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

On the Genetic efficiency of in situ conservation of germplasm

K K Vinod

In situ conservation, is the continuation of this traditional method of informal breeding that dis­tinguishes in situ from ex situ conservation on farms and in gardens. However, many developed countries now have legislation protecting plant breeders' rights, which effectively prevents the continuation of traditional agriculture and therefore in situ conservation of crop species. This section therefore applies to in situ conservation of wild species, and of crop spe­cies only in those countries where legislation permits.

Sites for inclusion in a network for in situ conservation must be chosen to maximize the diversity that can be maintained. This is similar in principle to choosing sites for collection to maximize diversity collected.

Sites should cover the entire ecological range of the species, with a bias towards its centre(s) of diversity and the ecological extremes of its distri­bution. There should be a stratified distribution of sites with several levels of clustering: each site should be large enough to encompass a cluster of several genetic populations; each site should be part of a cluster of several nearby sites, close enough for occasional gene flow between sites as a result of rare long-distance dispersal events; each cluster of sites should be part of a larger cluster; and so on to encompass the entire range of the spe­cies. Such stratification will serve a dual purpose of: (i) optimizing gene flow within and between populations; and (ii) encompassing the maxi­mum possible range of types of diversity. Within the general stratification, sites and clusters should be chosen to maximize diversity of environments and therefore of selection pressures within and between sites and clusters. As always, of course, diversity of selection pressures is taken to include artificial as well as natural selection, with diversity associated with varia­tion in local preferences and farmers' concepts of quality and agronomic value.

The shape of sites and clusters also needs consideration. For example, linear habitats may be useful to increase connectivity between clusters with minimal increase in areas of the region set aside for conservation. For rare species, there may be a need to create new popula­tions at sites with sufficient connectivity to existing populations to prevent loss of diversity through inbreeding.

Additional measures can be taken to increase biodiversity within the selected network of conservation sites, essentially by increasing the diver­sity of environments and selection pressures within and between sites.

For crop species, farmers can be actively encouraged to value the dis­tinctiveness of the traditional farming practices of the region; and the local customer community can be actively encouraged to value the distinctive­ness of local traditions and their consequent demands on local farmers. Important traditional farming practices can include factors such as con­scious selection by the farmer for genetic variation within and between varieties for tolerance to disease, drought, heat, etc. These traditions are based on utilizing high diversity to provide low-cost, sustainable, low-risk protection from environmental stresses and hazards. That is, they benefit not only conservation of biodiversity but also the farm economy.

For wild and some crop species, there can also be opportunities for increasing diversity by appropriately diverse management. Emphasis is on diverse management, as many management procedures, especially mech­anized ones, tend to reduce diversity. For example, cutting, liming, fertil­ization and control of weeds, pathogens and pests are usually applied uniformly across entire sites; in so doing they reduce environmental diver­sity and therefore the diversity of selection pressures and biodiversity at the scale of the site. If such procedures are also applied consistently from year to year, there will also be less temporal variation in selection pres­sures, again reducing biodiversity at the scale of the field. In contrast, management by grazing imposes cutting, trampling and fertilization that is spatially and temporally variable - to an extent that depends on the grazing behaviour of the selected herbivore.

Similarly, diversity of management should be encouraged at larger scales, including landscape and regional. The principal problem here relates to how to construct and implement a conservation policy. For example, a management policy may be implemented that maximizes bio­diversity within a field; but if that same policy is applied to all sites, the same range of biodiversity will be promoted at all sites, reducing biodiver­sity at the larger landscape and regional scales. If the policy is to be cen­trally established and imposed, it may be economically impossible to incorporate the larger-scale variation in management necessary to max­imize biodiversity at landscape and regional levels. A decentralized system is likely to be preferable, especially to incorporate regional varia­tions in traditions.

