Contact person for Genetic integrity: Jessica Rey, IRRI, Philippines
Contributors to this page: IRRI, Los Baños, Philippines (Jessica Rey, Kenneth McNally, Ruaraidh Sackville Hamilton); Bioversity International, Montpellier (Elizabeth Arnaud); CIMMYT, Mexico (Suketoshi Taba); CIP, Peru (David Tay); ICARDA, Syria (Kenneth Street); ICRISAT, Patancheru, India (Hari D Upadhyaya).
The magnitude of genetic differences between supposedly duplicate samples is disturbingly high, even unacceptably high. All causes of genetic change are significant contributing factors – genetic drift, unintentional selection, pollen contamination, seed contamination, and mislabelling. In particular, analysis has demonstrated an unexpectedly high rate of mislabelling, a risk that existing “best practices” for genebank management have failed to manage. Similarly, existing best practices to not address the loss of diversity of genes for flowering date noted in the maize dataset.
The greatest changes in genetic composition are apparent between duplicates maintained at different genebanks; the differences are such that “duplicates” at different genebanks should perhaps be described as “equivalents” rather than “duplicates”. Significant changes also occur during management within a genebank.
Thus it is important and urgent to improve both the handling of accessions within genebanks and the transfer of accessions between genebanks, and to develop a strategy and protocols to do so.
- Germplasm handling standards need to be significantly raised in all genebanks through implementation of appropriate quality management systems (linked to activity 1.1 in the risk management section and 2.3.1 regeneration procedures section).
- “Rationalization” among seed genebanks sharing crops in common, in the sense of eliminating or combining duplicates that have the same historical origin, is strongly deprecated. Eliminating or combining duplicates may only be considered if supported by DNA data demonstrating biological duplication, not only historical duplication. However, the cost of genotyping to demonstrate biological duplication still remains very high compared to the cost of seed conservation. This would be particularly true, if a large number of markers are required to capture minor differences in two seemingly duplicate accessions (in several instances even a phenotypically dissimilar accession (through having the same number) may be difficult to differentiate using markers. Systematic identification and elimination of biological duplicates therefore remains an ineffective, inefficient strategy for species with conventional seed that can be conserved long-term in cold stores.
- Other forms of “Rationalization” among seed genebanks sharing crops in common may be considered, provided they are not affected by the presence of genetic differences between equivalent accessions. For example, responding to seed requests could be rationalized by redirecting them to the most convenient source for the recipient, provided there is no specific reason for preferring the specific samples of equivalent accessions held at other genebanks.
- Eliminating duplicates within and among genebanks holding collections of clonal crops is justified because of the high cost of conservation, but only when supported by rigorous molecular characterization.
- Serious consideration should be given to the development of low-cost “DNA barcoding” technologies and to their incorporation as routine checks during genebank management. For example, ideally:
- Every new seedlot produced after a cycle of regeneration should be screened and compared against its parent or most original sample, to assure maintenance of genetic integrity. This is important, although it will add to the cost.
- Every transfer of seed between genebanks should be accompanied by data specifying the DNA barcode of the original seed source, to be verified by the receiving genebank on incorporating the seed into its collection.
- In terms of throughput and cost-effectiveness, SNP chips are now considered the most suitable technology for tracking germplasm and quantifying their genetic integrity.
- Low density (96 SNP) chips are effective for detecting mislabelling errors.
- Higher density chips are needed for the other factors contributing to loss of genetic integrity.
- Serious consideration should also be given to other methods to promote accurate germplasm tracking during genebank operations, such as barcoding all genebank operations and/or the use of high-precision GPS to identify plots without relying on plot labels.
Recommendations for clonal crops
- Using molecular markers combined with microscopic determination of ploidy level, check the classification of every newly received sample before introducing it into the collection.
- Verify before accessions are made available for distribution. DArT could be an appropriate tool, but the cost and the practicalities of DArT markers (in 96-well plates), for the moment, still do not make it appropriate for routine use.
- The new recommendation for Musa is now to test incoming material by SSR markers and ploidy determination.
- Regularly analyze accessions using SSR or DArT.
- Request recipients of germplasm to provide effective feedback on the comparison of the received material in the field with photos/descriptions of the original sample.
