited disorder called Li-Farumeni syndrome, whose victims are affected with a very incidence of certain cancers, including breast cancer and leukaemia (though there are separate and specific genes for breast cancer and leukaemia). Like individuals with the inherited form of retinoblastoma, persons with Li-Farumeni syndrome inherit only one functional copy of the p53 tumour suppressor gene and are thus highly susceptible to cancer as the result of random mutations that knock out the function of the remaining copy of the gene. Further, if we see the development of cancer in humans (for any type of cancer development), the possible sequence of genetic changes in a cell lineage are given below.

 

The complexity of cancer can be better understood if you read the above book. But in plant genetics we decide the genes based on the prediction methods and most of the gene predictions are based on the “breeder friendly phenotyping methods (whatever be the trait we have the simple means and many models to predict the genes). The advent of molecular marker technology made this simpler. Once there is co-segregation of DNA marker, the gene discoverer assumes he/she got gene for the phenotype (some where I read that there are 83genes identified for resistance to rice blast disease and we have 26 genes for brown plant hopper resistance in rice- just go back and read the situation of gene for Li-Farumeni syndrome). Gene identification based on breeder friendly phenotyping and pyramiding of those genes without knowing the functionality of the genes is a wasteful exercise. In recent times people started asking questions whether it is good to pyramid dominant genes or recessive genes without understanding the situation of obscurity prevailing over dominance or recessiveness. This can be remedied if every gene discoverer realizes the importance understanding the biology behind each of the phenotypes instead of evolving prediction methods.

My Professor stops his letter by saying “Predictions are always based on perceptions”.

Concern of my professor was of a genuine teacher, who attach paramount importance to imparting current and competitive knowledge to his students. He laments that except a very few teachers of plant genetics, none are ready to change the way science unfolds life’s mysteries. So instead of changing the dogma, we are often bending the data fit the dogma. Are we ruthlessly incompetent because we are afraid of deviating from the conventions?

A tribute to Damodar Dharmananda Kosambi

K.K.Vinod

Scientists and students of genetics studying linkage and recombination are familiar with Kosambi’s mapping function. Chances are that they never had heard of Kosambi before. The reason - Damodar Dharmanada Kosambi was not a geneticist by training and profession, but a mathematician. He was also a statistician, historian, marxist, linguist, writer – he was everything – a multi-faceted scholar. His famous mapping function was published in 1943, in Annals of Eugenics (Kosambi, 1943). How he got into this work is not known, but Kosambi’s works generally spanned across many disciplines from mathematics to children’s literature. At the time of this publication he was teaching mathematics at Fergusson College, Pune.

Recently, Professor Kosambi’s birth centenary was celebrated, in Pune mainly by the historians and scholars. I as a student of genetics, came to know more about Kosambi after this celebrations. Ignorant of a great scientist, whose name I would have used thousands of time while doing genetic map constructions, I decided to put this tribute.

Kosambi’s mapping function estimates the recombination fraction (c) between two loci as a function of the map distance (m) between the loci, by allowing some interference, as

c = ( e^4m -1) / 2( e^4m +1)

The estimate of the map distance between two loci can be obtained from

m = ln [ (1+2c)/(1-2c) ] /4

Kosambi’s function go intermediate between actual recombination fraction taken as map distance (no interference) and Haldane’s map function (Haldane, 1919), closely predicting recombination fractions especially when the loci are linked.

My article on Kosambi and the mapping function was published in the popular science journal Resonance in its Kosambi commemorative issue in June 2011. This article can be accessed from this link www.ias.ac.in/resonance/Volumes/16/06/0540-0550.pdf.

Damodar Dharmanand Kosambi (D.D. Kosambi) was born at Goa on 31 July 1907 to Acharya Dharmananda Damodar Kosambi and Balabai. After his early schooling, young Kosambi moved to Cambridge, MA (USA) and studied grammar and Latin. After successful schooling at Cambridge, he joined Harvard University in 1924 studying mathematics. He discontinued his studies for a brief period and returned to India, again to join back in 1926, where he was awarded with Bachelor of Arts degree. Returning to India soon after, he joined Banaras Hindu University as a professor, teaching German and mathematics. Here he started his personal research and started publishing his findings. He got married in 1931 with Nalini, and in the same year joined Aligarh Muslim University as the professor of mathematics. He continued his mathematical research more vigorously here, and publishing his papers regularly in European languages.

Two years after he joined Fergusson College in Pune, and continued to teach mathematics. His two daughters Maya and Meera were born here. It was during this period his famous paper on mapping function was published in 1943. Kosambi had done extensive research on many areas of mathematics and published many papers. However, many of his publications went unnoticed by Indian scholars and eventually a great scientist and a historian was getting ignored to a great extend. No students of genetics were told Kosambi was an Indian scientist.

It was probably Homi J Bhabha, who recognised his talents and made him to join Tata Institute of Fundamental Research (TIFR) in 1945. He was professor of mathematics and worked there for next 17 years. During this period, Kosambi published 40 research papers, mostly on mathematics. However, his interest was shifted to history and social sciences in the later years, extensively researching on ancient Sanskrit works, numismatics and ancient history of India. Probably these later works made him to be remembered as a historian rather than a mathematician.

Kosambi authored 9 books including edited ones and 127 articles. But this number is not authentic as there are many childrens’ stories written by him. As a prolific writer, thinker, mathematical genius, linguist and historian Professor Kosambi, as Dale Riepe wrote, ‘deserves to be remembered as one of the highly gifted and versatile scientific workers and indefatigable scholars of modern India for whom a relentless search for the highest human values was the only natural way of life’.

After leaving TIFR, in 1964, Kosambi was appointed as a Scientist Emeritus of the Council of Scientific and Industrial Research (CSIR) and worked in Pune. He got involved in many historical, scientific and archaeological projects, including stories for children. But most of his works that he produced in this period could not be published during his lifetime.

Professor Kosambi died at Pune, at the age of 59, on June 29, 1966. He was posthumously decorated with the Hari Om Ashram Award by the government of India's University Grant Commission in 1980.

A biographical sketch of Prof DD Kosambi written by Chintamani Deshmukh can be downloaded from here.

References (Click on the title to download)

Haldane, J.B.S (1919) The combination of linkage values, and the calculation of distance between linked factors. Journal of Genetics, 8: 299-309.

