A multi-species assessment of genetic variability in Nigerian Amaranthus accessions: potential for improving intra- and interspecies hybridization breeding

Amaranthus yields could benefit from our growing understanding of the genetic variability within and among individual species of local germplasm collections. Thirty-seven Amaranthus accessions representing three different species were evaluated for seed yield and yield components across two seasons. The objectives were to characterize the genetic variability of seed yield and its components across species and determine relationships among seed yield and its components by phenotypic correlation. Significant differences (P < 0.01) existed for all the evaluated traits among the accessions. Characters such as seed yield, stem girth and leaf length showed higher heritability estimates in A. cruentus (14) than in A. hypochondriacus (12) and A. dubius (11) accessions. Seed yield was significantly and positively correlated with stem girth and thousand seed weight. However, it was significantly and negatively correlated with number of days to flowering, plant height, leaf number, number of branches. The clustering of the accessions, which was partially in agreement with species grouping, will help in the identification of diverse parents for use in the breeding program. Enormous amount of genetic variability found in seed yield and its components among Nigerian Amaranthus accessions will offer opportunity for grain and leaf yield improvement through breeding.     

Delimitation of Amaranthus cruentus L. and Amaranthus caudatus L. using micromorphology and AFLP analysis: an application in germplasm identification

The ‘Morelos’ accessions of Amaranthus from Mexico demonstrate taxonomic ambiguity at the basic morphologic level. The main cause is the enormous morphological and genetic variation exhibited by the species in the genus. Although basic morphological criteria can be applied to herbarium specimens or germplasm collections for quick taxonomic identification, the morphological data alone can be misleading. To ascertain the taxonomic identity of the ‘Morelos’ accessions and their hypothesized species affiliation to Amaranthus caudatus or Amaranthus cruentus, we conducted a comparative analysis of phylogenetic relationships among these taxa/accessions using amplified fragment length polymorphism (AFLP) and micromorphology methods. Based on AFLP data, all the controversial ‘Morelos’ accessions can be consistently placed into a single A. cruentus species clade, which is clearly separated from the A. caudatus species clade. The AFLP-based phylogenetic relationship of ‘Morelos’ and delimitation of A. cruentus and A. caudatus are further supported by micromorphology, showing that the combination of these techniques can provide more reliable data for germplasm identification than each method used alone.     

Genetic diversity of <Emphasis Type="Italic">Nelumbo</Emphasis> accessions revealed by RAPD

Total 65 lotus accessions in genus Nelumbo mainly collected from China, were subjected to random amplified polymorphic DNA (RAPD) markers to estimate the genetic diversity and to test the genetic basis of the relationships between morphotypes and molecular markers. Seventeen primers generated a total of 195 highly reproducible and discernible loci, among which 173 were polymorphic. Percent polymorphism varied from 66.7 to 100 with an average of 88.72, and five primers out of them, OPC05, OPG10, OPN20, OPP09 and OPS17, showed 100% polymorphism. A relatively high genetic diversity was detected among all the samples with the similarity coefficient values ranging from 0.45 to 0.85, and Nei’s gene diversity (h) 0.30, and Shannon index (I) 0.46. The UPGMA dendrogram clustered 65 accessions in four clusters and the clustering pattern showed two groups, N. nucifera ssp. nucifera and those accessions related to the American lotus, and some special cultivars, landraces, hybrids and the American lotus. Principal Coordinate Analysis (PCA) further indicated that the genetic diversity of Nelumbo accessions was not evenly distributed, instead, was presented by a clustered distribution pattern. Similar to the results revealed by the dendrogram, two main groups representing the two subspecies of N. nucifera, as well as some special landraces, cultivars of Chinese lotus, the Japanese lotus and hybrids out of the two groups were obtained. Neither the UPGMA dendrogram nor the PCA analysis exhibited strict relationship with geographic distribution and morphotypes among the accessions.     

AFLP-based genetic diversity assessment among Chinese vegetable mustards (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.)

