Aspartate aminotransferase allozyme variation in a germplasm collection of the domesticated lentil (Lens culinaris)

Summary Variation at a polymorphic Aspartate aminotransferase locus was assayed in a sample of 298 accessions from the ICARDA germplasm collection of the domesticated lentil (Lens culinaris). Two alleles Aat-1 ^F and Aat-1 ^S were detected with global frequencies of 0.51 and 0.49, respectively. Fifty-nine percent of accessions were polymorphic for both alleles. The frequency of outcrossing was estimated from the observed heterozygosity to be about 1%. This is higher than direct estimates of outcrossing and implicates selection in favour of heterozygous gene combinations. Significant variation in allele frequency and in the occurrence of polymorphic accessions was observed between countries or geographic areas. Significant associations were observed between the allozymes and agronomic characters. In particular high frequency of Aat-1 ^F appeared to be associated with late flowering and maturity and low yield.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

Variability for restriction fragment lengths and phylogenies in lentil

Summary Thirty accessions of domesticated (Lens culinaris ssp. culinaris) and wild (L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans ssp. ervoides and L. nigricans ssp. nigricans) lentil were evaluated for restriction fragment length polymorphisms (RFLPs) using ten relative low-copy-number probes selected from partial genomic and cDNA libraries of lentil. Nei's average gene diversity was used as a measure of genetic variability for restriction fragment lengths within subspecies and a dendrogram was constructed from genetic distance estimates between subspecies. The wild lentils L. culinaris ssp. orientalis and L. culinaris ssp. odemensis showed the greatest variability for restriction fragment lengths and were closely positioned to domesticated lentil in the dendrogram. Little variability for restriction fragment lengths was observed within accessions of L. nigricans ssp. ervoides and L. nigricans ssp. nigricans. This observation is consistent with a previously published proposal that nigricans may have been independently domesticated. Estimates of genetic variability based on RFLPs tended to be greater than estimates from isozymes.     

中国花生核心种质中高油酸材料的分布和遗传多样性

利用代表花生基础资源的核心种质分析花生高油酸资源的分布和遗传多样性,结果表明：在花生核心种质中油酸含量高于57%的种质40份,主要分布在密枝亚种（普通型25份和龙生型8份）,少数分布在疏枝亚种（珍珠豆型6份和中间型1份）; 除了10份资源来源于国外（ICRISAT 7 份,美国1份,日本1份和韩国1份,其他种质资源来源于中国12个省市。 同时发现高油酸种质中3份资源的含油量在55%左右,分别是Zh.h4094（油酸66.70%,含油量54.99%）, Zh.h4029（油酸63.50%,含油量55.58%）和Zh.h4319（油酸59.70%,含油量56.04%; 小区产量超过3000 kg/ha 有10 份种质,前三位分别是Zh.h0883 （4086.06kg/ha）, Zh.h1182（3955.00kg/ha）和 Zh.h2910 （3741.00kg/ha）。基于植物学和产量性状分析,前5个主成份（PC）可以解释81.17% 的变异。聚类分析,在域值为0.1942时,可分为6个组。 因此中国花生核心种质中高油酸种质存在丰富的遗传多样性,而且分布较广,高油酸种质的获得对花生高油酸育种提供基础材料。     

园艺作物核心种质构建的研究进展

构建核心种质是植物遗传资源的研究热点和重点之一,对种质资源的鉴定、保存、利用与交流具有重要意义。本文简要介绍了植物遗传资源核心种质的概念及其构建方法,综述了园艺作物核心种质构建的研究新进展,并对今后该领域的研究趋势进行了展望。提出了种质分组及取样策略是园艺作物核心种质构建方法研究的重点;应及时构建一批大宗园艺作物以及我国原产和特产园艺作物的核心种质;高度重视基于重测序技术快速、精准、高通量地挖掘园艺作物核心种质优异基因的研究以及要加强科研管理与协作,切实提高我国园艺作物核心种质研究成果的共享性等观点,为园艺作物种质资源的深入研究与高效利用提供理论依据和技术参考。     

Genetic diversity estimation and core collection construction of Sinojackia huangmeiensis based on novel microsatellite markers

Sinojackia huangmeiensis is a critically endangered tree species in the Styracaceae endemic to China. To create tools to better evaluate the genetic diversity of this species, we used a modified genomic library enrichment method, the PETUER (Probe Extension and TEACl Universal EnRichment) method, to develop genetic markers. The resulting 18 microsatellite loci had high polymorphism with 3–12 alleles per locus and showed negligible stutter. Observed (H_O) heterozygosities were 0.057–0.724, and polymorphism information content (PIC) was 0.109–0.794. No significant linkage disequilibrium between pairs of loci was found. All 123 individuals found in the National Natural Reserve of Longgan Lake was genotyped at the 18 loci and used to estimate a UPGMA dendrogram. Using an iterative clustering method, a set of 18 individuals was established as a core collection to represent genetic diversity and would be useful to facilitate the conservation for S. huangmeiensis. In addition, we tested the 18 loci for cross-species amplification in three other Sinojackia species and the closely related Changiostyrax dolichocarpa. These polymorphic loci will be valuable for future population genetic and phylogenetic studies of S. huangmeiensis and congeneric species, as well as in closely related Styracaceae.     

