Cytological and molecular characterization for local and exotic accessions of Lathyrus spp. BY Walaa Taher Hassan EL-Salamony

Supervised BY Prof. Dr. Reda helmy sammour Prof. Dr. Salwa Fahmy Badr Dr. Abd El-Zaher M.A. Mustafa

Tribe: vicieae Family: Fabaceae Genus: Lathyrus The other members of this tribe are Pisum, Lens, Vicia and Cicer.

ECONOMIC IMPORTANCE Long history as cultivated plants Forage Human food Ornamental plants

Grasspea seeds oil is used in homeopathic medicine and as cathartic Duke 1981 & Ben Brahim et al., 2001

Geographic Distribution Kupicha 1983, Allkin 1985, Goyder Species 24 Species 78 Species 52 Species 24 Species

ORIGIN Unknown origin of Genus Lathyrus Some studies on L. sativus suggested that its origin is Southwest and central Asia Smartt et al & Hanelt et al. 1992

Taxonomy of Genus Lathyrus 160 annual and prennial species Allkin et al., 1986 and Plitmann et al., 1995 Controversy about its infra- generic classification for about 250 years. Conney et al., 1998

27.8% Lathyrus is a self pollinated plant, with a high out- crossing ratio (up to 27.8%) resulting in a wide range of genetic variation AnisurRhman et al. 2001

ECOLOGY Lathyrus plants can grow in a wide range of habitats They have adaptability nearly to all types of soils, adverse climatic conditions, and low input-environments Sharma et al., 2000

Diploid chromosome number (2n=14) L. pratensis and L. venosus are tetraploid (2n = 28) L. palustris is hexaploid (2n= 42) (Campbell, 1994). KARYOTYPE

There is a three-fold variation in nuclear repetitive DNA content and chromosome size This variation is accompanied with genetic divergence and evolution of Lathyrus species (Narayan and Rees, 1976)

neurotoxin in Lathyrus seeds causes Lathyrism to human and animals when consumed for 3-6 months (30-50% diet) Smarrt et al., 1994 Neurolathyrism

Forms of neurotoxin ODAP (Beta-N-oxalyl-alpha- beta-diamino propionic acid), BOAA (Beta-N-oxalyl amino- L-alanine) (Smartt et al., 1994 & William et al., 1994)

ODAP distribution in L. sativus In embryo (400 mg per gram) Cotyledons (126 mg per gram), Seed coat (81 mg per gram), Stem (64 mg per gram), Leaf (60 mg per gram) Pod (24 mg per gram) and root (14 mg per gram) Campbell et al., 1994

OBJECTIVES OF THE WORK 1.Study the genetic variation in a great number of accessions, collected from diverse environments and belong to a number of taxa, using karyological and molecular markers.

2. Study the phenetic relationships in both intra and inter specific species 3. Evaluation of genetic variability for some quality traits, especially the seed proteins and the neurotoxin ODAP.

The last objective was to study the genetic variation in L. sativus accessions (the cultivated species) using electrophoresis of seed proteins and isozymes.

KARYOTYPE ANALYSIS All 40 accessions have 2n = 14 chromosome. Satellites found in L. sativus from TUN and AFG B-chromosomes found in L. sativus from (USSR, BAN, CAN, PAK), L. gorgoni from Jordon and L. annus from Syria

This due to five fold variations in nuclear DNA amount. Some cytological variations scored in section Lathyrus rather than the other sections

Section Lathyrus L. SativusL. hierosolymitan

L. sativus

Karyotype idiogram by Levan et al., 1965 Section Clymenum

Section Linearicarpus

Genetic diversity among and within Lathyrus spp 100seed weight SDS-PAGE TPC The corelation between the seed weight and protein content

56 accessions of Lathyrus spp were studied using 100-seeds weight. It ranged from 57.5 to 315 / 100 seeds. The biggest was in L. sativus The lowest was in L. sphaericus.

