2.3. Description of the workpackages
Workpackage number: WP1 Genetic Resources
Phase: 24-36 months
Start date: February 2000
Completion date: January 2004
Current status: Ongoing
Partners responsible:
P1 C. Kik, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands: Tel: 31 317 477001, Fax: 31 317 418094, E-mail: c.kik@ plant.wag-ur.nl
P5 J. Auger. University F. Rabelais, IRBI, Faculty of Sciences and Techniques, Parc de Grandmont, 37200 Tours, France.Tel: 33 247 366970; Fax: 33 247 366966; E-mail:
P7: R. Kamenetsky. The Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel. tel: 972 3 9683511; fax:97239660589; e-mail:. Subcontractor: F.O. Khassanov, Institute of Botany, Uzbek Academy of Sciences, Khojaev Street 32, 7000143 Tashkent, Uzbekistan.
P10 H.D. Rabinowitch. Hebrew University of Jerusalem, Faculty of Agriculture, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot 76100, tel: 972 8481244, fax: 972 8 9468265, e-mail:
Person months per partner and total:
P1 – 8; P5 – 10; P7 – 30; P10 – 10. Total: 62
Already devoted person months per partner and total:
P1 - 7; P5 - 8; P7 – 23; P10 – 7 Total: 45
Objectives
The aim of this subproject is the collection of a large number of garlic accessions and close relatives in order to expand the current available short- and long-day garlic collections. The expanded collection is expected to have a much broader genetic variability related to the CSO content and the floral fertility. The construction of core collection is based on the level of similarity between accessions after DNA fingerprinting.
Workplan
Collection trips were carried out by P7 (Volcani, Israel) and P10 (Hebrew University, Israel) to the centre of origin of garlic (Central Asia). Approximately 300 accessions were sampled, grown, evaluated (P7and P10), and subjected to genetic fingerprinting (P1, The Netherlands) to determine the number of different genotypes/groups in the collection. The potential of these clones for fertility restoration was evaluated (P7, P10 and P1). Content of the organo-S compounds in selected clones was determined by P5 (France). On the basis of this a core collection of approximately 50 accessions will be constructed.
Progress during the third reporting period
Collection and evaluation of garlic accessions and close relatives (P!, P7& P10)
Phenological observations have been performed in Israel between January and June 2002.
Ca. 300 garlic accessions were separated into two major sub-populations: semi-bolters (producing blind scapes) and bolters (producing flower scape), which differ in leaf number, leaf length and bulbing ability under short-day conditions. The bolting accessions were subdivided into two subgroups according to flower/topset ratio. Most of flower-producing accessions had fertile pollen and receptive stigmas. Flower development was recorded in ca. 30 accessions. In seed-producing accessions, high seed viability was confirmed, and germination rate reached ca 90%, In the Netherlands, a total of 206 garlic accessions were evaluated. A large number of seeds were produced A provisional core collection of 53 accessions was established. 90 selected accessions were transferred for biochemical analysis to P5 and P3
Screening for CSO in the existing and the new collected garlic collections (P5)
Content of the organo-S compounds was determined in 85 accessions from Israel and The Netherlands. PCA analysis shows that the accessions of sativum group differ from the accessions of the longicuspis group. Cv. Morado is closer to the sativum group, as compared with Cvs. Morasol and Thermodrom.
List of deliverables for WP1DP: Plant / Deliverable title / Delivery date / Participant N° / Completion
DP. 3 / Collected bulbs for screening for CSO, fertility and clonal identity (finger printing). / 9 / P1, P5, P7, P10 / +
DP. 7 / Overview of the variation in CSO, fertility and clonal identity in the already existing collections and establishment of the core-collection / 12 / P1, P5, P7, P10 / +
DP. 10 / Establishment of the G&H garlic core collection and writing a paper / 21 / P1, P5, P7, P10 / In progress
DP. 31 / Maintenance and distribution of garlic clones for further use in the project / 21 / P1, P10 / +
In addition:
1. Paper on establishment of the method for SCO analysis for the "Journal of Chromatography" (P5 and P15).
2. Paper “Garlic (Allium sativum L.,) and its wild relatives from Central Asia: evaluation for fertility potential" (P7, P1 and P10) submitted to Acta Horticulturae, 2003
Milestones
Year 1 Collection trip to Central Asia (P7). Documentation, planting and quarantine inspection of collected material (P10). Screening for fertility (P7), CSO (P5) and clonal identity (finger printing) in the existing and the new collected garlic collections (P1).