We have seen that biodiversity is a scale-dependent phenomenon and that for its efficient conservation we need to include all scales from a few square centimetres to thousands of square kilometres. We have also shown that the distribution of genetic diversity of any spe­cies depends on its life cycle and consequent evolutionary characteristics. Efficient conservation depends on having a good knowledge of population structure and the life cycle characteristics that determine this - dispersal profiles, breeding system and longevity. The same principles apply not only to wild species but also to crop species, the major difference being that crop species have dispersal profiles determined largely by the farmer and mar­ket, and are subject to artificial selection by the farmer as well as natural selection.

References:

Forman, R.T.T. (1995) Land Mosaics: the Ecology of Landscapes and Regions.Cambridge University Press, Cambridge.

On the Genetic Efficiency of ex situ germplam conservation

K K Vinod

When we plan for an expedition for germplasm collection from a region following objectives are need to be set.

1. Acquire the maximum genetic diversity of targeted taxa within the region, within the constraints of limited available resources.

2. Acquire germplasm with the maximum novelty value with respect to a collection already held ex situ; i.e. the greatest number and diversity of genes and genotypes that have not previously been collected.

3. Combat genetic erosion.

4. Acquire genes or genotypes most likely to benefit a particular breeding or research objective.

5. Acquire germplasm for analysis of agro-ecogeographic patterns of the distribution of biodiversity.

In the case of first objective, limiting resources vary from expedition to expedition, and may be time (time to travel to a site, time to collect overall site data, time to collect each seed or plant at a site, speed of returning live plants to base); space available in the collecting vehicle; or labour and facilities to process samples at base. Resource limitations influence optimal collecting strategy in a way that depends on population structure.

For the objective 2 above, except that additional information is needed on the diversity and origins of the pre-existing collection.

Where the primary objective is to combat genetic erosion (objective 3 above), sampling strategy can and should still be designed to satisfy objec­tive 1. However, painstaking planning to maximize the diversity collected may be counterproductive where the rate of erosion is so high that diver­sity is lost whilst planning is in progress. In these circumstances, speed of undertaking a collecting expedition is of overriding importance.

Objective 4, a breeder-driven collection to support a particular breed­ing objective, requires a totally different sampling strategy, to locate par­ticular genes rather than maximize diversity of genes. Nevertheless, knowledge of evolutionary patterns can aid identification of sites most likely to contain the desired genes or genotypes.

Objective 5, collection for agro-ecogeographic analysis, requires yet another strategy, namely an appropriately randomized sampling proce­dure. This fact is not sufficiently recognized, as many published analyses are based on collections made for conservation or breeding purposes. Yet any sampling strategy that aims to maximize diversity or to target specific genes can generate incorrect and misleading estimates of components of variance. For example, suppose two collections are undertaken in two dif­ferent regions, both with a sampling strategy to maximize the diversity sampled within each region. A comparison of both collections will then incorrectly suggest that there is less difference between them and more variation within each region than is really the case.

A specific example of an erroneous conclusion may be the widely accepted latitudinal cline in genetic diversity of Trifolium repens across Europe, according to which southern populations are believed to be highly diverse and northern ones uniform. This is likely to be spurious, and at least partly a consequence of collections being specifically targeted at well-managed pastures in the north but at highly diverse habitats in the south. A northern collection targeted at diverse hab­itats contained as much diversity as southern populations (Hamilton, 1980).

Appropriate randomization for agro-ecogeographic analyses need not mean full randomization. Collecting for maximum diversity can be com­patible and even beneficial to agro-ecogeographic analysis. Seeking to col­lect maximum genetic diversity by targeting maximum environmental diversity improves the sensitivity of analysis of the relationship between genetic diversity and environmental diversity. This of course requires col­lection of all relevant environmental data so that they can be incorporated as independent variables in statistical analysis.