- Every ten years, verify the morphology of the accessions maintained in vitro (medium-term conservation), by growing them on the field. This would thus represent 1/10th of the collection each year.
- So far, molecular methods have not been effective for identification of off-types, or somaclonal variants.
- Morphological observations are required to detect these.
Thus during management of Musa, there is a need for more regular, specific morphological and molecular characterization to ensure adequate quality control.
Recommendations for seed crops
Existing FAO/IPGRI (1994) recommended standards for genebank management include these recommendations for seed increase:
- Regenerate only when required, either because of a loss of viability below a critical threshold (typically 85%) or because of a reduction in seed stocks below a critical threshold.
- Minimize the frequency of seed increase by ideally producing just enough seed to satisfy all seed requests before they begin to lose viability, so that regeneration will not be necessary until seed viability tests reveal a reduction in viability below 85%.
- Every cycle of seed increase almost inevitably results in some loss of genetic integrity, but we can and should avoid accumulating losses of genetic integrity over multiple generations of seed increase. As a general rule, avoid more than three successive generations.
These standards are supported, but should be more rigorously enforced and operationalized. It is recommended that the following elements of best practices be added:
- If seeds need to be multiplied early because seed stocks have been depleted sooner than 50% of the projected life span of seed in storage, double the plot size to store more seed from the next cycle.
- If seeds need to be multiplied because stocks have been depleted (not because of a loss of viability), then:
- Keep remnants of the parental seed for future seed increase – do not use them for distribution.
- For seed distribution, always use the youngest available seed lot of an accession.
- For seed multiplication, always use the oldest available viable seed lot of an accession.
- Conduct a final seed multiplication using the oldest seed lot at the moment its viability falls below 85%.
The maize data demonstrate a loss of diversity following regeneration, particularly in genes controlling flowering date; but they revealed no relation between the number of ears saved in the regenerated cycle and loss of genetic integrity among the accessions studied. This suggests that gene-specific selection is more important and random drift less important, (but it exists with inbreeding of the population, and should be preferably known with respect to the numbers of ears saved. However, it is usually assumed that outbreeding maize populations has a minimum level of inbreeding. Thus, a theoretical sampling strategy can still be employed to ensure conservation of the diversity present in the population. Inbred lines may need another study at SNP diversity level at various inbreeding cycles (as maize inbred can diverge in seed stocks maintained at the different seed genebanks and breeding stations) than has previously been supposed. It is concluded that the maize regeneration protocol should be modified to maintain the diversity of flowering dates unchanged.
In chickpea and rice, increases in average heterozygosity, as measured with Arlequin 3.0, were observed following regeneration. The results indicate that the consequences of cross-pollination and seed contamination are greater than have previously been supposed. It is common practice to ignore both factors, for example growing adjacent plots of self-pollinating crops with no control over cross-pollination. The results show this is inappropriate.
In rice it is known that almost all of the limited cross-pollination occurs over very short distances, between adjacent plots. Since 2007, the extent of cross-pollination between rice regeneration plots has been reduced in IRRI by sowing adjacent plots to varieties differing by more than two weeks in flowering date, so that a plot is never releasing pollen at the moment its neighbours have receptive stigmata: it has not been possible to evaluate the consequence of this management change during the current activity. Other methods need to be designed and introduced to reduce seed contamination.
A range of innovations is urgently needed to prevent the rapid losses of genetic integrity noted here. These include the introduction of new technologies such as barcoding, “DNA barcoding” with SNP chips and high-precision GPS. Morphological verification remains essential, both for somaclonal variants or other mutants that are difficult to identify with molecular methods, and for efficiently screening out obvious errors. In addition, we need a thorough re-evaluation of genebank management standards.
References and further reading
FAO/IPGRI. 1994. Genebank standards. Food and Agriculture Organization of the United Nations, Rome and International Plant Genetic Resources Institute, Rome. Available in English, Spanish, French and Arabic.
Hirano R, Jatoi SA, Kawase M, Kikuchi A, Watanabe N. 2009. Consequences of ex situ Conservation on the Genetic Integrity of Germplasm Held at Different Gene Banks: A Case Study of Bread Wheat Collected in Pakistan. Crop Science 49:2160-2166.