Determination of genetic variation and taxonomy in lentil (Lens Miller) species by chloroplast DNA polymorphism

Summary Chloroplast DNA restriction fragment lenght polymorphisms (RFLP) were used to examine the taxonomic relationships of cultivated and wild lentil (Lens Miller) species and identify the extent of genetic variation in this genus. Twelve accessions representing all Lens subspecies were digested with four hexanucleotide-recognizing restriction endonucleases. These digests randomly surveyed 540 base pairs, or 0.4% of the approximately 125 kilobase lentil chloroplast genome. A high degree of gragment length conservation was seen among members of crossability group I, i.e., L. c. ssp. culinaris, L. c. ssp. orientalis and L. c. ssp. odemensis. Accessions of the two subspecies comprising crossability group II, i.e., L. n. ssp. nigricans and L. n. ssp. ervoides, showed the greatest amount of variation when compared to the cultivated lentil, L. c. ssp. culinaris. Limited variation was observed within subspecies except for L. n. ssp. nigricans, where accessions of the normal cytotype were highly polymorphic to those of the differentiated cytotype. Chloroplast DNA RFLPs reaffirm hypotheses that propose L. c. ssp. orientalis as the progenitor to the cultivated lentil. The implications of this study on taxonomy and genetic resources is also discussed.     

Genetic variation for isozyme genes and proteins in spanish primitive cultivars and wild subspecies of Lens

Summary An analysis of the variability for genes encoding seven isozyme systems and storage proteins in a collection of cultivated and wild accessions of Lens is reported. The collection, which is part of the Spanish INIA Cenebank, contains the ssp. culinaris, orientalis, odemensis, nigricans and ervoides, and presents a high degree of genetic diversity both within and between the accessions. A total 25 loci were examined; of these, 18 were polymorphic (the 7 genes encoding storage proteins, and the following isozyme loci: Acp-1, Acp-2, Cpx-1, Cpx-2, Aat-p, Aat-m, Lap-1, Mdh-2, Mdh-3, Mdh-4 and 6pgd-p) and 7 were monomorphic (Aat-mb, Aat-c, Mdh-1, Mdh-5, 6pgd-2, Pgm-c and Pgm-p). The phylogenetic relationships between subspecies were analyzed using the allelic frequencies. The study suggests that orientalis and odemensis share more biochemical characters than the other subspecies, and that those subspecies keep an intermediate position between Lens culinaris and Lens nigricans.     

Reaction of wild species of the geneus Lens to drought

Summary Susceptibility to drought stress is a key factor in dry land lentil (Lens culinaris Medikus) production in the Mediterranean region of West Asia and North Africa. This study examined the response to drought stress of 121 accessions representing all subspecies of the genus Lens; cultivated, and the wild L. culinaris ssp. orientalis (Boiss.) Ponert, L. culinaris ssp. odemensis (Ladiz.), L. nigricans M.B. Godr. ssp. nigricans Godr. and L. nigricans ssp. ervoides (Brign.) Ladiz. for their potential use in breeding for dry land conditions. Accessions were grown under two moisture regimes (dry land and dry land plus supplemental irrigation) at Breda, Syria during the 1990–91 and 1991–92 seasons. The cultivated lentil had markedly superior seed and straw production than did the wild Lens species. Time to flowering accounted for less than 10% of the variation in yield of wild accessions under rainfed conditions in the two seasons, showing that, in contrast to the cultivated germplasm, drought escape was relatively unimportant in wild lentil. Performance under drought in wild lentil, measured in terms of dry land seed yield or drought susceptibility index (S), was randomly distributed among collection locations with little relation to collection site aridity. Direct selection of wild lentil germplasm for biomass yield under dry conditions is of little value and an evaluation of wild accessions in hybrid combination is needed.     

Pollen and pistil morphology, in vitro pollen grain germination and crossing success of Lens cultivars and species

Morphological and in vitro germination characteristics of pollen altogether with pistil and style length were analysed, so as to carry out a morphological comparison between different lentil (Lens culinaris Medik.) cultivars and wild species, as well as to correlate the morphological and functional statistics with data regarding crossing success. Pollen length and width in the different genotypes were found to have a positive and highly significant correlation with pistil and style length and in vitro pollen tube length. A cluster analysis detected two different plant groups, namely, (i) large‐seeded L. culinaris (macrosperma), and (ii) a second group formed by two subgroups, a first subgroup with L. ervoides standing alone and a second subgroup including small‐seeded L. culinaris (microsperma) and the wild L. culinaris ssp. orientalis, L. odemensis, L. nigricans. A high and significant correlation was detected between crossing success and phenotypic similarity (r > 0.98; P < 0.05), therefore implying that phenotypic similarity, based on pollen morphology and in vitro pollen length together with pistil and style length, is a good predictor of hybridization success.     