Collections targeting spatial scale of biodiversity and overall distribution of sites

The general scaling properties of biodiversity have two imme­diate implications. First, it is important to cover as large an area as pos­sible. Second, adjacent sites should not be further apart than the genetic patch size, since increasing the geographical distance beyond this does not increase the expected genetic distance between two populations. For this purpose, genetic patch size should be measured using neutral genes, to provide a general baseline sampling strategy that is not influenced by any particular pattern of environmental diversity

At the lower end of the scale, the genetic neighbourhood area defines the minimum possible scale for taking distinct population samples, at least of the seed population. At smaller scales mating is random and Hardy-Weinberg equilibrium is expected, with no possibility of division into genetically distinct subpopulations. This applies only to seeds: the population of adult plants may show genetic subdivision at smaller scales if the environment is heterogeneous at smaller scales, imposing smaller scale heterogeneity of pressures within the genetic neighbourhood. Thus there may be merit in finer-scale sampling of adult populations than seed populations.

However, the genetic neighbourhood area of most wild plant species is remarkably small, far smaller than the unit regarded as one population by the collector. For the insect-pollinated self-incompatible perennial Trifolium repens the reproductive genetic neighbourhood area is 2m²; for the wind-pollinated self-incompatible per­ennial Lolium perenne it is 8.4 m². In practice, therefore, each sample of a wild population in an ex situ collection almost invariably comprises genotypes from what were originally numerous dis­tinct genetic populations.

The genetic neighbourhood area of crop plants is closely related to the type of farming. In primitive farming communities it is generally much smaller than for modern agriculture. Farmers in such communities usually maintain and select their own seed, with limited 'dispersal' (by seed exchange) between isolated communities or even between farmers within communities. It is essential, when collecting, to determine what are the local customs in relation to seed selection and exchange, especially (i) whether a formal centralized system exists for exchanging seed, or whether exchange is informal and centralized through the market, or informal and localized to individual farmer-farmer interactions; and (ii) how much farmers rely on their own farm-saved seed, and if so whether they consciously make their own selections. Only with such local knowledge can the collector judge the probable scale of distribution of diversity.

Collections targeting by habitat and adaptation to environment

Targeting the maximum diversity of habitats for collection will maximize the diversity of genes contributing to adaptation to the selection pres­sures imposed in the environments sampled. It will also maximize diver­sity of genes closely linked to the adaptive genes and of pleiotropic characters. It will have no effect on the diversity of genes that are neutral for the particular environmental diversity sampled - this includes not only genes that appear neutral with respect to all known selection pres­sures, but also genes that are non-neutral for different types of environ­mental diversity.

Effective environmental targeting in this manner depends on the col­lector having good knowledge of environmental diversity in the region, and of the distribution of the target taxa in relation to environmental diver­sity. Much of the planning phase of a collection should be devoted to iden­tifying contrasting environments, using as many sources of information as possible, preferably in map form: not only conventional geographical maps, but also maps of surface geology, soil, temperature, rainfall, vegeta­tion and land use. Much additional information is not available in map form, and may not be readily available prior to the expedition, being in the knowledge domain of local extension scientists and farmers. Relevant local knowledge covers not only natural variation between fields but also diver­sity in farmer-selection pressures resulting from variation in crop usage and variation in preferred crop characteristics.

Collections targeting centres of diversity

It is now widely accepted that evolution does not progress at a uniform rate, but involves periods of relative stability interspersed with periods of rapid change. Exactly how and how much the rate of evolution changes is still the subject of debate. Nevertheless, for most species and genera it is possible to identify centres of diversity, associated with a phase of rapid diversification at some stage in their evolutionary history.

Centres of diversity are most strongly developed for crop species, leading to the famous pioneering work of Vavilov (1951). These centres are associated with early agricultural developments. They are attributed to disruptive selection caused by the simultaneous action of natural selection for fitness and artificial selection for agronomic value, combined with diverse artificial selections applied by different farmers in different envi­ronments, and with introgression between conspecific crop and wild rela­tive.

By definition, a collecting expedition will obtain the greatest diversity if it is located within the centre of diversity of the target taxa. The content of ex situ collections should therefore contain a bias in favour of popula­tions from the centre of diversity.