Year 2 Collection trip to Central Asia (P7). Screening for fertility (P7). CSO (P5) and clonal identity (fingerprinting) in the existing and the new collected garlic collections (P1). Maintenance of core collection (P1, P10).
Year 3 Screening for fertility (P7), Maintenance of core collection and distribution of material (P1, P10).
Year 4 Maintenance of core collection and distribution of material (P1, P10).
Methodology and study materials
In 2000-2001, the collection trips were made to Central Asia, the main centre of garlic diversity. Collection sites and local environmental conditions were recorded for each accession in compliance with IPGRI plant passport regulations (Astley et al., 1982). In 2002, plant material was evaluated for morpho-physiological traits and floral fertility. Phenological observations have been performed in Israel between January and June 2002. The main parameters were leaf number and maximal leaf and scape length at bolting; date of spathe opening; topset/flower ratio in the inflorescence, and bulb structure.
DNA fingerprinting was employed to reduce the risk of redundancy in the collection. On the basis of the above results, a core collection of ca. 50 accessions representing maximum variability will be established and maintained both in Israel (short and intermediate day plants) and in the Netherlands (intermediate and long day plants), thus complementing each other. When fertility regained or molecular introgression becomes possible, they will serve as the main sources for expanding the genetic variation by crossing the most remote genomes (SD and LD types), thus widening the resources for breeding and improvement of garlic.
Selected accessions from the Israeli and Dutch parallel collections were screened for CSO (S-alk(en)yl cysteine sulphoxide) content (Tables 1 and 2).
Table1. List of 32 garlic accessions, grown in Israel (P7 and P10) and transferred to France (P5) in July 2002 for CSO analysis
Acc. / Group* / Acc. / Group / Acc. / Group / Acc. / Group2505 / S II / 2540 / S II / 2560 / S II / 2455 / S II
2507 / S II / 2541 / S II / 2568 / S II / 2456 / S II
2510 / S II / 2544 / S II / 2439 / S II / 2460 / S II
2512 / S II / 2546 / S II / 2441 / S II / 2462 / S II
2517 / S II / 2554 / S II / 2442 / S II / 2464 / S II
2528 / L I or IV / 2556 / S II / 2445 / S II / 2466 / S II
2537 / S II / 2557 / S II / 2450 / S II / 2468 / S II
2539 / S II / 2558 / S II / 2451 / S II / 3026. / L I ?
Table 2. List of 55 garlic accessions, in The Netherlands (P1), and transferred to France (P5) in September 2002 for CSO analysis
Acc. / Group* / Acc. / Group / Acc. / Group / Acc. / Group1006 / L I or IV / 73081 / L IV / 97069 / L IV / 99005 / S c d
2000-1 / nc Morasol / 73095 / S II / 97072 / L IV / 99006 / nc Morado III
2000-2 / nc Morada / 73097 / S II / 97073 / L I / 99008 / O III
20173 / L I or IV / 73100 / S II / 97075 / L IV / 99067 / S II a b
20174 / L I or IV / 73 105 / S II / 97076 / L IV b / 99069 / L I
73005 / S II / 73107 / S II / 97080 / nc / 99071 / S II a b
73022 / nc / 73110 / L I or IV / 97081 / L IV b / 99073 / nc
73024 / L I / 73113 / L I or IV / 97084 / L IV b / 99074 / L IV
73025 / L IV / 73117 / L I or IV / 97085 / L IV b / 99077 / S II a b
73028 / L I / 73120 / L I or IV / 97089 / L IV / 99078 / L I
73029 / S II / 73122 / L I or IV / 97090 / L I or IV / 99081 / L I
73077 / L IV / 96039 / Thermidrome / 97091 / L IV / 99082 / L IV
73078 / L IV / 97015 / L IV b / 97353 / L I
73080 / L IV / 97068 / S II a b / 97356 / L I
* Classification of garlic accessions (for details, see the second annual report G&H)
L I: Longicuspis group I S II: Sativum group II
L I or IV: Longicuspis group I or IV S II ab: Sativum group II a,b
L IV: Longicuspis group IV S II cd: Sativum group c,d
L IV b: Longicuspis group IV b O III: Ophioscorodon group III
nc: not clear
CSO Analysis
Lichtwer's method
The method used is a technique without derivatization. Chromatographic separations were performed using Waters instrument with Hypurity Elite C18 column (150mm X 3mm). The detection is an UV detector with a wavelength fixed at 208nm.