Variation in the ribosomal RNA units of the generaLens andCicer

Summary Investigation of the organization of the tandemly repeated 5S and 18–25S ribosomal RNA (rRNA) genes was carried out on members of the generaLens andCicer using restriction endonuclease digestion and Southern hybridization. The 5S rRNA unit (gene+spacer) inLens (0.38 to 0.50 kb) andCicer 0.50 to 0.44 kb) is the largest among the legumes. InL. nigricans andL. culinaris ssp.odemensis a second repeat size was detected. Restriction maps for the 18–25S units ofLens andCicer species were developed. The 18–25S rRNA unit varied from 8.3 to 9.8 kb inLens and 10.5 to 11.4 kb inCicer. The only detectable variability in theLens andCicer 18–25S rRNA unit was in the length of the intergenic spacer (IGS) region, except for a HindIII site in the IGS region ofL. nigricans ssp.nigricans and in allCicer species.Similarities in the size of both 5S and 18–25S rRNA units were noted betweenL. culinaris ssp.culinaris andL. culinaris ssp.orientalis, which supports previous evidence thatL. culinaris ssp.orientalis is the progenitor of the cultivated lentil. Differences in the arrangement of both 5S and 18–25S rRNA units were noted betweenL. nigricans ssp.nigricans andL. nigricans ssp.ervoides, indicating either divergent evolution of these subspecies or alternatively incorrect taxonomy.On the basis of the size of the 5S rRNA unit,C. bijugum, C. chorassanicum andC. echinospermum formed a group with a slightly smaller unit than the otherCicer species. Similarities in the length of both 5S and 18–25S rRNA units ofC. arietinum andC. reticulatum possibly confirmC. reticulatum as the progenitor ofC. arietinum. Cicer cuneatum has a smaller 18–25S rRNA unit than the otherCicer species as a result of a smaller intergenic spacer.     

Attempts to overcome postfertilization barrier in interspecific crosses of the genus Lens

Wild relatives are a potential source of genetic diversity to lentil (Lens culinaris Medik). The objective of this research was to obtain viable interspecific hybrids between the domesticated lentil and its wild relatives. The paper details the results of a number of interspecific crosses among L. culinaris, L. orientalis, L. odemensis, L. ervoides and L. nigricans. Viable hybrids were produced between L. culinaris × L. orientalis, L. culinaris × L. nigricans, L. culinaris × L. ervoides and between L. culinaris × L. odemensis. Further viable hybrids were obtained between L. culinaris and L. ervoides, which have the potential to be a ‘bridge’ in hybridization to L. culinaris for specific L. nicrigans lines which proved recalcitrant in L. culinaris × L. nigricans crosses. This is the first time that four wild species of lentils have been used successfully in hybridization with cultivated lentils, and viable hybrids produced. This paper also suggests that the artificial supplement of GA₃, hormone is needed after fertilization for the normal growth of the hybrid embryo, possibly as the natural GA₃ production is restricted with alien pollinations in cultivated lentils in both F₁ and backcross hybrids.     

The origin of lentil and its wild genepool

Summary Following hybridization experiments and cytogenetic analysis of interspecific hybrids three chromosome interchanges were found between the cultivated lentil L. culinaris and L. nigricans, and only one between the cultivated species and L. orientalis. This indicates that the latter species is more likely to be wild progenitor of lentil. The partial fertility of the interspecific hybrids indicate further that both L. nigricans and L. orientalis should be included in the wild genepool of lentil, and their variation can be exploited by relatively simple hybridization techniques. The wild lentils L. orientalis and L. nigricans are morphologically very similar but reproductively strongly isolated from one another by the albino seedling of their hybrids. It has been suggested that the populations of L. orientalis that gave rise to the cultivated lentil still possess a similar chromosome arrangement as in L. culinaris and are also capable of forming normal hybrids with L. nigricans. According to these considerations it is unlikely that lentil originated from populations at the south western corner of the distribution area of L. orientalis.     

The genetics of hard seed coat in the genus Lens

Summary Seeds of the cultivated lentil are capable of germinating shortly after maturation. The seed dormancy of wild lentil species is due to a hard seed coat. In crosses between the cultivated species L. culinaris and its wild progenitor L. orientalis the hard seed coat of the wild species was controlled by a single recessive gene in homozygous condition. In a cross between the wild species L. ervoides and L. culinaris the hard seed coat of L. ervoides was controlled by a single dominant gene. The significance of the genetics of seed coat hardness in the domestication of lentil is briefly discussed.     