Sampling targeting environmental heterogeneity: stratified sampling

The environment is a multidimensional entity. Genetic adaptation to environment is correspondingly multidimensional. Different environmen­tal variables show different patterns of variation in space and time. Therefore genetic variation for adaptation to different environmental vari­ables also shows different patterns. For example, Hamilton (1980) collected Trifolium repens from an area of high diversity of soils and grass­land management but uniform climate. Relative to the global diversity pre­sent in the entire gene pool of T. repens, genetic diversity between populations was high for vegetative and morphological characteristics important for adaptation to soils and management but low for time of flowering. More generally, it may be, for example, that populations from adjacent fields differ mainly in genes affecting response to management; ones from nearby fields differ mainly in genes for response to aspect; ones further apart differ mainly in genes for response to soils; ones from differ­ent altitudes differ mainly for response to temperature; ones from differ­ent villages for local human preferences; ones from different latitudes for response to day length; and so on.

Given this situation, a stratified sampling strategy will not just max­imize the genetic diversity collected; it will maximize the diversity of dif­ferent types of genetic diversity collected. A particular advantage of stratified sampling is that it does not depend on prior knowledge of the different scales of heterogeneity of different envi­ronmental attributes. Although such knowledge helps, nevertheless the fact that different environmental variables show different scales of hetero­geneity is itself sufficient to make a stratified sampling procedure more efficient in obtaining qualitatively different types of genetic diversity.

For some purposes, the stratification of sampling procedure should be extended to sampling individuals within sites, at least for natural popula­tions. Certainly this will maximize within-accession diversity sampled from such populations. For example: sampling several individuals from a single genetic population will sample diversity in genes that are truly polymorphic at the genetic population level; sampling from different quadrats within a field will acquire diversity in genes responsible for micro scale adaptation to patchiness of the vegetation, soil characteristics, and microflora, microtopography, etc.; and samples from the boundaries of the field are more likely to contain immigrant genes from nearby, differ­ently adapted populations.

Stratification of sampling procedure within a population is rarely appropriate for crop populations and market populations. Even for natural populations it may not always be appro­priate. In particular, by maximizing within-population variance, a stratified sampling procedure will invalidate agro-ecogeographical comparison of dif­ferent populations. If this is important, a random sampling procedure is more appropriate, unless the individual plants are maintained separately.

For species where it is difficult, or even impossible as routine practice, to distinguish plants from each other - as with most perennial herbaceous species communities - it may be impossible to take a truly random sam­ple. In these species, only inflorescences, or leaves, or some other part of the plant, can be sampled at random. This inevitably introduces a size bias into the sample in favour of those plants with the most inflorescences, leaves, etc.

Sampling targeting breeding system and adjustment of sampling procedures

The breeding system has a major influence on the distribution of genetic diversity. A number of mechanisms operate to fix par­ticular variants in lines: inbreeding fixes it through homozygosity; apo­mixis fixes variants even in heterozygotes; some complex chromosome linkages, like those in Oenothera, operate to minimize recombination. All these cases reduce within-population variance, so that a correspondingly increased proportion of the total gene pool is represented by variation between populations. In contrast, outbreeders show higher within-population variance. Sampling procedures must be adjusted corres­pondingly, to take relatively few individuals from many populations of inbreeding, apomictic and similar species, and many individuals from each of fewer populations of outbreeding species (Marshall and Brown, 1975).

Vegetative propagation (by stolons, bulbs, rhizomes, etc.) is function­ally equivalent to apomixis in that it can generate numerous genetically identical plants. However, vegetative propagation is often associated with outbreeding, generating a complex two-level population structure. There is high genetic variance among the individuals originating by sexual reproduction through different zygotes, and zero genetic variance (ignor­ing somatic mutations) among the vegetative progeny derived solely by mitotic division from a single zygote.

In many such vegetatively reproduced species it is impossible to know at a glance whether two plants are derived from the same or from differ­ent zygotes. In these species the two-level population structure can present very considerable problems for collection for efficient conservation. The commonest approach is to ensure a large enough distance between sam­pled individuals so as to be reasonably confident that they are genetically distinct. However, a single clone of even small herbaceous species can cover hundreds or thousands of square metres. The distance between adjacent samples therefore has to be undesir­ably large, in that it eliminates sampling the genetic diversity that is expressed at smaller scales. For many species there is no satisfactory reso­lution to this problem, as the only resolution may be intensive sampling followed by genetic fingerprinting to determine the genotypic composition of the population sample, which of course is unjustifiably labour intensive.