Kosambi DD (1943) The estimation of map distance from recombination values, Annals of Eugenics, 12(3): 172-175

Marker Assisted Selection

K K Vinod

Primary objective of any plant breeding programme is selection of the best genotype from an array of breeding lines (genotypes). Conventionally, selection involves various traits that are expressed in plants (phenotypes) and the selection tools employed by the breeders involve qualitative (visual or perceivable traits) and/ or biometrical (quantitative) traits. However, since gene expression is always modified by the environment due to several adaptive reasons, selection of traits is not always as successful as it should be. This led to the thinking of selecting directly for the genes themselves, as the genes are coded on the DNA molecules that are free of environmental interference. However, selection of genes was not an easy job as it appeared to be, because of their obscure locations on the genome. Hence, this is achieved indirectly by selecting detectable DNA variations in the individual genomes, which are either associated or closely linked to the target genes, as detected by the co-segregation of these fragments with phenotype. Such detectable DNA fragments are called markers. Use of these molecular markers has opened a novel way for selection known as marker assisted selection or marker aided selection (MAS). Extensive use of molecular markers by the present day breeders has opened up a separate field of study, the molecular breeding.

Principles of MAS

As the name indicates, MAS is not a breeding method but markers are used as a selection aid. We consider that traits may be typically controlled by single or many genes. Although in strict sense no trait is controlled by a single gene but a group of genes, the practical consideration of mono, oligo and polygenes revolves around the number of genes that produce the perceivable quantum effect of the trait. Any detectable DNA fragment lying around or within these genes can report the presence of this gene in its carrier. This forms the fundamental idea behind MAS. MAS therefore can involve in selection for both qualitative and quantitative characters. However, MAS is not generally advocated for the selection of easily selectable traits, especially those qualitative traits with high heritability and penetrance, while for traits with low heritability and low expressivity MAS may be a good option.

Since markers tag for genes, MAS is useful mostly in transfer of genes from a donor to a recipient. Therefore the donor should have a distinguishable marker allele that is linked to the target gene. This marker allele should be distinctly different from the allele produced by the same marker in the recipient genotype. Now while crossing both donor and recipient it is easy to determine which progeny carry the gene by identifying the linked marker allele. The target allele of the gene may be either a dominant one or recessive. Most commonly used procedure in transfer of single gene from donor to recipient is backcross breeding. Since the recipient genotype is used repeatedly in crossing steps in the backcross process it is also called recurrent parent (RP). Using markers, the backcross breeding proceeds by selecting progenies that are carriers of the target allele until a stable homozygous genotype is obtained which has almost entirely of the recipient genome carrying the target allele. This is called marker assisted backcross breeding (MABB). Depending on the transfer of dominant or recessive allele backcross procedures may suitably vary.

Basic Concepts of Selection in Plant Breeding

K.K.Vinod

Generally breeders in two ways can change variability in their working population. Firstly by allowing individuals to produce next generation within limits of breeders preference or by natural choice in which which some of these individuals may be allowed to produce many offsprings and some of which may be allowed to produce which few or no offsprings. This non-random reproduction in a breeding population is called selection. The two agencies involved in the selection, one is by the natural choice is called natural selection and the other that is done by the breeder is the artificial selection. Secondly, breeders can decide how the selected individuals will be mated to each other either by inbreeding and cross-breeding.

Following basic principles operates on selection:

1. Selection works only on heritable variation

Variation that is governed by genetic makeup of the individual alone can be recovered in the progenies, not the variation that is environmentally dependent.

2. Selection works only in existing variability ie., it cannot create new variation. 

Hence, selection can only shuffle the variation and also can combine variations of different individuals of the population, which is however, pre-exist within the population itself.

3. Selection works by providing preference to certain individuals to reproduce, therefore certain set of genes get preferential inheritance at the expense of the unselected genes. Asa consequence gene and genotypic frequency  get altered. 

4. Continuous selection leads to loss of variability, because unselected genes get eliminated in the course of generation advancement.

5. When a new variability is created in a population either by mutation or by movement of genes from outside (from another unrelated population), new selection pressure is generated on the introduced genes.

6. Purpose and consequence of selection varies from type of selection, individual genes, mating methods etc.

Plant breeding employs artificial selection to generate plant populations that are cultivated for the benefit of the mankind. Artificial selection is fundamentally aimed at the following facts:

1. To change the phenotype of the selected individual in the most desired way to the man

2. To bring down the variability in the selected population

3. To increase uniformity in the selected population

4. To increase number of populations with different level of variability between them

5. To increase the range of uniform populations

Various artificial selection methods used in plant breeding are, 

In self pollinated crops,

1. Mass selection

2. Pureline selection

3. Pedigree selection

In cross pollinated crops,

1. Mass selection

2. Progeny selection (ear to row methods, modified ear to row methods etc)

3. Family selection (half sib, full sib family selection and their modifications)

4. Recurrent selection

Clonally propagated crops,  

1. Clonal selection.

Crop improvement in Para rubber tree (Hevea brasiliensis)

K.K.Vinod

Para rubber tree (Hevea brasiliensis) belong to Euphorbiaceae. It is the major source of natural rubber in the world. The genus Hevea occurs naturally through out the Amazon basin and in parts of Matto Grosso, Upper Orinoco and the Guianas (Schultes, 1970). Hevea brasiliensis (2n = 36) is a monoecious perennial tree belonging to Euphorbiaceae. There are ten species recognised under this genus, H. brasiliensis, H. benthamiana, H. camporum, H. camargoana, H. nitida, H. pauciflora, H. guianensis, H. microphylla, H. spruceana and H. rigidifolia. These species are highly cross-pollinated and putative hybrids of natural intercrossing between the species occur in the natural habitat. Experimental crosses between species show no hybridisation barriers (Webster and Paardekooper, 1989).

Genetic resources

Though there are few reports of introduction of rubber plants to other areas of the world, all of them lack evidence to substantiate such claims. Recorded history of introduction the rubber from its natural habitat starts with the collection of rubber seeds by H.A. Wickham near Santarem during 1872. In 1876, Wickham collected about 70,000 seeds of H. brasiliensis from near Boim on the Rio Tapajoz and from the well-drained undulating areas near Rio Madeira and despatched them to Royal Botanic Gardens at Kew, England. About 2800 seedlings raised at Kew, 2397 were despatched to Sri Lanka and few to Malaysia, Singapore and Indonesia. Virtually all the rubber trees cultivated in Asian countries originated from this collection.