Genetic variability in Lycopersicon species and their genetic relationships

Summary Genetic diversity has to be described and measured in order to establish breeding strategies and manage genetic resources. It is also fundamental to develop a comparative intraspecific study before attempting to discuss and conclude any phylogenetic relationship. The genetic variability of Lycopersicon species was studied using starch gel electrophoresis of 11 enzymatic systems in a hierarchical fashion. The species with the greatest genetic variability are L. chilense, L. peruvianum and L. pennellii, mainly due to the within-line component. L. chmielewskii, L. parviflorum and L. pimpinellifolium show an intermediate total variability and their between-component clearly predominates over the within-component. The least variable species are L. cheesmanii and L. esculentum. Cluster analysis resulted in three main groups: one formed by the cultigen, L. pimpinellifolium, L. cheesmanii and L. peruvianum;another by two species with self-incompatibility systems, L. pennelli and L. chilense; and another by two autogamous species L. chmielewskii and L. parviflorum. With respect to L. esculentum the farthest related species is Solanum rickii and the closest, L. pimpinellifolium.     

Allozyme evidence for the origin and diversification of Gossypium barbadense L.

Summary Gossypium barbadense L. is a commercially important cotton species of tropical South American origin presently grownin many regions of the world. The species is morphologically diverse, consisting of a wide range of wild (or feral), commensal, landrace, and highly improvedcommercial forms. We performed allozyme analysis on 153 accessions representing the spectrum of G. barbadense diversityto ascertain the geographic origin of the species, its patterns of diffusion subsequent to domestication, and to reveal infraspecific relationships. Levels ofgenetic variation in G. barbadense are moderate. Of 59 loci scored, 24 were polymorphic, with a mean number of alleles perlocus of 1.69 and an average panmictic heterozygosity of 0.062. Principal component analysis revealed geographic clustering of accessions into six relativelydiscrete regions. Gene frequencies at many loci are significantly heterogeneous among these regions, with an average G _STof 0.272. Northwestern South America contains the greatest genetic variability; we suggest that this region is the ancestral home of the species. The data indicate separate diffusion pathways from this region into Argentina-Paraguay and into eastern and northern South America east of the Andes. Caribbean Island and Central American forms appear to be derived from the latter. These diffusion pathways are in accordance with morphological evidence and historical record. In contrast to expectations based on geographic proximity, Pacific Island forms have their closest affinity to accessions from eastern South America. Advanced cultivated stocks seem largely derived from western Andean material, but also contain introgressed G. hirsutum germ plasm. Introgression was relatively high (22%–50% of accessions) in commercial stocks and in forms from Argentina-Paraguay and various Pacific Islands, but was conspicuously low or absent in material from Central America and the Caribbean, where commensal and commercial forms of both species are sympatric.     

Pedigree analysis for composing a core collection of modern cultivars,with examples from barley (Hordeum vulgare s. lat.)

A method for analyzing the pedigrees of cultivars is developed that allows for the calculation of the effective number of origin lines (n _OL ). The n _OL is defined as the average number of alleles, not identical by descent, per locus in a set of lines. Its relationship with the commonly used coefficient of parentage is clarified. A related quantity, the effective overlap of origin lines (r _OL ) is defined as the average number of alleles, not identical by descent, per locus common in two sets of individuals. A set of 85 modern barley cultivars is used to illustrate the application of n _OL and r _OL . This set originated from 153 mutually unrelated ancestors. The degree to which each ancestor contributed was quantified, and the result was a n _OL of only 43.1. In the set were 51 spring and 34 winter cultivars, with a n _OL of 25.0 and 21.0, respectively. Consequently, the r _OL of these two groups was 2.9, indicating that the two groups can be considered to be nearly distinct genetically since they have only 2.9 origin lines in common. How the effective number of origin lines can be used to create a core collection of cultivars with known pedigrees by maximizing the n _OL in a set of cultivars of given size is also discussed.     

Distribution of genetic variability in a durum wheat world collection

Summary A durum wheat world collection of 349 entries has been used to study the amount and distribution of genetic variability based on isoenzymatic characters involving a minimum of 13 loci. Genetic variability has been studied in a hierarchical fashion: between origins and within origins, further divided into between entries per origin and within entries. Factorial analysis of correspondences and chi-square distance were the basic statistical tools. The effect of domestication is deduced by comparing isozymic frequencies between wild emmer and durum wheat. It involves changes in frequencies mainly towards the accumulation of null alleles. The richest origins of genetic variation for durum wheat were Iran, Mexico, Ethiopia, Egypt and Afghanistan. Generally, between-entry variability was larger than the withinentry component. Exceptions were the accessions from Mexico, Greece, Argentina and Cyprus. The relationships between origins were greatly affected by their within-variability, the logic in the grouping is mostly along geographical or political lines. Egypt might be considered a microcenter of diversity for durum wheat within the Mediterranean center, although it is certainly related to Ethiopia (included in the Abisinic center). Mexico has become a new microcenter of diversity, quite likely man-made, and is distant from other centers of durum wheat diversity as far as gene frequency is concerned.The experimental part of this study was carried out at the Department of Genetics, Fac. Biologia, Universidad Complutense, Madrid, Spain     