Populations of these species often show a highly skewed distribution of physical size of genotypes, with a few large genotypes and many small ones (Hamilton et al., 1996). When population sam­ples are based on a random selection of inflorescences or leaves, the sam­ple will be strongly biased in favour of the few large genotypes.

Collections targeting temporal scale of biodiversity

Little attention has been paid to the temporal scale of biodiversity for con­servation purposes. Whilst the importance of cyclic fluctuations, chaotic changes and continuous directional shifts are all well acknowledged and documented, it has rarely if ever been considered justifiable for conserva­tion purposes to return to the same sites for repeat collections. The only common reason for returning to a site or region is to test specific hypothe­ses, for example to test the extent of genetic erosion.

References:

Hamilton, N.R.S. and Chorlton, KH.C. (1995) Collecting vegetative material of forage grasses and legumes. In: Guarino, L, Ramanatha Rao, V and Reid, R. (eds) Collecting Plant Genetic Diversity: Technical Guidelines. CAB International, Wallingford, UK, pp. 467-484.

Hamilton, N.R.S., Jones, D., Cresswell, A. and Fothergill, M. (1996) Genetic diversity and sustainability of clover-based pastures: 2. Size hierarchies and sampling bias. In: Younie, D. (ed.) Legumes in Sustainable Farming Systems. Symposium of the British Grassland Society, Aberdeen. pp. 179-180.

Marshall, D.R and Brown, AH.D. (1975) Optimum sampling strategies in genetic conservation. In: Frankel, O.H. and Hawkes, J.G. (eds) Crop Genetic Resources for Today and Tomorrow. Cambridge University Press, Cambridge, pp. 53-80.

Vavilov, N.I. (1951) The origin, variation, immunity and breeding of cultivated plants (translated by KS. Chester). Chronica Botanica 13, 1-366.

Characterization of variability and population structure

K K Vinod

The state of variability within and between populations can be determined by application of both biometrical and molecular procedures. Whilst the former can describe a population in terms of means and variances and the underlying mode of gene action, if an appropriate biometrical design is applied as part of the characterization of a population (Kearsey, 1993), it is only the procedures of molecular genetics which allow a measure of population structure at the level of the gene and genome to be gained. Various molecular tools are available for the characterization of popula­tions. The most widely used to date is electrophoresis of isozymes, but direct DNA methods, including restric­tion fragment length polymorphisms (RFLPs), randomly amplified poly­morphic DNA and amplified fragment length polymorphism (AFLP), are now being exploited. The application of these procedures allows several different parameters of variability within and between populations to be determined. These include the percentage of polymorphic loci, average and effective numbers of alleles per locus, heterogeneity and heterozy­gosity indices and the various measures based upon the F statistics of Wright (1965).

More recently the application of DNA-based technologies, particularly 'fingerprinting', has provided a wealth of infor­mation on the diversity of wild populations and the relatedness of cultivars. For wild species these techniques have been applied to answer­ing specific questions particularly in relation to breeding systems. A com­parison of Plantago spp. using RFLP analysis revealed by the M13 probe showed that the inbreeding P. major possessed little variation within pop­ulations but marked differentiation between populations whilst the out­breeding P. lanceolata possessed high variability within populations but only moderate variability between populations. Similarly, in an analysis of cultivar differentiation in three species of Bermuda grass (Cynodon), using DNA amplification fingerprinting (DAF), were able to distinguish some closely related cultivars. On the basis of this technique's discriminatory power they recommend that it be used as a method for seed certification and registration purposes. These are just two examples of the many that have been carried out on a wide range of species exploiting the 'fingerprinting' capacity of the molecular methods.