After Wickham, there were several attempts to introduce H. brasiliensis to Asian countries, but these introductions were confined to very few in number did not contribute much to the introduced genetic base. Later wild species and few cultivated cloned were introduced to Rubber Research Institute of Malaysia from Brazil. Besides few other organised attempts by International Rubber Research And Development Board (IRRDB), a major collection of wild Hevea Germplasm was carried out in 1981 by IRRDB team of scientists from major member countries, in collaboration with Brazil collected 64736 seeds from the states of Acre, Rondonia and Mato Grosso and budwoods from 194 high yielding trees free of major diseases like abnormal leaf fall (Phytophthora sp.) and South American leaf blight (SALB) caused by Microcyclus ulei (Ong et al., 1983). Of these collections, India introduced about 9000 accessions from Malaysia, of which about 6000 are surviving and being conserved in ex situ gardens. Besides, India also introduced 127 commercial clones from other countries where Hevea is grown on commercial scale.

Apart from H. brasiliensis, SALB affects only three more species, H. benthamiana, H. guianensis and H. spruceana (Langford, 1945; Chee and Holliday, 1986). Others species are free from infection. Eleven physiological races are reported to be identified for this disease. Though H. brasiliensis is totally affected by this disease immune reactions are shown by some H. benthamiana, H. spruceana and H. pauciflora derivatives. Sporadic attempts to use these immunities, by crossing with H. brasiliensis had produced clones with transient and non-durable resistance (Simmonds, 1989). Evaluation of wild Hevea germplasm for sources for biotic and abiotic stresses are in progress at major rubber growing countries.

Crop improvement

The rubber tree is introduced into cultivation very recently. The genetic base of the cultivated germplasm is very narrow, converging to a very few seeds collected from upper Amazon basin near river Tapajos in Brazil. Though rubber tree is infected by many diseases, the plantation industry suffers mainly from only one really devastating pathogen causing South American leaf blight (SALB). This disease in still confined to American sub-continent and all the rubber growing areas of South and Southeast Asia are free from it. However, it is already proved that none of the cultivated varieties in Asia are resistant to this disease when tested at various locations of South America. However, in recent years, another disease Corynespora leaf fall (CLF) has gained importance for wiping out entire RRIC 103 plantations of Sri Lanka, which had almost gained its name SALB of Asia.

Clonal selection is the most important procedure followed in breeding rubber. Clones can be evolved at any stages of different breeding steps. Usually selective hybridisation of promising parents is done among themselves and also with wild germplasm lines. The progenies are directly selected from seedling nurseries and cloned for further evaluation. Also, natural seedling population or half-sib population are also screened for desirable characters including resistance. Susceptible and poor performing clones are generally discarded. Polycross gardens comprising of pre-potent clones are also utilised and the selection is generally exercised in the polyclonal seedling orchards, even at the stage of maturity. The selections are directly carried forward for clonal evaluation and selection. A general scheme of rubber improvement is provided in Fig.

A general scheme of Hevea breeding

The major thrust of quality improvement in Hevea does not orient to the quality of rubber, but on the quality of secondary products like Hevea wood. Rubber trees produce enormous quantity of semi-hardwood at every replanting cycle. The shrinking availability of natural timber from the forests has made this so valuable. The wood on appropriate chemical treatment could be used as a best substitute for timber for furniture making and for similar uses. The treated wood is now being used to produce very high quality furniture, panel boards, house-hold articles and for flooring purpose.

Owing to the growing importance of rubber wood, the improvement in the direction of developing timber – latex clones is in progress. The clones combining better yield, high vigorous growth, short life span, high quality strong wood, free from diseases and with good branching habits are preferred.

Rubber tree is a prolific producer of honey. Honey is produced on extrafloral nectaries located on the Hevea leaves. High honey production can give additional income to plantation sector, and population that yield more honey are preferred.

Though rubber products are being used for so many decades, recently, the problem of latex protein allergy has emerged mainly in America. The allergy is reported to be caused by the proteins present in the latex, which are found in traces in the finished products. However, the allergy reported is mainly of type IV allergy of cutaneous origin. There are several processing methods available for the deproteinization of the rubber products. However, it would be better to look out for genotypes, which do not accumulate harmful proteins in the latex.

Biotechnology rubber crop improvement

The one area of biotechnology with clear applications in rubber is that of molecular markers. Marker-assisted selection offers prospects of accelerating the process of long term breeding objectives offered by the conventional approaches. An important first step towards developing linked markers is the construction of a linkage map. Maps have been published for rubber (Seguin et al, 1996). A map allows the selection of markers which are evenly distributed over the genome, thus enhancing the probability of finding markers linked to quantitative trait loci (QTL). Molecular markers that are linked to these QTL will co-segregate with the genes involved in desirable traits and could be used efficiently to follow introgressions and accumulation of favourable traits during recombination cycles.

Besides, interfering with the biochemical pathway are also being contemplated, in producing many biomolecules including antibiotics. However, the lesson is that a single, apparently simple, change in a synthetic pathway may have unexpected side effects. Successful transformation programmes have been those where the transformation work is integrated into a conventional breeding programme, allowing individuals with the required phenotype to be developed from a range of transformants.

References

Chee, K.H. and Holliday, P. (1986) South American leaf blight of Hevea rubber. Malaysian Rubber Research and Development Board, Kuala Lumpur. 50p.

Langford, M.H. (1945) ) South American leaf blight of Hevea rubber trees. USDA Technical Bulletin, 882: 31.

Ong,S.H., Ghani, M.N.A. and Tan, H. 1983. New Hevea germplasm: Its introduction and potential. Proceedings of the RRIM Planters Conference, Kuala Lumpur. pp. 3-17.

Seguin, M., Besse, P., Lespinasse, D., Lebrun, P., Rodier-Goud, M., and Nicolas, D., 1996. Hevea molecular genetics. Plantations, Recherche, Développement 3(2): 77-87 

Shultes RE (1970) The history of taxonomic studies in Hevea. Bot. Rev., 36: 197-276.

Simmonds, N.W. 1989. Rubber Breeding. In: Rubber (C.C. Webster and W.J. Baulkwill, eds). Longman Scientific and Technical, U.K. pp. 85-124.

