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.     

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 diversity and taxonomic relationships within the genus Lens as revealed by allozyme polymorphism

Summary A survey of allozyme polymorphism at 11 loci was carried out on 439 accessions from the genus Lens. This comprised 153 Lens culinaris subsp. orientalis, 35 L. odemensis, 117 L. ervoides, 32 L. nigricans, 2 of a differentiated cytotype of L. nigricans and 100 landrace accessions of the cultivated lentil (L. culinaris subsp. culinaris), from 10 different countries. The aim of the survey was to determine intra-specific genetic diversity and species relationships, based on phylogenetic and phenetic analyses, particularly regarding the position of L. odemensis and the differentiated cytotype of L. nigricans. Diversity was described by three statistics. The level of diversity in the cultivated taxon was lower than in any of the wild species according to two of these statistics, the percentage of polymorphic loci and mean number of alleles per locus. For the third measure (Nei's mean genetic diversity) it was only greater than L. ervoides. Genetic diversity statistics of the wild species indicated differences in the nature of between-population genetic diversity within the different taxa. Phylogenetic analysis suggests that L. odemensis and L. ervoides evolved from a common ancestor, and L. culinaris subsp. orientalis subsequently evolved from L. odemensis. Phenetic analysis, however, places L. odemensis closer to L. culinaris subsp. orientalis than to L. ervoides. Nei's mean genetic distance of L. odemensis from both L. culinaris subsp. culinaris (0.204) and L. culinaris subsp. orientalis (0.110) was greater than the distance between them (0.062). This evidence is not conclusive in determining whether L. odemensis should retain its specific status. Further crossability studies should be carried out on a range of genotypes to assess the potential for gene flow. The evidence presented shows the differentiated cytotype of L. nigricans to be quite distinct from other L. nigricans accessions, both phenetically and phylogenetically. This indicates that the differentiated cytotype of L. nigricans may constitute a new taxon. Discriminant function analysis reveals that isozymes may be useful in validating species classification.     

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.     

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.     

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.     

The BamHI site in the Internal Transcribed Spacer Region of Mungbean ribosomal RNA gene is partially methylated

The 18s-5.8s-25s ribosomal gene (18s-25s rDNA) in higher plants is present in multiple copies, on different chromosomes, as tandemly repeated units. Among the multiple BamHI sites that occur in the repeat unit, only the site in the middle of the 25s rRNA coding region is methylated in most cereals and pulses. BamHI restriction enzyme analyses of the mungbean 18s-25s rDNA showed the presence of two populations. Nearly 50% of the 18s-25s rDNA population had BamHI site situated in the internal transcribed spacer region (ITS-BamHI) resistant to cleavage by BamHI. The amplified ITS fragment was completely digestible by BamHI showing that partial cleavage by BamHI is not due to variation in the recognition sequence but most probably due to methylation. The complete cleavage of the ITS-BamHI site by BstYI that recognizes BamHI site but is insensitive to methylation confirms that the ITS-BamHI site is methylated. Methylation is probably due to the presence of a guanosine residue adjacent to the 3′ cytosine in the recognition sequence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the yield potential and yield components of F1 and F2 hybrids of crosses between wheat (Triticum aestivum L.) and spelt (Triticum spelta L.)

Summary Crosses of wild barley (Hordeum vulgare ssp. spontaneum and Hordeum vulgare ssp. agriocrithon) with Hordeum vulgare ssp. vulgare were used to select high yielding grain types under dryland Mediterranean conditions. No special difficulties were faced in making the crosses, in eliminating the brittle rachis genes from the grain types or in selecting 6-rowed types in crosses between 2-rowed wild barley and 6-rowed ssp. vulgare varieties. Brittle rachis genotypes, present in the segregating populations were used in developing self-reseeding permanent pastures for dry areas. The best selections were tested in seven trials during 1989–92 and some of them outyielded their parents and also the best improved check variety by 13–22%. Indications for transgressive segregation were obtained for grain yield, straw yield, total biological yield, harvest index and volume weight. The crude protein content of some of the selections was significantly higher than that of the checks. For breeding programs aiming at large seeds, special ssp. spontaneum lines should be used as parents. High grain yield was positively correlated with high straw yield, total biological yield, earliness in heading date, high harvest index and negatively with volume weight. It was concluded that unexploited useful genes, even when not directly observed in wild barley, could be transfered easily into high yielding genotypes by breeding.     