Interspecific Hybridization in the Genus Lens Applying in Vitro Embryo Rescue

Summary Four different hybridization experiments were carried out to obtain interspecific hybrids with Spanish cultivars of lentil (Lens culinaris M.). In hybridization experiments I and II, undertaken with only pollination and pollination with the addition of gibberellic acid after fertilization, respectively, no lentil hybrids were recovered. A single interspecific hybrid with L. odemensis was obtained in experiment III using the embryo rescue protocol of Cohen et al. (1984), in this case, a crossing efficiency of 0.11% and a rescue efficiency of 2.5% were obtained. Hybridization experiment IV used a specific embryo rescue protocol developed in this study. In this experiment, ovule-embryos of 18 DAP were cultured on MS salts with 1% sucrose and 1 μ M IAA + 0.8 μ M KN; after two weeks, embryos were released from the ovular integuments and cultured on the same medium for another two weeks in upright position. Afterwards, the embryos were transferred to test tubes containing the same medium and one month later plantlets were obtained. Using the above protocol, out of a total of 1707 pollinations, 6 interspecific hybrids with L. odemensis, 2 with L. nigricans and one with L. ervoides were recovered, yielding on average a crossing efficiency of 0.53% and an average rescue efficiency of 8.26%. Taking into consideration only the interspecific crossing blocks in which hybrids were recovered, the crossing efficiency with L. odemensis was 9%, while with both L. nigircans and L. ervoides the crossing efficiencies were 3%. Rescue efficiencies based on hybrids recovered per number of ovules cultured ranged between 50–100%.     

Genetic analysis and linkage study of seed weight in lentil

Summary The genetics of seed weight was studied in crosses between the cultivated lentil L. culinaris and the wild species L. orientalis and L. ervoides. Seed weight was found to be under polygenic control with additive and dominant gene action and with partial dominance of low seed weight alleles. High heretability estimates were obtained in the analysed crosses. Nuclear-cytoplasmic interaction was found to affect seed weight in crosses involving accession No.138 of L. orientalis. Factors affecting seed weight were linked to morphological and DNA markers distributed over several linkage groups. High seed weight in segregating generations were usually associated with alleles of marker loci originated from the cultivated parent. Factors enhancing seed weight were detected in accession No.138 of L. orientalis, indicating the potential of wild genetic resources for seed weight improvement in lentil.     

Screening and selection of lentil (Lens Miller) germplasm resistant to seed bruchids (Bruchus spp.)

Seed weevil (Bruchus spp.) is one of the most damaging pests to lentil crop, and the current chemical treatments to control this insect present numerous disadvantages. In this work, we have searched for resistant or almost resistant lentil cultivars to be used as potential sources of genes for resistance to bruchids as a more sustainable and environmentally friendly approach to diminish harvest loss. Thus, a lentil germplasm collection of 571 accessions from 27 different countries, and including wild species was screened for susceptibility to seed bruchids under natural field conditions in Central Spain. Infestation was evaluated in comparison with a check (lentil cultivar ??Lyda??) according to the number of eggs per pod and the infestation rate in seeds (IR). A large variation of IR (0?C70?%) was observed among accessions although, in general, wild species were consistently less affected by bruchids than the check. A total of 32 accessions, including Lens culinaris Medikus subsp. culinaris, Lens culinaris Medikus subsp. orientalis (Boiss.) Ponert, Lens nigricans (M. Bieb.) Godr., and Lens lamottei Cezfr., showed lower infestation rates than the check and were selected as potential sources of resistance to this pest. Potential mechanisms of resistance of selected accessions and their inclusion in a breeding program are outlined.     

Inheritance of new genetic markers in lentil (Lens Miller)

Summary We report on the inheritance of 11 morphological markers and 17 isozymes in lentil (Lens culinaris). The monogenic inheritance of 11 morphological markers and 11 isozymes is confirmed. The inheritance of six isozymes (Aco-2, Enp, Est-3, Est-4, Lap-3, and Mdh-m) is reported for the first time in lentil. This brings the total number of described genes in lentil to 78. Cases of disturbed segregation were more frequent than expected by chance. It is suggested that disturbed segregation was in most cases caused by linkage with a piece of chromosome that showed preferential elimination in crosses between Lens culinaris ssp. odemensis and other subspecies. The prevalence of disturbed segregation in crosses with Lens culinaris ssp. odemensis could limit the usefulness of this subspecies in genetic and linkage studies.     