Webster,C.C and Paardekooper. 1989. Botany of the rubber tree In: Rubber (C.C. Webster and W.J. Baulkwill, eds). Longman Scientific and Technical, U.K.

Marker assisted selection in rice breeding

K K Vinod

Classical plant breeding is primarily of phenotypic selection of superior individuals, among segregating progenies following a hybridization, induced variability created through mutation, polyploidisation etc., and from a native outbreeding mixture of individuals. Though the idea seems simple, success of choosing a right kind of genotype is often hindered by genotype x environment interaction and the genetic nature of trait of interest itself.  In addition to testing procedures for selection of target traits in target environments are difficult, unreliable and expensive due to the nature of the traits themselves like biotic and abiotic stresses.

Latest advents in molecular marker technology, using tiny DNA fragments, that can distinguish individuals with slightest genetic variation, and unaffected by the environments, became a handy tool in providing information on selecting individuals possessing target trait genes. These DNA fragments are known as DNA markers.  These markers can be established through various molecular marker systems viz., restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellites or simple sequence repeats (SSR), inter-simple sequence repeat (ISSR), retrotransposon based polymorphisms, sequence characterized amplified regions (SCAR), sequence related amplified polymorphisms (SRAP), single nucleotide polymorphisms (SNP) etc. Each of these systems has its own merits and demerits.

Molecular markers which are stable, unique and abundant can help us in study the linkage among themselves relating to their positions in the target genome.  When subjected to classical genetic analysis based on the segregating pattern of markers among individual segregating progenies obtained by crossing two homozygous individuals (purelines) which are genetically different, linkage distance between each of the segregating markers can be determined and drawn on a linkage map. When many markers are employed in this attempt covering the entire genome, we will be able to reconstruct individual linkage groups or chromosomes of that particular genome. This framework of chromosomes will provide us the information where individual markers are located and in consultation with classical cytogenetic maps we can assign individual chromosome designations to the constructed linkage groups. These populations used for molecular map construction are called mapping populations.

Further extending the marker segregation pattern and the linkage disequilibrium among themselves, on to the phenotypic segregation of quantitative traits, help us in identifying markers that closely follow the pattern of segregation of the target traits.  The simplest possible analysis for this is to carryout the simple regression between marker and phenotype segregation pattern. Alternatively we can use simple factor analysis of variance (ANOVA). This analysis is called single marker analysis (SMA). Extending further these analysis the model can be allowed to include more than one marker interacting, so that we can identify marker-by-marker interaction significantly influencing the phenotype. These markers can either be on the same chromosome (linked) or on different chromosome (epistatic). Locations of these markers which significantly influence the phenotype is called quantitative trait loci (QTL). Locations on these markers can be traced to the molecular linkage map, thereby the genomic location of the QTL.

However, one may be very conscious while referring the QTL as the gene responsible for the trait. Actually QTLs are putative locations of genes responsible for influencing the trait.  Actual gene may, therefore, be away from this location or at this location.  Nevertheless, the function how these genes influence the trait is also unknown.  Besides, there may be different degree of influence of QTLs to the traits. Some QTLs which show larger and conspicuous influence are called major QTLs, and those with minor effects are called minor QTLs.

Further extending concept of linkage analysis on multipoint mapping, the methods called interval mapping is designed. There are two types viz., simple interval mapping (SIM) and composite interval mapping (CIM). Compare to SIM, CIM is statistically robust and help in predicting more accurate QTL positions.  The procedures of interval mapping include extensive step-wise regression and predictions based on maximum likelihood ratios and/or best linear unbiased prediction (BLUP).  Mixed model mapping incorporating models of additive x additive, additive x epistatic interactions and phenotyping under varying environments are also employed currently. Many statistical predictions and sub-sampling are done using Bayesian methods or jackknifing procedures.

Location of QTLs, help us in closely looking at the chromosomal locations using closely placed markers. This is known as fine mapping of the target genomic locations, and once the exact location of the QTL is identified this genomic location can be sequenced to see whether this location code for a known or unknown protein that influence the trait. By doing this the exact influence of the gene located at this QTL can be understood.  If found extensively useful this QTL can be cloned and used for further genetic engineering programmes.

However, more useful and practical approach a breeder is interested in is using the QTL information and the looking for presence of them in a population using the linked markers, help him in selecting the target QTLs carrying individuals which will in turn contain the target trait itself.  This procedure of selection is called marker assisted selection (MAS).  However, marker assisted selection is not much confined to QTLs as it can be extended to any molecular marker linked to any major gene. Best examples are single genes conferring resistance to diseases. Also there can be the involvement of more than one major genes to which MAS can be targeted.  Another avenue MAS is extensively used is the selection among the transgene derived populations. Here, when the MAS is exercised on the target trait directly (herbicide tolerance) it is called foreground selection and when done on the marker trait (antibiotic resistance) it is called background selection.

The success of MAS depends on location markers with respect to the gene of interest. Three kinds of relationships are common, (i) marker lie within the gene, which is most favourable situation for positive selection (ii) marker is in linkage disequilibrium with the gene of interest in the whole population, where there will be the tendency of the marker to inherit closely with the gene of interest, and (iii) the marker in linkage equilibrium with the gene of interest, in which case the success of MAS is unpredictable.  Another challenge in MAS using QTL is the interaction of QTL with the target environments. QTL x environment (QE) interaction is a serious problem in MAS.  Here it is more prudent to look for environment specific QTL or widely adapted QTLs depending upon the objective of the selection programme.

Marker assisted selection in rice

MAS has been successfully employed in rice crop. Successful marker assisted screening and selection for root traits (Price and Curtois, 1999) resulted in better drought tolerance in upland rice. Selection was done based on RFLP and SSR markers for QTLs that determined root traits. Successful MAS based backcrossing also was done to transfer early season drought resistance and aroma from a japonica variety Azucena to Kalinga III, a high yielding height grain quality indica variety (Steel et al., 2002).  Participatory plant breeding using MAS bulks and purelines were successfully carried out in backcross progenies of Kalinga III x Azucena, following schemes given below:

Another approach was to develop purelines out of the BC3 population and evaluating them for the presence of individual root QTLs and also for the combination of more than one QTLs. As many as four root QTLs were pyramided in successful lines.