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

Summary Germination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatum Lad. and C. cuneatum Rich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F₁ plants.     

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%.     

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.     

Analysis of durable resistance to stem rust in barley

Summary Since the mid-1940's, barley cultivars grown in the northern Great Plains of the USA and Canada have been resistant to stem rust caused byPuccinia graminis f. sp.tritici. This durable resistance is largely conferred by a single gene,Rpg1, derived from a single plant selection of the cultivar Wisconsin 37 and an unimproved Swiss cultivar. At the seedling stage, barley genotypes withRpg1 generally exhibit low mesothetic reactions at 16–20° C and slightly higher mesothetic reactions at 24–28° C to many stem rust pathotypes. This resistance is manifested by a low level of rust infection and mostly incompatible type uredia on adult plants.Rpg1 reacts in a pathotype-specific manner since some genotypes ofP. g. f. sp.tritici are virulent on cultivars carrying this gene in the field. Several factors may have contributed to the longevity of stem rust resistance in barley, a) since barley is planted early and matures early, it can sometimes escape damage from stem rust inoculum carried from the south; b) one or more minor genes may augment the level of resistance already provided byRpg1; c) the cultivation of resistant wheat cultivars and eradication of barberry have reduced the effective population size and number of potential new pathotypes ofP. g. f. sp.tritici, respectively; and d) virulent pathotypes ofP. g. f. sp.tritici andP. g. f. sp.secalis have not become established. This situation changed in 1989 when a virulent pathotype (Pgt-QCC) ofP. g. f. sp.tritici became widely distributed over the Great Plains. However,Rpg1 may still confer some degree of resistance to pathotype QCC because stem rust severities have been low to moderate and yield losses light on barley cultivars carrying the gene during the last four seasons (1989–1992). Several sources of incomplete resistance to pathotype QCC have been identified in barley. To facilitate the transfer of resistance genes from these sources into advanced breeding lines, molecular marker assisted selection is being employed.     

Possibility of genetic improvement of pigeonpea (Cajanus cajan (L.)Millsp.) utilizing wild gene sources

Summary Various wild relatives of pigeonpea,Cajanus cajan, namely some species ofAtylosia andRhynchosia, possess desirable characteristics that could be utilized for effecting genetic improvement of this crop. In total 73 cross combinations among two cultivars ofC. cajan and one accession each of eightAtylosia species and one ofRhynchosia were attempted. Twelve hybrids were obtained. Seven of these were analysed for F₁ fertility and their utility for agronomic improvement of theC. cajan. Fertility behaviour of the different F₁ hybrids varied and indicated that potential of gene transfer between the two genera,Atylosia andCajanus, was as good as within the genusAtylosia. From F₂ and F₃ families ofC. cajan × A. scarabaeoides andC. cajan × A. albicans, plants were selected with greater physiological efficiency and agronomic superiority. The prospects of transferring pod borer resistance and higher seed protein content from someAtylosia species to pigeonpea are discussed.     

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.     

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.     

Breeding of the cultivated potato species Solanum x juzepczukii Buk. and Solanum x curtilobum Juz. etBuk.