Seeds protein content It ranged from mg/g in L. sylvestris (USA) to mg/g in L. cicera (NOR).

Corelation between protein content and seed weight

SDS-PAGE Bands in L. marmoratus, L. inconspicous and L. annus showed some correlations with geographical origin and certain seed characteristic (seed weight).

Species of section Clymenum are generally closely related to one another, So the separation of species in section Clymenum conflicts with our data and with morphology.

The previous study which indicated that L. sativus are closely related to L. cicera and L. marmoratus is incongruent with our data. In our data each species is separated in a different cluster.

we do not support that section Linearicarpus seems to be an natural groups we suggest that a hybrid between L. sylvestris and L. latifolius bear out the generally accepted view that the species are closely related to one another.

The similarity matrix by Jaccared’s was tacked, with similarity coefficient for all accessions was 0.5 The principal component analysis of seed protein shows that the first three components accounted for of total variation.

Genetic variation in L. sativus Seed weight protein content Isozyme

TPC in L. sativus range from 28.7 to 41% with a mean value of 34.2%. The lowest TPC was found in Ethiopia The highest in accession from Italy.

The scatter plot for the 100-seeds weight Accession No. Protein content (mg/g) EUROPEAN AND CANADIAN ACCESSIONS ASIAN ACCESSIONS AFRICAN ACCESSIONS

we suggest that the center of origin of Lathyrus sativus is Southern and Eastern Asia (Indian Subcontinent) and Eastern Africa (small seeds). There is more variation among accessions from a given sub- region, e.g. South African sub- region

Absence correlation between protein content and seed weight Protein Content 100- seeds weight

Figure This showed a sort of correlation between SDS-PAGE data and geographic origin.

The jaccard’s similarity coefficients ranged from 0.95 (between accession from Tunisia and accessions from USSR) to (between accession from Egypt and accession from USSR, Iran and Afghanistan) with an average of 0.428

The genetic variation of L. sativus based on isozyme was studied. Four isozyme systems were examined, they were Amylase, Phosorelase, β- esterase and α- esterase.

All enzymes were polymeric, all with 12 loci and also all isozyme migrated anodally. A total alleles of isozymes was 34. The total number of alleles ranged from 24 to 32 with a mean of 27.5 (total = 34).

Amy-2 Amy-1 Amy-2 Amy-1

Est-4 Est-3 Est-2 Est-1 Est-4 Est-3 Est-2 Est-1

Est-4 Est-3 Est-2 Est-1 Est-4 Est-3 Est-2 Est-1

We studied the heterogenesity by Hardery Weinbring expectations test Genetic structure indicat by using Nei genetic diversity statistics. F statistics indicated that the mean breeding index was significantly higher than zero (0.399).

The relationship between the population size and the mean number of alleles per locus was significant on the contrary the proportion of polymorphic loci and the heterozygosity observed with population size were non significant.

The lowest ODAP levels (< 0.2%) were recorded in seeds of L. inconspicous from Turkey. The analyses of toxic mater ODAP

The highest value (> 6%) was recorded in seeds of L. sylvestris collected from USA. L. sativus accessions showed neurotoxin levels varying between 0.33% and 3.26%.

C.VMaxMinMean ± SETrait ±53.09 Protein content (%) ± Ash content (%) ± Seeds Weight (9) ±1.036 ODAP content (%)

PRIN2PRIN1Trait Protein content Ash content Seeds Weight ODAP content Eigen – value Variation (%) Variation comm(%)

The principle analysis Protein, ash contents and ODAP show higher coefficients in the first component (PRIN1). The second component (PRIN2) shows significant shares of variation to associate with the 100 seeds weight.

We found that the accessions belonging to L. inconspicuous, L. aphaca, L. hierosolymitan, L. tingitanus had an amount of ODAP less than that found in L. sativus and L. cicera.

with all my respects Thank you Walla Taher