A great advantage of these selections is that many are done at farmers holdings and selections were done by the farmers themselves.  Predominantly lines selected by the farmers were accumulating the targeted QTLs confirming the success of MAS. As many as 24 lines from upland, 12 from medium upland and 16 lines from lowland conditions were selected by these participatory plant breeding approach using MAS from crosses using Kalinga III as one parent, and IR 64, Radha 32, IR 36 and Vandana as other parent (Steele et al., 2002).

Many successful attempts for MAS in rice is reviewed in Babu et al., (2004). These include resistances to blast, bacterial blight, rice tungro virus, gall midge, brown plant hopper and green leaf hopper, tolerance to submergence and salt accumulation, wide compatibility, temperature sensitive male sterility, garin aroma, amylase content, photoperiod sensitivity, semi-dwarf stature and shattering tolerance.

References

Babu, R., Nair, S.K., Prasanna, B.M., and Gupta, H.S. (2004) Integrating marker-assisted selection in crop breeding – Prospects and challenges. Current Science, 87: 607-619.

Steele, K.A., Virk, D.S., Prasad, S.C., Kumar, R., Singh, D.N., Gangeswar, J.S., and Witcombe. J.R. (2002) Combining PPB and marker assisted selection: Strategies and experiences in rice. In: Quality Science in participatory plant breeding. Workshop at IPGRI, Sept 30- Oct 4, 2002,Rome, Italy.

Price, A.H., and Curtois, B. (1999) Mapping QTLs associated with drought resistance in rice: Problems, progress and prospects. Plant Growth Regulation, 29: 123-133.

Price, A.H., Steele, K.A., Moore, B.J., and Wyn-Jones, G. (2002) Upland rice grown in soil filles chambers and exposed to contrasting water deficit regimes. II. Mapping QTLs for root morphology and distribution. Field Crops Research, 76:25-43.

About Rice (Oryza sativa L.)

K K Vinod

[Following is the text of my short presentation made to my class at UNIVERSITY OF NEBRAKSA-LINCLON]

Rice is a semiaquatic annual grass belonging to the genus Oryza. The genus oryza includes 24 species, of which 22 are wild and two namely Oryza sativa and Oryza glaberrima are cultivated. O. sativa is grown all over the world while Oryza glaberrima has been cultivated in West Africa for about 3500 years. There are more than 120,000 varieties of cultivated rice (IRRI, 2001). It is believed that rice domestication occurred independently in China, India and Indonesia, thereby giving rise to three races of rice: sinica (also known as japonica), indica and javanica (also known as bulu in Indonesia).

Cultivated rice is diploid (2n=24) and belongs to AA genome. The sativa rice varieties of the world are commonly grouped into three subspecies namely indica, japonica and javanica. Rice grown in India belongs to the indica subspecies. They are characterised by having leaves slightly pubescent and pale green in colour. Indicas are awnless or possess short and smooth awns. The rice grown in Japan belongs to japonica subspecies. Japonicas are adapted for cultivation in the subtropical and warm temperate regions. Japonica varieties mostly have oval and round grains. They may be awned or awnless. Leaves are narrow and dark green in colour. Subspecies javanica is characterised by a stiff straw, long panicle with awned grains, sparse tillering habit, long duration and low sensitivity to difference in day length. These are found mainly in Indonesia.

Rice was domesticated more than 10,000 years ago is possibly one of the oldest domesticated species. Huke and Huke (1990) observes that the domestication of rice ranks as one of the most important developments in history, for this grain has fed more people over a longer period of time than has any other crop. Rice is the staple cereal for more than 50% people (~3.25 billion) around the world, cultivated in about 9% of the earth's arable land, which is the largest single use of land for producing food. Rice provides 25 to 85 percent of the calories in the daily diet and 15% of per capita protein (IRRI, 2002). In Asia, where rice is the major energy providing food, it accounts for 50-80% of daily caloric intake, especially among the poor (IRRI, 2001). Unlike other major cultivated grains like wheat and corn which are also used for feeding livestock, rice is exclusively used for human consumption.

With China, India and Indonesia producing the most of the world’s rice, Asia accounts for over 90% of the world's production of rice. Only 6-7% of the world's rice crop is traded in the world market. Production of rice in The United States accounts to 1.5% of the world's production, with Arkansas, California and Louisiana producing 80% of the U.S. rice. Thailand, Vietnam, China and the United States are the world's largest exporters (IRRI, 2002).

Rice is the only cereal that can be grown for long period in standing water. Even though predominantly semi-aquatic, rice is grown under many different conditions and production systems, including upland and dry conditions. (FAO, 2004a). 57% of the world’s rice is grown on irrigated land, 25% on rainfed lowland, 10% on the uplands, 6% in deepwater, and 2% in tidal wetlands (Chopra and Prakash, 2002). The flooded rice paddy sustains rich aquatic biodiversity, providing a home for fish, plants, amphibians, reptiles, mollusks, and crustaceans (FAO, 2004b).

Rice has many characteristics, making it useful in various ways to be included in cereals, snack foods, brewed beverages, flour, oil, syrup, flakes and religious ceremonies. Rice grains can be short, medium and long or waxy (sticky) or non-waxy. Some are aromatic (Alford and Duguid, 1998; Chaudhary et al., 2001), some are colored including brown, red, purple and black (FAO, 2004c) and some are of medicinal value. The variation in characteristics makes one variety more popular in one region of the world than another.

Rice breeding

The primary breeding objective in rice growing countries has been high yield potential. Plant breeders have greatly contributed to the development of high-yielding crop varieties and have changed the morphology and physiology of crop plants, and incorporated desirable traits and resistant gene(s) into traditional varieties while stabilizing or increasing crop production. Dramatic advancement in productivity has achieved by incorporation of the semi-dwarf gene from Dee-Gee-Woo-Gen into traditional tall, leafy rice. The semi-dwarf rice varieties are now planted in 60% of the world's rice land.

High-yielding varieties have made a great contribution to the world's food supply, but they also have several major problems. The high yields of these varieties can only be attained with a high level of inputs, in particular heavy applications of fertilizer. This has led to problems associated with pest outbreaks in certain areas, while increased rice production has resulted in lower rice prices.

In rice breeding, the ideal plant type sought by breeders have been high yield potential; resistance to major diseases and insects; and improved grain and eating quality. However, there are few conflicting objectives like, high grain quality tends to result in unstable yields and also, too much emphasis on disease and insect resistance and stable yields leads to poor grain quality. Hence, breeding efforts should be fashioned in a way to sustain the yield under unfavorable conditions, and to maximize yields when conditions are favorable. 