Broadening the genetic base of lentil cultivars through inter‐sub‐specific and interspecific crosses of Lens taxa

Wild Lens taxa are invaluable sources of useful traits for broadening genetic base of cultivated lentil. Nine inter‐sub‐specific and interspecific crosses were made successfully between cultivated (Lens culinaris ssp. culinaris) and wild lentils (L. culinaris ssp. orientalis, odemensis, lamottei and ervoides). The effect of species groups, day length and temperature on crossability in lentils was evident under normal winter sowing in New Delhi and in summer Himalayan nursery at Sangla in Himachal Pradesh, India, although pollen fertility assessed in all the cross‐combinations showed no significant variation. True hybridity of nine inter‐sub‐specific and interspecific crosses was confirmed through morphological and molecular (ISSR) markers, in which three of 120 primers could confirm the hybridity of all the crosses. All cross‐combinations were also studied for important quantitative traits related to yield. The range, mean and coefficient of variation were estimated in parental lines, F₁ and F₂ generations to determine the extent of variability generated in cultivated lentils through the introgression of genes from wild L. taxa. A high level of heterosis was observed in F₁ crosses for important traits studied. Substantially higher variations for seed yield and its attributing traits were exhibited in F₂ generations indicating transgressive segregation. The results of the present investigation revealed that wild L. taxa can be successfully exploited for lentil improvement programmes, and the variations generated could be easily utilized for broadening the genetic base of cultivated lentil gene pool for improving the yield as well as wider adaptation.     

Widening the gene pool of cultivated lentils through introgression of alien chromatin from wild Lens subspecies

Wild Lens species/subspecies are a potential source for increasing genetic diversity in cultivated lentil. Four intraspecific crosses were attempted between cultivated and wild lentils. Viable hybrids were produced between L. culinaris ssp. culinaris × L. culinaris ssp. orientalis and L. culinaris ssp. culinaris × L. culinaris ssp. odomensis. Normal meiosis and pollen fertility were observed in the first set of crosses, whereas chromosomal abnormalities and reduced pollen fertility were observed in the second set of crosses. These crosses were also studied for some quantitative traits. The range, mean and coefficient of variation were calculated in parents, F₁, F₂ and BC₁ generations to determine the extent of variability generated in the cultivated lentil through introgression of genes from wild lentil. The cultivated lentil × L. culinaris ssp. orientalis crosses showed substantially higher variability for all the traits than crosses involving cultivated lentil ×L. culinaris ssp. odomensis. The results of the present study indicated that these wild subspecies can be exploited for breeding purposes and their variation can easily be utilized to widen the genetic base of the cultivated lentil.     

Genetic diversity in tef [Eragrostis tef (Zucc) Trotter] and its relatives as revealed by Random Amplified Polymorphic DNAs

Tef is one of the staple cereal crops in Ethiopia. To evaluate genetic diversity of tef and its relatives, 47 accessions of tef, three accessions of E. pilosa, and six accessions of E. curvulawere analyzed using random amplified polymorphic DNA (RAPD) markers. The level of polymorphism among the wild species was extremely high, while low polymorphism was detected among tef accessions. All cultivars and wild species under study could be distinguished with the help of different primers, thereby indicating the potential of RAPD in the genetic fingerprinting of tef. Accessions from E. curvula and E. pilosa can be differentiated by a single selected primer. In spite of low polymorphism within tef, accessions under study could be distinguished by a combination of selected primers. Cluster analysis indicated that tef is a very closely related species to E. pilosa with 45%similarity, supporting the hypothesis that tef originated from E. pilosa based on morphological data. Given that RAPD are relatively quick, simple to use, and are not subjected to environmental influences, they provide a valuable new approach for the genetic fingerprinting and study of genetic diversity in tef. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effectiveness of evaluating wild species: searching for sources of resistance to bruchid beetles in the genus Vigna subgenus Ceratotropis