The separation requires an elution gradient with two solvents A and B. The solvent A constitution is 20mM sodium dihydrogen phosphate + 10mM heptane sulfonic acid at pH of 2,1. The solvent B constitution is 50%A+50% of acetonitrile. The flow-rate of the eluant is 0,4 ml/mn.
Gradient:
Time (min) / %A / %B0 / 100 / 0
5 / 70 / 30
25 / 46 / 54
26 / 0 / 100
28 / 0 / 100
30 / 100 / 0
40 / 100 / 0
CSO concentrations in garlic accessions were analysed and graphically presented by Principal Component Analysis (PCA) (Gomez and Gomez, 1984; Wold et al., 1987)
P1, P7, and P10 - Collection and evaluation of garlic accessions and close relatives
Garlic is an important condiment with proven nutraceutical values. It is used world wide both as a fresh herb and processed spice, and also as an important constituent of preventive and curative medicine. The lack of sexual phase prohibits classical breeding and improvement in garlic, and consequently the genetic variation available within the commercial clones is rather limited. In the Central Asian center of evolution, however, the introduction of plants from the wild repeated numerous times during the last 10 millennia. In the rare occurrence of restored fertility, there was a continuous flow of genes from the wild population into the domesticated plants. The genetic diversity in this region is therefore expected to be higher than in any other region.
The collection of garlic and its relatives in the Central Asian center of diversity is thus important not only for the G&H project, but also for the future of Allium improvement. It is expected that the variation within this genepool includes some (or many) economically important traits, such as fertility, high concentration of nutraceutics, high concentration of dry matter (for the dehydration industry), tolerance to biotic and abiotic factors, size, shape, and more.
Evaluation of collected plant material
Israel
In the first and second years of the project (2000-2001), we have collected ca. 300 landraces of garlic from a limited number of ecological niches. This collection plus 11 and 27 clones of garlic accessions [obtained in 1997 and 1999, respectively, before the beginning of the project], were documented.
In Israel, garlic collections were sorted and planted in December 2001, at the experimental farm of the Faculty of Agricultural, Food and Environmental Quality Sciences in Rehovot, and at the experimental plot of the Volcani Center in Bet Dagan. Before planting, bulbs were stored at low temperatures for floral induction and development.
Phenological observations have been performed in Israel between January and June 2002. The main parameters were leaf number and maximal leaf and scape length at bolting; date of spathe opening; topset/flower ratio in the inflorescence, and bulb structure.
Garlic accessions were separated into two major sub-populations: semi-bolters (producing blind scapes) and bolters (producing flower scape), which differ in leaf number, leaf length and bulbing ability under short-day conditions. The bolting accessions were subdivided into two subgroups according to flower/topset ratio. Most of flower-producing accessions had fertile pollen and receptive stigmas.
In Israel, flower development was recorded in ca. 30 accessions from Central Asia. However, hot spells during seed maturation (May-June) in Israel hampered seed development, and only a small number of garlic seeds were thus harvested in July 2002.
The Netherlands
A total of 206 garlic accessions were planted in November 2001 in the field to assess for their general performance and for multiplication. Furthermore a total number of 35 accessions were planted outdoors in pots during the beginning of November 2001. These latter accessions were used for analysing seed set. The plants used to assess their seed-set capacity were selected on basis of their AFLP fingerprint and previously obtained info on flowering. Both general performance and seed-set assessment should lead to a garlic core collection which is highly variable and which has a high seed-set potential.
In March 2002 the 35 accessions were transferred to an unheated greenhouse, placed with their pots in the soil and assigned randomly to 7 large transparent cages (2x4x2 m3). During the season bulbils in the inflorescence were removed every morning. In contrast to the last years, a large number of seeds were produced (around 1700) and this is most probably due to the better cultivation technique used, namely placement of the plants in an unheated greenhouse in the soil (better buffering against temperature and drought), giving the plants nutrient solution during the season, adding plenty of blowflies during flowering and daily removal of bulbils. We found that 7 accessions produced most seed. On the basis of this, the assessment of general performance and the AFLP fingerprinting pattern a provisional core collection of 53 accessions was established. Currently all accessions have been planted in the field and in pots for vegetative and generative multiplication and all garlic seeds went into a germination cycle.