The following breeding approaches should be emphasized in producing varieties for sustainable rice production.

·         - High-yield potential under low inputs.

·         - Heterotic F₁ hybrid

·         - New plant type

·         - Premium grain and eating quality to meet consumer demand, and to provide grain suitable for processing.

·         - More genetic diversity.

·         - Durable host resistance to major diseases and insects.

·         - Wider range of growth duration for various purposes.

·         - Proper levels of tolerance to environmental and climatic stresses in specific areas.

Common breeding method used in rice is pedigree breeding method.  Other than the introduction of semi-dwarf gene (sd1), popularization of male sterile systems in early 1980’s, hybrid rice production has met dramatic increase in rice yields in China. Three line breeding of hybrid rice carrying wild-abortive cytoplasmic male sterility has been utilized in commercial scale (Kim and Rutger, 1988). The advent of environmentally sensitive male sterility systems (TGMS and PGMS) paved way for the development of two-line hybrid breeding in rice.  Transfer of cytoplasm from wild species to cultivated backgrounds used backcross procedures widely.

Host resistance to various biotic stresses is a very important aspect of high yields, and can be expected to play a significant role in sustainable rice production. There are now numerous varieties resistant to rice blast, bacterial blight, various virus diseases, and plant hoppers and some possess multiple resistance to diseases and insects. Varieties with the Xa4 gene resistant to bacterial wilt have been grown in the Philippines for the last 15 years, and continue to be resistant. It is extremely difficult to identify polygenic resistance and incorporate it into improved germplasm (Khush and Virmani, 1985). Current studies on host resistance to crops emphasize the durability of resistance (Ikehashi and Kiyosawa, 1981; Ahn, 1982; Lee et al., 1989). Polygenic traits rather than absolute resistance would be preferable in sustainable agricultural production (Hauptli et al., 1990).

Improvements in rice quality are very important in meeting the demands of consumers for healthy, high-quality food. Many traditional varieties in both the tropics and the temperate zone have excellent cooking and eating quality, but a low grain yield (Khush and Juliano, 1985). For many years, breeders have focused their attention on quality improvement, but there seems to be some unknown genetic barrier to incorporating this trait into high-yielding varieties.

Biotechnological Advances

Modern day crop breeding in rice is supplemented with biotechnological tools.  Success stories are fast emerging with the development of golden rice (Ye et al., 2000), and many efforts are on to develop transgenic rice with various incorporated traits, including resistance to pests, herbicides etc.  Successfully the Xa21 gene conferring resistance to Bacterial leaf blight has been cloned. The deciphering the entire rice genome has been completed. Marker assisted frameworks of quantitative trait loci are being developed intensively which will help in developing strong target trait directed marker assisted selection programs.

References:

Ahn, S.W. 1982. The slow blasting resistance. Proceedings, Symposium on Resistance to Rice Blast. IRAT/GERDAT, Montpellier, France, pp. 343-70. 

Alford, J. and N. Duguid, 1998. Seductions of Rice. Artisan Publishers, NY, NY

Chaudhary, R., et al., eds., 2001. Speciality rices of the world. Science Publishers, Inc, NH, USA. 

Chopra, V.L. and S. Prakash, 2002. Evolution and Adaptation of Cereal Crops. Science Publishers Inc, NH, USA. 

Food and Agriculture Organization, 2004a. Rice and water: a long and diversified story, International Year of Rice, 2pp.

Food and Agriculture Organization, 2004b. Aquatic biodiversity in rice fields, International Year of Rice, 2pp.

Food and Agriculture Organization, 2004c. Rice and human nutrition, International year of rice, 2pp. 

Hauptli, H., K. David, B.R. Thomas, and R.M. Goodman. 1990. Biotechnology and crop breeding for sustainable agriculture. In: Sustainable Agricultural Systems, A. Edwards, R. Lal, P. Madden, R.H. Miller,and G. House. (eds.). Soil and Water Conservation Society, U.S.A., pp. 142-156. 

Huke, R.E. & Huke, E.H. 1990. Rice. then and now. Manila, International Rice Research Institute. 44 pp.

Ikehashi, H., and S. Kiyosawa. 1981. Strain-specific reaction of field resistance of Japanese rice varieties revealed with Philippine strains of rice blast fungus, Pyricularia oryzae Cav.. Jap. J. Breed. 31, 3: 293-301. 

International Rice Research Institute, 2001. Rice Research and Production in the 21st Century. 

International Rice Research Institute, 2002. Rice Almanac, 3rd Edition. 

Khush, G.S., and B.O. Juliano. 1985. Breeding for high-yielding rices of excellent cooking and eating qualities. In: Rice Grain Quality and Marketing, International Rice Research Institute, College, Laguna, Philippines, pp. 61-69. 

Khush, G.S., and Virmani. 1985. Breeding rice for disease resistance. In: Progress in Plant Breeding. Vol. 1. Butterworths, United Kingdom, pp. 240-279. 

Kim, C.H., and J.N. Rutger. 1988. Heterosis in rice. In: Hybrid Rice. International Rice Research Institute, College, Laguna, Philippines, pp. 39-54. 

Lee, E.J., Qi Zhang and T.W. Mew. 1989. Durable resistance to rice disease in irrigated environments. In: Progress in Irrigation Rice Research. International Rice Research Institute, College, Laguna, Philippines, pp. 93-100. 

Ye, X, Al-Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P and Potrykus, I. 2000. Engineering the Provitamin A (b-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm, Science, 287: 303-305.

Hybrids or Improved Populations for Poor Farmers : A Breeder's Debate

K K Vinod, Javed Sidiqui, Konnie Frederick, Jorge Venegas, Raquel Guedes, Scott Matthew Dworak, Mauricio Erazo-Barradas and Brian Patrick Bresnahan

During one of our threaded discussions, my Professor  of University of Nebraska-Lincoln, Dr Stephen Baenziger was asking us of the choice of recommending improved populations or hybrids for the poor farmers of a country. Following is a note prepared on the discussion that went on the board.

There were thirteen messages of discussion. There were arguments favoring hybrids and improved populations but, general opinion largely favored the latter. 

The first respondent, Javed Sidiqui, had the preference to choose and release the improved seed to a the poor and small farmer's community considering the facts like, hybrid seeds are expensive and poor farmers may be unable to buy it in every season for cultivation due to its high price and also they can not use the seed from one year to another while having access to improved seed offer them the opportunity to save their own seed for next cultivating season. Furthermore, hybrid seed requires more dose of fertilizers, much greater amount of water and technology.