Methods of developing a core collection of annual Medicago species

A core collection is a subset of a large germplasm collection that contains accessions chosen to represent the genetic variability of the germplasm collection. The purpose of the core collection is to improve management and use of a germplasm collection. Core collections are usually assembled by grouping accessions and selecting from within these groups. The objective of this study was to compare 11 methods of assembling a core collection of the U.S. National collection of annual Medicago species. These methods differed in their use of passport and evaluation data as well as their selection strategy. Another objective was to compare core collections with sample sizes of 5%, 10% and 17% of the germplasm collection. Core collections assembled with evaluation data and cluster analysis better represented the germplasm collection than core collections assembled based solely on passport data and random selection of accessions, The Relative Diversity and the logarithm methods generated better core collections than the proportional method. The 5% and 10% sample size core collection were judged insufficient to represent the germplasm collection.     

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H₂O₂ content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.     

Plant regeneration of the legume Lens culinaris Medik. (lentil) in vitro

Until recently, grain legumes in general have proven recalcitrant at de novo regeneration in vitro. By culturing portions of lentil (Lens culinaris) shoot meristems and epicotyls on a medium containing kinetin and gibberellic acid, callus tissue was produced which could be induced to regenerate shoots in relatively large numbers, even after several subcultures. The shoots could be rooted in a mist chamber to yield whole, fertile plants.     

Isozyme variation in germplasm accessions of the wild oat Avena sterilis L.

Summary Optimal exploitation of crop genetic resources requires a knowledge of the range and structure of the variation present in the gene pool of interest. Avena sterilis L., the cultivated oat progenitor, contains a store of genetic diversity that is readily accessible to the oat breeder. The objectives of the present paper were: (1) to evaluate isozyme polymorphisms in a sample of A. sterilis accessions from the U.S. National Small Grains Collection, (2) to analyze the distribution of isozyme diversity across the geographic range of the accessions, (3) to classify the accessions into groups based on isozyme variation, and (4) to suggest strategies for efficient sampling of this germplasm collection. One thousand and five accessions from 23 countries and 679 collection sites were screened for variation using 23 enzyme systems. Due to limited information about the genetic relationship among individual members of families of isozymes in hexaploid oat species, data were recorded solely for band presence. The frequencies of bands in accessions from the various countries were used to calculate the probability of genotypic identity (I_x.y), the probability of a unique genotype (U_x.y), and an adjusted polymorphic index (H_x). Accessions from Turkey and Lebanon had the largest polymorphic index values, Turkish and Moroccan accessions displayed the greatest numbers of bands. Accessions from Iran, Turkey, Iraq, and Lebanon had the largest mean probabilities of containing unique genotypes. Based on isozyme data, Turkey appeared to represent the center of diversity in this germplasm collection. Band frequencies calculated among countries were used in a principal component analysis. Accessions from Israel and Morocco clustered together; accessions from Iran, Iraq, Turkey, and Ethiopia formed another group; and Algerian accessions formed an outlying group. Several isozyme bands had a regional distribution. These results suggested that choosing accessions from countries based on their groupings in the principal component analysis should secure a greater range of diversity than sampling from the collection at random. Cluster analyses based on Jaccard's distances calculated for all pairwise combinations of the 1005 accessions revealed six broad genetic groups of accessions. Groups 1 and 6 contained accessions from many countries and encompassed half of all accessions. Groups 2 and 4 were heavily populated by accessions from Israel and Morocco. Groups 3 and 5 were composed almost exclusively of accessions from Iran, Iraq, and Turkey. By selecting representative accessions from these six groups, oat breeders could most effectively sample the range of genetic variation in this A. sterilis collection.     

Evaluation of five strategies for obtaining a core subset from a large genetic resource collection of durum wheat

The use of plant genetic resources contained in a large collection may be enhanced by specifying subsamples, called core samples. Five strategies for selecting a core sample from a collection of 3000 durum wheat accessions were applied and evaluated using four qualitative and eight quantitative spike characters. Each of the following strategies generated about 500 accessions for the core sample: random, random-systematic according to chronology of entry of the accessions into the collection, stratified by countryof-origin, stratified by log frequency by country-of-origin, and stratified by canonical variables. The first three strategies produced samples representative of the whole collection, but the remaining two produced the desired effect of increasing frequencies from less-represented countries-of-origin for several characters. The stratified canonical sample increased phenotypic variances. The quality of core samples is dependent upon good passport and evaluation data to partition the collection. The multivariate approach is extremely useful, but requires considerable data from the whole collection. Ecogeographic origin may be used in the absence of evaluation data on several characters to select useful core samples.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Paternal plastid DNA can be inherited in lentil

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F₁ progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F₁ progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F₁ plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F₁ progeny.     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of a recalcitrant grain legume,lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T₀, T₁, T₂ and T₃ plants. PCR assays of T₁, T₂ and T₃ progenies confirmed the stable transmission of the transgene to the next generations.