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic relationships and diversities of Chinese vegetable mustards. Fourteen pairs of primers generated a total of 366 scorable fragments among 16 accessions of Brassica juncea studied, of which 296 bands were polymorphic with an average of 21.1% polymorphic bands per primer combination. Genetic similarities were obtained using Nei and Li similarity coefficients, and a dendrogram of the 16 accessions was made by UPGMA clustering method. The Nei and Li Similarity coefficient value ranged from 0.63 to 0.88. This result indicated that the 16 accessions of B. juncea possessed high level genetic variations. The cluster analysis showed that the vegetable mustards could be grouped into two main groups and some minor rami, which was partially in accordance with the traditional classification that based on different edible organs of vegetable mustards. The incongruity between morphological and molecular classification might be attributed to the high selection pressure during domestication of Chinese vegetable mustards, producing some accessions with similar genetic backgrounds evolving into abundant morphological variations. The great diversification among Chinese vegetable mustards not only provides an excellent object for molecular evolution research of B. juncea but also is of great value for widening the genetic basis of breeding programs and breeding materials selection. Besides, our study also indicates that AFLP are informative and can provide significant insights for genetic diversity research in B. juncea.     

Phylogenetic relationships of Cajanus cajan (L.) Millsp. (pigeonpea) and its wild relatives based on seed protein profiles

Total seed storage protein of 9 accessions of cultivated C. cajan and 10 wild Cajanus species was reported and compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A considerable variation was detected among the protein profiles of different accessions of C. cajan while those of wild species were very specific and distinctly different from each other. Relative similarities between various taxa were estimated by Jaccard's similarity index and cluster analysis was performed to produce a UPGMA dendrogram. The clustering of 10 wild species and C. cajan more or less agrees with their sectional classification and available data based on morphological characteristics, crossability, genome pairing in hybrids and nuclear RFLPs. The species closest to C. cajan is C. cajanifolia although the accessions of C. cajan also share some bands present in the profiles of C. scarabaeoides, C. goensis, C. lineatus, C. acutifolius and C. volubilis. This points towards polyphyletic origin of the cultigen which has been discussed in this paper.     

Genetic diversity of the African hexaploid species Solanum scabrum Mill. and Solanum nigrum L. (Solanaceae)

Two hexaploid species of Solanum sect. Solanum are present in Africa: Solanum scabrum and S. nigrum. Solanum scabrum is a widely cultivated species and is used as a leafy vegetable, as a source of medicine and as a source of ink dye. In previous studies a wide range of morphological diversity has been reported in this species and in some studies subspecies have been proposed. Subspecies are also recognized in S. nigrum. However, it has not been established whether or not the morphological differences are reflected at the genomic level. The present study applies AFLPs to study the genetic diversity in S. scabrum and its relationship to geographical provenance, morphological differences and the possible existence of subspecies within S. scabrum and S. nigrum. The data obtained were analyzed with cluster analysis (using UPGMA and NJ). The results indicate that the genetic variation within S. scabrum was higher within accessions than between accessions. Accessions did not cluster according to their geographical provenance, indicating that accessions from different geographical areas were not significantly different genetically. The clustering reflected neither morphological differences nor domestication status (cultivated or wild). The morphological differences exhibited by S. scabrum could be due to selection by farmers for different plant types. The AFLP derived clustering pattern did not segregate the subspecies recognized in S. scabrum and S. nigrum into separate subclusters.     

Amaranthus genetic resources in Indonesia: morphological and protein content assessment in comparison with worldwide amaranths

The morphological variations (growth habit, leaf shape, leaf color, height, stem diameter, number of branches and internodes, leaf area, blade ratio, leaf thickness, number of leaves, and total leaf area) and protein content of 53 Indonesian amaranths (Amaranthus spp.), consisting of weedy-, vegetable-, and ornamental-types, were assessed. The extent of variation in the Indonesian Amaranthus accessions were compared with the worldwide collection (26 accessions from United States Department of Agriculture (USDA) and 5 ornamentals from Nepal) in the experimental field of the University of Tsukuba, Japan. The variation in average values of most morphological traits and protein content in the Indonesian accessions were similar to those of the worldwide germplasm, but the important parameters that influence vegetable yield (e.g., number of leaves and stem diameter) were superior in the Indonesian accessions. Protein content showed a positive correlation with the number of leaves, whereas a negative correlation was observed with leaf thickness. The Indonesian accession of A. viridis and A. dubius showed a great potential to be further selected as parental lines for high protein content and number of leaves. The relatively high protein content of the leaves of Celosia may also potentially serve as an alternative protein source in the tropics.     