Improved seeds have to be tested at different phases to be adoptable in the region where it is cultivated in view of tolerance of drought, disease resistance, and other abiotic stress conditions. Javed concludes saying, as plant breeders we are responsible for producing improved seed based on specific farming conditions and needs of the poor farmers, because they may be dwelling in marginal farm environments (e.g., poor soils, and little rainfall) and my not be having adequate money to buy, fertilizers and pesticides; for they depend mostly on plants that survive and produce under adverse conditions year after year. 

Konnie Frederick however, suggested in favor of hybrids arguing, if the poor small farmer gets a hybrid he can select the best plants prior to pollination to improve his crop for next year.  He can then save seed and trade seed with another farmer who has a different hybrid and cross those.  By being able to barter with other farmers in his surrounding area, he can improve his crop yield.

Jorge Venegas while respecting Konnie’s ideas cautions that, reality in our poor countries is different. Commonly, our poor farmers do not have access to this technology; of course, that is simply to us, but they do not have education and funds to give to this hybrid its requirements. Therefore, if we want to implement a hybrid production program in a poor country, we have to be sure of the complete adoption of these hybrids in the poor farmers. The support of government and nongovernmental organisms is a main point in this technology implementation. Jorge adds that we must think that these hybrids require optimal conditions to produce very much. However, commonly the poor farms have strong conditions or marginal farm environments as Javed said previously. His experiences in Honduras and Ecuador, both poor Latin countries, where poor farms are localized in the most difficult terrains, on very inclined slopes and poor soil make him to suggest in favor of improved populations.

Scott Matthew Dworak, however, fully backed Javed’s ideas, adding that many of small-scale farmers who farm mainly for their own food supplies are unfortunately ignored by giant seed companies, who typically release hybrid seed, because the poor farmers aren’t viewed as attractive customers to these giant firms.  Market-based solutions are not an effective means in this aspect; poor farmers, like Javed said, lack the resources to pay for hybrid seed and manage it via cultural practices.  These farmers, located in rural areas, continue using farm-saved (improved) seed simply because they are not integrated into the market economy.   Distribution and/or allocation of resources may need to be addressed.

Konnie however, argues that if several of the smaller farmers’ pool their seed order they may be able to get a better deal on hybrid seeds than if they bought it by themselves.

I chose to complement Javed and Scott for their comments and presented my views focusing on a country where there are predominantly poor farmers, where we can expect these farmers to have low yielding crop varieties, mostly may be landraces. Agro-management also may be poor. However, these varieties may be highly locally adapted, having better quality, better resistance to biotic and abiotic stresses and good genetic variation. They may have less genetic purity due to outcrossing and unscientific propagation practices. In a situation like this, introduction of hybrids is not advisable due to following reasons.

a. High cost of hybrid seeds, which farmers may not be able to afford

b. Poor agro-management practices may not be suitable to exploit full potential of the hybrids

c. Farmers have the practice of advancing the seeds of his crop to next crop, which will result in a mixture of segregating materials if he uses a hybrid.

d. May not be suitable to his taste of quality

It is more prudent to go for population improvement under such situations. He emphasizes on subsistence farming rather than a market based approach, as a need to adopt under such situations. Different landraces can be improved separately by mass selection or recurrent selection procedures, and the traits can be combined if required using hybridization and selection. Once the yield levels are pulled up combining with good quality, pedigree breeding can be looked into. This will definitely improve the farmers returns also and his financial positions. He need to be taught about good agro-management practices and made aware of them. 

When farmers become self sufficient and are looking for a market, the hybrids can be introduce to him, which he would be able to buy, and grow as per the needs of the market, while adopting good management.

Raquel Guedes discussed that if he was to working in a country with poor farmers he would choose hybrids. Poor farmers who have lack of money to buy expensive hybrid seeds, they can buy double cross or three-way cross hybrids that are less expensive than single cross hybrids. These seeds are also more adapted to adverse soil and climate conditions and more resistant to diseases. He believes development is reached with high technology. If open-pollinated varieties (OPVs) would be the solution, developed countries would not be using 100 % of hybrid seeds.

Approximately 58% of the maize area in developing countries is planted to improved maize: 44% to hybrids, 14% to improved OPVs, and 42% to unimproved OPVs. In contrast, nearly 100% of maize area in the developed countries is planted to hybrids. Improved OPVs are easier to develop than hybrids; their seed production is more simple and relatively inexpensive (CIMMYT, 1994; Pandey and Gardner, 1992). The farmers who grow them can save their own seed for planting the following season, reducing their dependence on external sources. However, OPVs do not produce as much as hybrids. 

Crossing the progeny of a single cross with an unrelated inbred results in a three-way cross hybrid [(A x B) x C]. Crossing the progeny of two unrelated single crosses results in a double-cross hybrid [(A x B) x (C x D)]. Single-cross hybrids result from crossing two unrelated inbreeds (A x B). Single-cross hybrids generally have higher grain yield and less variability in appearance and maturity than do the three-way and double crosses because they are genetically uniform and they also cost more (Extension Service of Mississippi State University, 1914). 

Furthermore, governments would also need to make sure that there is some assurance that farmers are going to receive a fair price for their product at harvest time, and this price must reflect the international price for that commodity.

Mauricio   Erazo-Barradas prefered to release an improved population rather than a hybrid. While agreeing partially with the answer/argument provided by Raquel, Mauricio would stick to the idea of releasing an improved population. This improved population would be a "better" open pollinated population (better OPV) that would be developed using two different approaches/methodologies proposed by Pandey and Gardner (1992) and CIMMYT (1994), briefly described as;

a. Regardless of the recurrent selection scheme employed, 8-10 superior families should be identified based on their performance in multi-location tests. Using their remnant seed, the selected families should be intermated by making plant-to-plant diallel crosses among them to form an OPV. Diallel crossing among 10 or fewer genotypes is easily accomplished, permits more complete recombination, and reduces inbreeding (Hallauer and Miranda, 1988). In the crossing block, if a family looks different from other families during any stage of its growth and development, it can be discarded before or after pollination. Plants of other families fertilized with pollen from the undesirable family must also be discarded. 

b. Superior OPVs can also be developed by recombining elite inbred lines not derived from a population improvement program. In this case, it is desirable to select 8-10 lines with high general combining ability and intermate them as described before. High-yielding OPVs have also been developed by crossing among four or five single- or two or three double-cross hybrids. It is recommended that the parents of the hybrids- that is, the inbred lines- be selected and used instead of the hybrids themselves to form an OPV. This is because general combining ability is more important in the performance of OPVs than specific combining ability (which plays a greater role in the performance of hybrids).