In the majority of applications of these DNA-based technologies com­parisons are based upon statistical analyses of the number of 'bands shared'. Such a procedure can be fraught with problems both on a techni­cal and a genetic level. Some of the technical problems and other sources of error which can be encountered in the preparation, running and read­ing of gels have been considered by Weising et al. (1995), who emphasize the need for technical care and caution in scoring closely spaced bands. In addition there is also the question as to whether two DNA fragments which migrate to a common position on a gel are homologous. This may only be ascertained by extraction of the bands and comparative cross-­hybridization. In RAPD analysis of the Lolium/ Festuca complex of species it was found that four out of six amplification products were homologous when tested by Southern hybridization. Although this is a small sample and ranged across genetically diverse species, it does emphasize the need for caution in assuming homology.

The use of individual isozyme loci and similar genetic markers, as measures of variability, is limited in that they give no indication of the genomic associations that are of importance in the maintenance of co-­adapted gene complexes. It is only by looking at combinations of markers that such information can be ascertained. In Avena spp. and Hordeum spp., for example, Allard and his coworkers (Allard, 1990; Allard et al., 1993) have shown by comparisons involving up to 14 discrete loci that popula­tions are made up of individuals containing differing multilocus assem­blages of favourable epistatic combinations of alleles. These have arisen by rare outcrossing followed by inbreeding to near homozygosity. In A. hir­tula, for example, a majority of Spanish populations were found to be poly­morphic for different multilocus genotypes, which suggests that 'interactions at the interplant level may contribute to adaptive significance' (Allard et al., 1993). In addition, in this species, polyploidy is present which, in its own right, can lead to a greater allelic diversity and potential for differing multilocus associations. Here again the breeding system rein­forces the stabilization of such associations by restricting the degree of recombination that takes place.

The current procedures using molecular markers for assessing popula­tion differentiation consider both expressed and non-expressed parts of the genome. Given the ease with which genetic maps may now be con­structed, it seems likely that, in future, measures of population differentia­tion will take into account genome organization and will target those regions of interest. Already this problem is being addressed as a means of determining identity by descent as part of the statutory procedures in determining 'essential derivation' of cultivars (Dillmann et al., 1995).

Recent developments in QTL analysis of crop plants in defined popu­lations, such as F₂s and recombinant inbred lines, are now bringing together the power of genome analysis at the molecular level and biomet­rical procedures which will eventually allow a more detailed understand­ing of the genetic architecture of traits of both agronomic interest and of importance to evolutionary fitness.
These various studies of population structure provide an insight into the manner in which variability is distributed within a species and some of the factors controlling that pattern. From a conservation aspect it is now neces­sary to consider how these mechanisms interact to determine the spatial dis­tribution of variability and their implications for collection and conservation.

References:

Allard, R.W. (1990) Future directions in plant population genetics. In: Brown, AH.D., Clegg, M.T, Kahler, A.L and Weir B.S. (eds) Plant Population Genetics, Breeding, and Genetic Resources. Sinauer Associates, Sunderland, MA, pp. 43-63.

Allard, R.W., Garcia, P., Saenz-de-Miera, L.E. and Perez de la Vega, M. (1993) Evolution of multilocus genetic structure in Avena hirtula and Avena barbata. Genetics 135, 1124-1139.

Dillmann, C, Charcosset, A, Bar-Hen, A, Goffinet, B., Smith, J.S., Datte, Y. and Guiard, J. (1995) The estimation of molecular genetic distance in maize for DUS and ED protocols: optimisation of the information and new approaches of kinship. BMT /3/6. UPOV; Working Group on Biochemical and Molecular Techniques and DNA Profiling in Particular. Upov, Geneva, pp. 2-27.

Kearsey, M.J. (1993) Biometrical genetics in breeding. In: Hayward, M.D., Bosemark, N.O. and Romagosa, I. (eds) Plant Breeding: Principles and Prospects. Chapman & Hall, London, pp. 163-183.

Weising, K., Nybom, H., Wolff, K and Meyer, W. (1995) DNA Fingerprinting in Plants and Fungi. CRC Press, Boca Raton, FL, 322 pp.

Wright, S. (1965) The interpretation of population structure by F statistics with special regard to system of mating. Evolution 19, 395-420.