Morphophysiological variation in some Mexican species of vegetable Amaranthus: evolutionary tendencies under domestication

Summary Amaranth (Amaranthus spp.) is an important food resource for indigenous peoples of México. Grain-producing species are mostly cultivated plants, while the species used as vegetables are commonly encouraged in maize plots. Notable biological differences exist between these two types of amaranths. This study analyzes the morphophysiological variation in some Mexican species of Amaranthus and its relationship to the use and management of this taxa. A sample of individuals grown from 14 field collections representing 3 species and putative hybrid derivatives were grown in experimental plots and 18 morphological and physiological characters were measured. A statistical multivariate analysis was performed on these data. The results show that the grain-producing plants tend to allocate a high proportion of energy to the production of inflorescences while the plants used as vegetables allocate a higher proportion of biomass to the foliage. This suggests thatdomestication of Mexican species of Amaranthus have taken two different evolutionary paths depending upon the form of use and management by humans.     

Measurement of nuclear DNA content of the genus <Emphasis Type="Italic">Trifolium</Emphasis> L. as a measure of genebank accession identity

The collection, identification and maintenance of genebank accessions of the genus Trifolium is a major task because of the large number of genera and their occasional morphological similarity. We investigated whether the measurement of nuclear DNA content can serve as an additional criterion for identification of mislabeled accessions. Relative nuclear DNA content was determined by flow cytometry measurements for a total of 151 genebank accessions of 23 Trifolium species with notable agronomical value. Among 23 species analyzed, 15 were found to possess a uniform relative nuclear DNA content, with intraspecific variability of the majority of analyzed species lower than 5%. Within six Trifolium species, 1–2 atypical accessions with outstanding differences in relative DNA content, chromosome number and morphological features were found. In T. hybridum and partially in T. ambiguum these outstanding differences could be ascribed to variations in accession ploidy level. For the remaining atypical accessions, the determined nuclear DNA content, chromosome number and morphological features were not related to those characteristic of the species. Additionally taxonomic identity of atypical accessions was determined using ITS region sequencing and morphological observations. We propose flow cytometric measurements of nuclear DNA content as simple and reliable technique which can be used at seedling stage to verify identity and genetic stability of Trifolium genebank accessions.     

The Weedy Rice Complex in Costa Rica. I. Morphological Study of Relationships Between Commercial Rice Varieties,Wild <Emphasis Type="Italic">Oryza</Emphasis> Relatives and Weedy Types

Weedy rice is a complex of Oryza morphotypes widely distributed in commercial rice fields, which interfere with rice cultivation, seed production, industrial processing and commercialization of this crop in several countries. The objective of this study was to characterize the weedy rice complex of Costa Rica by comparing it with the cultivated and wild rice species found in the country. A collection of weedy rice accessions, representative of the morphotypes found in the country, was established and characterized. Their morphometric relationships were established by comparing 27 morphological traits with commercial rice cultivars, landraces and wild Oryza species and by performing a multivariate analysis. Twenty-one weedy rice morphotypes were identified among 735 weedy accessions by using a three-digit code based on seed characters. Three principal components (PCs) explained 66.25% of the variation observed. The first PC accounted for 36.21% of the variation and separated CCDD genome type Oryza latifolia and O. grandiglumis from AA genome species O. sativa, O. glumaepatula, O. rufipogon and O. glaberrima. The second (18.9%) and third (11.14%) PCs separated the weedy morphotype groups from the AA genome species O.sativa, O. glaberrima and O. rufipogon. The weedy morphotypes were scattered between the indica commercial rice varieties, the cluster landraces–glaberrima and O. rufipogon. Additionally, a group of morphotypes showed intermediate characteristics between O. sativa and O. rufipogon, suggesting that hybridization could have taken place in the past between these species. None of the morphotypes collected in Costa Rica clustered with the allotetraploids CCDD species or O. glumaepatula.     