Summary This study investigated the possibility of recombining anew the genomes of the wild and cultivated progenitors of triploid S. juzepczukii and pentaploid S. curtilobum by following the known evolutionary pathway of these two species. Before starting the actual breeding work, the natural variation of S. juzepczukii, S. curtilobum and their wild progenitor S. acaule was studied from the point of view of morphology, quantitative and qualitative tuber glycoalkaloid content and frost resistance. The morphological study was supplemented by a study of the soluble tuber proteins employing polyacrylamide slab-electrophoresis. From 137 accession of S. juzepczukii only 19 morphotypes were identified, 18 of which were also different in their protein spectra. The only red-tubered S. juzepczukii revealed a protein spectrum identical to that of the largest white-tubered group. On phylogenetic grounds, the occurrence of a red-tubered S. juzepczukii cannot be explained. It is concluded that this red clone is a somatic mutant for tuber colour which arose from a whitetubered clone. S. curtilobum was restricted in its variation to just two morphotypes differing only in tuber colour which are, however, identical chemotypes. This would be the case if one of the clones was a somatic mutant for tuber colour from the other one. The glycoalkaloids -solanine, -chaconine, tomatine, demissine and - and -solamarine are shown to be useful taxonomic characters which confirm earlier hypotheses on the origin of S. juzepczukii and S. curtilobum. Laboratory tests showed the two cultivated species to be resistant to about –3°C whereas S. acaule is resistant to temperatures sometimes below–5°C. The diploid progenitor of S. juzepczukii, S. stenotomum, also has forms resistant to –3°C. The results of this study demonstrate that the proposed breeding scheme is possible.     

Components of rust resistance in lentil

Four components of resistance to Uromyces viciae-fabae, namely, latent period (LP), infection efficiency (IE), pustule size (PS) and spore production (SP) were evaluated on lentil (Lens culinaris) in glasshouse experiments. Four cultivars, Gudo, R-186, FLIP-87-66L and FLIP-89-60L, with different levels of resistance, and the susceptible check EL-142, were included in this study. The cultivars were also compared for area under the disease progress curve (AUDPC), area under the pustule density curve (APDC), apparent infection rate (r_G), disease severity (DS), pustule density (PD), LP and PS under field conditions. Gudo and R-186 had significantly smaller and fewer pustules, lower spore yield and longer latent period than EL-142. FLIP-87-66L was intermediate for infection efficiency and pustule size. In addition, significant differences were found between cultivars for AUDPC, APDC, DS and r_G. Estimates of AUDPC, APDC, DS and r_G were reduced in Gudo, R-186 and FLIP-87-66L compared with the susceptible check EL-142. FLIP-89-60L also showed low AUDPC, APDC and DS. Some of the components obtained in the field were significantly correlated with each other (r = 0.92–0.99, P 0.05) and those measured in the glasshouse. Most of the components studied in the glasshouse were significantly (P 0.05) correlated with AUDPC and DS. Data indicated the existence of incomplete [partial (PR)] resistance in the test cultivars, and the possibility of using IE, LP, SP and PS as selection criteria in the evaluation of PR to rust in lentil. Since there was an interdependence of the components, selection based on more than one component should help obtain lines with higher levels of PR. The AUDPC, DS and r_G could also be used for selecting lines with PR in the field.     

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.     

Mutagenesis of faba bean (Vicia faba L.) and the identification of five different genes controlling no nodulation,ineffective nodulation or supernodulation

Summary Mutagenesis ofVicia faba L. using ethyl methanesulfonate has yielded three mutants (f13, f73, f245) with ineffective nodulation (Nod⁺Fix^–), one mutant (f48) unable to nodulate (Nod^–) and one supernodulating mutant (f32) having 3 to 5 times more nodules than the control in the absence of combined nitrogen, and expressing nitrate tolerant nodulation (Nod⁺⁺, Nts). All of the mutant phenotypes were controlled by single and distinct recessive genes designatedsyml tosym5, respectively. Grafting experiments showed that the (Nod⁺Fix^–) and (Nod^–) phenotypes were both under root genotype control and that the (Nod⁺⁺, Nts) phenotype was under shoot genotype control.     

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.