Konnie  continued to emphasize on the importance of hybrids, says that the Green Revolution has done a lot to help poor and the underdeveloped countries become sustainable in its own food production.  The large seed companies have also jumped into help out, however the rapid progression of biotechnology has done little to aid in putting a curb on world hunger.  Biotechnology may be helping the developing countries, but it has done little to help the poor as they can not afford to purchase seeds to advance their crops thru technology.  Hybrids are less expensive and more beneficial to the poorer farmers as soil conditions and rainfall all play a big role in increased production; whereas an improved population variety may not do as well in the adverse conditions that may be presented in specific area.  

The farmers in question are not producing corn to sell on the open market, but for their own food consumption.  They have very few resources that are available to them and the big seed companies overlook the very small producers who may only buy one bag of seed corn a growing season.  These farmers are more likely to save their own seed from year to year to cut expenses, so the hybrid would be the best choice to begin with.

Scott Matthew Dworak went ahead with his idea by taking alfalfa (an autotetraploid) as a good option, if the seed can be made inexpensively enough for the farmers.  Segregation is restricted to a great degree in alfalfa varieties.  Not all genotypes can occur in early generations of seed increase, and several generations are required for all segregates to appear.  For example, in a 6-parent variety more than 17,000 distinct genotypes are formed at a locus in the third generation, while the first generation is relatively uniform.

Plant-to-plant variation is limited in the early generations of seed increase.  The greatest change comes in the Syn 2 generation, and variety stabilizes in the Syn 4 (Busbice and Gurgis, 1976).  Early generations may differ dramatically from later generations.  The Syn 1 and Syn 2 represent the breeder and foundation seed generations, respectively, and often are tested under experimental designations.  It is the Syn 3 and Syn 4, which represent the third and fourth generations of seed increase, respectively, that are sold to farmers as planting (certified) seed.  Based on alfalfa’s autotetraploid genetics, the Syn 1 and Syn 2 generations are more uniform than the commercial variety and higher yielding than the commercial variety (Busbice and Gurgis, 1976).  This means that all traits influenced by heterosis or genotypic structure such as yield, plant height, and persistence are confounded by the generation of seed increase.  For these traits, commercial varieties must be compared using commercial seed samples, not experimental ones.

If Syn 1 or Syn 2 seed could be sold to the farmers at an inexpensive price, farmers would get relatively uniform yields, which would be ideal.  Furthermore, alfalfa is a leguminous species, so the crop would freely add nitrogen to the soil, reducing expensive fertilizer costs in the future for the poor farmers.

Brian Patrick Bresnahan, is focused on the problem how he as a plant breeder would train his efforts on what is feasible, desirable among those who are going to be his customers, the recipients of the breeding program. His experience in Iraq forces him to think more of populations rather than hybrids. He calls that the question one should ask himself in addressing the problems of poor farmers is that, "what are my objectives? What do the farmers need and want in this poor, rural country?". Sure, they could use hybrids, especially if hybrids were available which fit the specific growing conditions and agronomic conditions of their area.  He recalls of a dozen corn hybrids he had seen in some of the salty, drought prone, sandy, high pH soils of Southwest Nebraska, western Kansas, and the Panhandle of Texas that would have been interesting to try in the fields west of Fallujah, Iraq where he worked for some time.  Although the staple grain was wheat in that area, some corn was planted and the potential for more corn did exist.

However, in reality, with regard to corn seed, none of the small farmers he worked with in Iraq had the money for hybrid seed.  They were subsistence farmers, just trying to feed themselves another year.  Thus, the corn they planted was open pollinated, saved seed.  At times they were provided one of two hybrids (one from Iraq the other from Jordan) if the government provided them that for the year, but they mostly relied on their own, saved seed, or saved seed they purchased elsewhere in their villages.  So, to fit that group, as a breeder, he would work on improved populations because they couldn't afford hybrid seed and could at least stand a chance of improving yields over time.

Additionally, in many of the countries he visited did not have the infrastructure to support a government funded breeding and seed production program for distribution to their country's farmers. That leaves the seed industry, which has been pointed out by others, is not likely to invest in such small, unstable, likely unprofitable markets. So, again, efforts would have to focus on improved populations as a cheaper alternative because it would have to be assumed that funding for the research and the distribution of seed in a poor country would be limited.

He went ahead of suggesting that, if funding were available, say through a USAID funded program implemented by a land grant university, he could start a corn breeding program in the poor country, long term though, developing hybrids to fit the farms which are owned and operated by the few elite/rich farmers in the country, something that seems to be consistent. With hybrids, the agronomic and production challenges the farmers face are much easier/quicker to overcome than with improved populations. Over time, if the government stabilizes, the older hybrids might be made available to the poor farmers through a government program. 

If the market/acreage among this group was initially large enough and potentially profitable enough, there could be a possibility for commercial funding, or at least continued U.S. federal funding as long as the political interests deem it a priority.  Which in and of itself might be another reason to focus efforts, and limited resources, on improving populations because political priorities are sure to change and multi-national corporations are fickle when it comes to profitability.    

References

Busbice, T. H. and Ramzy Y. Gurgis. 1976. Evaluating parents and predicting performance of synthetic alfalfa varieties. USDA, ARS-S-130. June 1976.

CIMMYT. 1994. CIMMYT 1993/94 World Maize Facts and Trends. Maize seed industries, revisited: Emerging Roles of the Public and Private sectors. Mexico, D.F. 

Extension Service of Mississippi State University, cooperating with U.S. Department of Agriculture. Published in furtherance of Acts of Congress, May 8 and June 30, 1914.

Hallauer, A.R., and J. B. Miranda Fo. 1988. Quantitative genetics in maize breeding. 2nd ed. Iowa State University Press, Ames, IA.

Pandey, S., and C. O. Gardner. 1992. Recurrent selection for population, variety, and hybrid improvement in tropical maize. Advances in Agronomy 48: 1-87