The genetic characterization of Turkish watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis>) accessions using RAPD markers

Genetic diversity of the Turkish watermelon genetic resources was evaluated using different Citrullus species, wild relatives, foreign landraces, open pollinated (OP) and commercial hybrid cultivars by RAPD markers. The germplasm was consisted of 303 accessions collected from various geographical regions. Twenty-two of 35 RAPD primers generated a total of 241 reproducible bands, 146 (60.6%) of which were polymorphic. Based on the RAPD data the genetic similarity coefficients were calculated and the dendrogram was constructed using UPGMA (Unweighted pair-group method with arithmetic average). Cluster analysis of the 303 accessions employing RAPD data resulted in a multi-branched dendrogram indicating that most of the Turkish accessions belonging to var. lanatus of Citrullus lanatus (Thunb.) Matsum et Nakai were grouped together. Accessions of different Citrullus species and Praecitrullus fistulosus (Stocks) Pangalo formed distant clusters from C. lanatus var. lanatus. Among 303 accessions, a subset of 56 accessions was selected representing different groups and a second dendrogram was constructed. The genetic similarity coefficients (GS) within the Turkish accessions were ranged from 0.76 to 1.00 with 0.94 average indicating that they are closely related. Taken together, our results indicated that low genetic variability exist among the watermelon genetic resources collected from Turkey contrary to their remarkable phenotypic diversity.     

Biodiversity in the barnyard millet (<Emphasis Type="Italic">Echinochloa frumentacea</Emphasis> Link,Poaceae) germplasm in India

One hundred ninety four accessions of barnyard millet collected from different eco-geographical regions of India were evaluated for 14 quantitative traits during kharif 2003 and 2005. These accessions were grouped into 5 groups on the basis of place of collection. These groups differed significantly in their mean values for quantitative traits and magnitude of correlation among traits indicates origin based association. The group ‘C’ (source of origin is unknown) was found most diverse group (mean coefficient of variation 17.67%), while rest of the groups recorded mean coefficient of variation between 12 and 13%. Correlation studies indicate that flag leaf width, number of racemes along with internode length should be considered while performing selection in segregating generations. The plotting of first and second principal component axes scores suggests that presence of three distinct morphotypes in the present study. These morphotypes are quite similar to three botanical varieties viz., var. robusta, var. intermedia and var. stolonifera in their morphological traits, while botanical variety laxa (endemic to Sikkim area of India) was completely absent from the present study.     

Genetic variation within and among species of five sections of the genus Arachis L. (Leguminosae) using RAPDs

Some Arachis species are widely used as commercial plants, e.g. the groundnut A. hypogaea, an important source of good quality protein and oil, and A. pintoi and A. glabrata, that are utilized as forage species. Germplasm of most Arachis species is available in germplasm banks. However, little it is known about the genetic attributes of this germplasm, and mainly about its genetic variability, which is very important for its maintenance. In the present study RAPDs were used to assay the genetic variation within and among 48 accessions of five sections of the genus Arachis and to establish the genetic relationships among these accessions. Ten of 34 primers tested were selected for DNA amplification reactions since they yielded the largest numbers of polymorphic loci. A dendrogram was constructed based on data from the 10 primers selected. Eighty RAPD polymorphic bands were analyzed among the accessions studied. The relationships among species based on RAPDs were similar to those previously reported based on morphological, cytological and crossability data; demonstrating that RAPDs can be used to determine the genetic relationships among species of the different sections of the genus Arachis. In general, wide variation was found among accessions and low variation was found within the accessions that had two or more plants analyzed. However, higher polymorphism was found in the section Trierectoides and in one accession of A. major, indicating that generalizations should be avoided and each species should be analyzed in order to establish collection and maintenance strategies.     