A species level germplasm collection representing 76% of known taxa in the genus Vigna subgenus Ceratotropis was evaluated for resistance to two species of bruchid beetles, Callosobruchus chinensis and C. maculatus. Seven taxa consisting of 29 accessions were found to be resistant to C. chinensis and 4 taxa consisting of 24 accessions were found to be resistant to C. maculatus. This compared with no resistant accessions being found in several hundred landrace accessions of mungbean, V. radiata var. radiata, in the same subgenus. Sometimes resistance was found in all accessions of a particular taxon, such as complete resistance to both C. chinensis and C. macualtus in V. umbellata. Other taxa showed intra taxon variation for resistance such as V. reflexo-pilosa andV. minima. The levels and patterns of resistance among taxa were diverse. The results suggest that various factors cause resistance to bruchid in the subgenus Ceratotropis. While the number of eggs laid on seeds generally reflected seed size, one small seeded cultivar of V. mungo var. mungo, black gram, had an unusually high number of eggs laid per seed. No correlation was found between seed size and levels of resistance. The species level germplasm collection, which reflects the core collection concept in trying to maximize genetic diversity in a limited number of accessions, has enabled a large number of potentially useful sources of resistance to bruchid beetles to be found efficiently. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity of wild coffee (Coffea arabica L.) using molecular markers

Genetic diversity was studied using RAPD markers among119 coffee (Coffea arabica L.) individuals representing 88 accessions derived from spontaneous and subspontaneous trees in Ethiopia, the primary centre of species diversity, six cultivars grown locally in Ethiopia, and two accessions derived from the genetic populations Typica and Bourbon, spread in the 18^th century, which gave rise to the most currently grown cultivars. Twenty-nine polymorphic fragments were used to calculate a similarity index and construct dendrograms. The Ethiopian material was separated from the Typica- and Bourbon-derived accessions and classified in four groups: one with most of the collected material from southwestern Ethiopia and three from southern and southeastern Ethiopia. Almost all detected diversity was found in the southwestern group while the southern and southeastern groups presented only 59% of identified markers. The genetic distances were low between the southwestern group and the southern and southeastern groups, and between the southwestern group and the Typica- and Bourbon-derived accessions. The cultivated coffee derived from the genetic populations Typica and Bourbon appeared little differentiated from wild coffee growing in the southwest. The results supported the hypothesis that southwestern Ethiopian coffee trees could have been introduced recently in the south and southeast. A separate analysis of the 80accessions classified in the southwestern group allowed identifying particular spontaneous- and subspontaneous-derived accessions and redundancies in the collected material from southwestern Ethiopia. RAPD markers did not detect any within-collection polymorphism except for two trees that were identified as off-types in the CATIE field genebank. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity of a global collection of maize genetic resources in relation to their subspecies assignments,geographic origin,and drought tolerance

The genetic diversity among an international collection of 40 maize accessions has been evaluated using DNA ISSR fingerprinting. Among the 180 ISSR markers scored by 15 primers, 161 markers (89.59%) were polymorphic and 19 were unique in 16 accessions. A cluster tree based on the average distance coefficients and the Dice similarity indices divided the accessions into three major groups, each including clusters of accessions assigned to their subspecies. However, a low level of genetic differentiation among the accessions was demonstrated by the STRUCTURE analysis of ISSR data in agreement with the low gene flow (Nm) value among the accessions. A scatter diagram of the principal component analysis (PCA) based on ISSR data analysis revealed that the accessions were differentiated into three groups comparable to those produced by the cluster analysis, in which some accessions of the same subspecies showed a close similarity to each other. A scatter diagram of the principal coordinate analysis (PCoA) based on the drought tolerance indices (DTIs) showed that nine genetically similar accessions share drought tolerance characteristics; these include four of subsp. indurata, three of subsp. everata, and two of subsp. indentata. An abundance of unique ISSR alleles found in the 16 accessions, including the nine drought-tolerant accessions, represents rich untapped genetic resources and these accessions may be exploited in the future breeding of maize commercial lines.     

Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of a partial reproductive barrier between wild and cultivated pearl millets (Pennisetum glaucum)

Summary The occurrence of seed malformation in association with reduced thousand grain weight and germination ability has been observed in crosses between cultivated female plants and wild male plants. A survey of 16 cultivated accessions (P. glaucum subsp. glaucum) and 11 wild accessions (P. glaucum subsp. monodii) ranging over the whole species diversity showed this postzygotic incompatibility was general, but its intensity varied greatly with the cultivated female accession used and very little with the wild male parent origin. About 15% of the 123 cultivated x wild crosses observed gave normal seeds. Seed malformation has never been observed in crosses between cultivated accessions and appeared independent of genetic distances between the parents. The reciprocal crosses between wild female plants and cultivated male plants gave normal-looking seeds with good germination but consistently reduced thousand grain weight. Both seed malformation and seed small size are an expression of a genetic imbalance. These slight reproductive barriers seem to have been built during the domestication process.Abbreviation ICRISAT International Crop Research Institute for the Semi-Arid Tropics