Molecular phylogeny of Chinese vegetable mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequence variation of nuclear internal transcribed spacer regions of ribosomal DNA (ITS1, 5.8S rRNA and ITS2) from Chinese vegetable mustards (AB-genome) and its putative parents Brassica rapa (the A-genome) and Brassica nigra (the B-genome) were used to investigate the molecular phylogeny and the probable evolutional pattern of this amphidiploid species that uniquely formed in China. Totally, 16 accessions of Chinese vegetable mustard those covering nearly all the diverse variations were included in this study, and together with three accessions of B. rapa and one accession of B. nigra. The results disclosed two strongly supported clades, one containing four accessions of vegetable mustard which have closer relationship with B-genome species “B.nigra” lineage and the other containing 12 accessions of B. juncea and three A-genome accessions. This classification was in disagreement with the evidence from chloroplast DNA, mitochondrial DNA, nuclear DNA restriction fragment length polymorphism (RFLP), which suggested that B. juncea was closely related to the A-genome type. For the incongruence, we speculated that the B. juncea crops derived from Chinese have evolved through different recombined events of the diploid morphutypes and evolved unidirectional concerted evolution. The traditional phenotypic classification of B. juncea was not wholly supported by ITS results, and hence the phylogenetic relationships among these subspecies need to be reconsidered on molecular level.     

Analysis of genetic diversity in Piper nigrum L. using RAPD markers

RAPD analysis was conducted in 22 cultivars of P. nigrum(black pepper) from South India and one accession each of P. longum and P. colubrinum. Twenty-four primers generated 372 RAPD markers of which 367 were polymorphic. Jaccard's similarity between pairs of accessions ranged between 0.11 and 0.66 with a mean of 0.38. Among P. nigrum cultivars, the similarity ranged between 0.20 and 0.66 and the mean was 0.42. The existence of wide genetic diversity as revealed in the present study is supported by earlier reports of extensive inter- and intrapopulation morphological variability in pepper cultivars from South India. UPGMA dendrogram and PCO plot revealed P. colubrinum to be most distant of the three species. Genetic proximity among P. nigrum cultivars could be related to their phenotypic similarities or geographical distribution. Greater divergence was observed among landraces than among advanced cultivars. Landraces grown in southern parts of coastal India and those grown in more northern parts were grouped in separate clusters of the dendrogram.     

Crop–weed hybrids are more frequent for the grain amaranth ‘Plainsman’ than for ‘D136-1’

White-seeded cultivated amaranth [Amaranthus caudatus L., A. cruentus L., and A. hypochondriacus L.] grain is less valuable if contaminated with brown off-type seeds from the hybrid weedy progeny of spontaneous crop–weed crossing. Crop–weed crossing frequency was estimated by using two grain amaranth cultivars. Both cultivars were planted at the same nine locations in the Midwest United States and exposed to pollen from local weedy Amaranthus species. Harvested seeds were grown in a greenhouse, and the frequency of crop–weed hybrids was determined by observing dominant weed traits in the progeny. The cultivar ‘Plainsman’ was approximately tenfold more likely to hybridize with weedy amaranths than was ‘D136-1’. These are the first amaranth cultivars to be evaluated in this way. The reduced hybridization potential of ‘D136-1’ or similar material can be exploited to reduce the occurrence of off-type seed contamination in grain amaranths.     

RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Collection of Lupinus angustifolius L. Germplasm and Characterisation of Morphological and Molecular Diversity

Lupinus angustifolius L. is a Mediterranean species, domesticated in the 20th century, representing an important grain legume crop in Australia and other countries. This work is focused on the collection of wild germplasm and on the characterisation of morphological and molecular diversity of germplasm accessions. It reports the collection of 81 wild L. angustifolius accessions from the South and Centre of Portugal, available at the ‘Instituto Superior de Agronomia Gene Bank’, with subsequent morphological and molecular characterisation of a selection of these and other accessions. A multivariate analysis of morphological traits on 88 L. angustifolius accessions (including 59 wild Portuguese accessions, 15 cultivars and 14 breeding lines) showed a cline of variation on wild germplasm, with plants from Southern Portugal characterised by earlier flowering, higher vegetative development and larger seeds. AFLP and ISSR molecular markers grouped modern cultivars as sub-clusters within the wider diversity of wild germplasm, revealing the narrow pool of genetic diversity on which domesticated accessions are based. The importance of preserving, characterising and using wild genetic resources for L. angustifolius crop improvement is outlined by the results obtained.