Genetic diversity among wild sugarcane germplasm from Laos revealed with markers

A group of 35 wild Saccharum complex clones, collected in Laos(ECL), was studied for its nuclear genetic diversity by RFLP revealed by 10dispersed low copy probes. Representatives of the main germplasmdiversity, regularly used in introgression, were also included. A preliminarycharacterisation of the new clones was performed by Erianthus specificprimers. A set of the 11 ECL clones most outstanding for their diseaseresistance and vigor and basic germplasm members were also characterisedby 9 cytoplasmic probes. Nuclear diversity evidenced that ECL clonesrepresented an independent genetic pool consisting at least of 3 differentgroups. A group of bands was exclusively exhibited by ECL clones. Theoccurrence of genetic recombination among them was suggested by thepresence of the new bands in 2 or 3 clonal groups. Erianthus specificamplification was positive for 7 ECL clones from different nuclear groups.Dendrogram analysis based on cytoplasmic diversity divided genotypes into2 major cytoplasmic types comprising 5 groups. The ECL clones separatedin 2 distinct groups: one located in the same cytoplasmic type as Erianthus clones; the other one, in an intermediate position, closer to Saccharum species. Basic clones groups were consistent with theirtaxonomic classification. The diversity revealed by nuclear singlecopy/nuclear repetitive/cytoplasmic patterns of the ECL germplasmcontrasts with what is usually observed and points to an active populationevolution. Further studies needed to confirmed this hypothesis arediscussed.     

Molecular diversity and genome structure in modern sugarcane varieties

Summary RFLP analysis was performed on 40 sugarcane cultivated varieties. Twenty-two maize low copy DNA clones located on different regions of the 10 maize chromosomes were used as probes to survey variability among the sugarcane varieties. A total of 425 fragments, 411 of which were polymorphic, were identified for 22 probe/enzyme combinations. Each variety displayed an average of 7.28 fragments per combination, revealing the complex polyploid origin of modern sugarcane varieties. The average genetic similarity between sugarcane varieties was 0.61. Although cultivated varieties appear closely related to S. officinarum clones, the genes of S. spontaneum seem to constitute the principal component of varietal diversity. A very weak global structuring among the 40 varieties is observed, in agreement with the profuse exchanges of parental materials between sugarcane breeding stations. Traces of linkage disequilibrium can be attributed to the distribution of S. spontaneum chromosomes among sugarcane varieties. The possibility of using modern varieties as a population for detecting associations between molecular markers and agronomic traits is suggested.     

Diversity in selected wild and cultivated species of pigeonpea using RFLP of mtDNA

Diversity in 28 accessions representing 12 species of the genus, Cajanus arranged in 6 sections including 5 accessions of the cultivated species, C. cajan, and 4 species of the genus Rhyncosia available in the germplasm collection at ICRISAT was assessed using RFLP with maize mtDNA probes. Cluster analysis of the Southern blot hybridization data with 3 restriction enzymes – 3 probe combinations placed the genus Rhyncosia in a major group well separated from all the species belonging to the genus Cajanus. Within the genus Cajanus, the 4 accessions of C. platycarpus belonging to section Rhynchosoides formed a separate group in contrast to those in other sections of pigeonpea. In the section, Cajanus all the 5 accessions of C. cajan were grouped together and C. cajanifolius belonging to the same section was in a subgroup by itself closer to the main group. The four accessions of C. scarabaeoides, were together and the other species belonging to section Cantharospermum were in different subgroups. The intra-specific variation was seen even within accessions of certain pigeonpea wild species such as C. scarabaeoides, C. platycarpus, C. acutifolius, and even the cultivated species of C. cajan. This study suggests that RFLP of mtDNA can be used for the diversity analysis of pigeonpea and it gives some indications on the maternal lineage among the species. The variations in the mitochondrial DNA hybridization patterns also suggest the extensive rearrangement of the organelle genome among the Cajanus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of tissue culture explant source on sugarcane yield components

Clonal propagation of sugarcane(interspecific hybrids of Saccharum)is conducive to spread of systemicdiseases, such as ratoon stunting disease,caused by Leifsonia xyli subsp. xyli. This important disease iscontrolled by obtaining and plantinghealthy seed-cane. In Louisiana, commercialseed-cane initially produced through tissueculture is available to sugarcane farmersand is being widely planted. Long-termacceptability of this seed-cane productionmethod depends on the production of healthyplants that do not differ significantly inphenotypic and yield characteristics fromthe clones originally selected and releasedas commercial cultivars. To determinewhether tissue culture affects yield or itscomponents, three cultivars, CP 70-321, LCP85-384, and HoCP 85-845, were compared inthree successive crops initially plantedwith stalks from three sources: plantsderived from callus culture of the leafroll above the apical meristem, directregeneration from the apical meristem, andconventional bud propagation. Stalks ofplants derived from both explant sourceswere typical of seed-cane farmers wouldpurchase for planting that had beenpreviously rogued for phenotypic variantsand increased by bud propagation.Differences in yield components amongtissue culture explant sources and budpropagated cane only occurred in CP 70-321.Stalk diameter and stalk weight were lowerand stalk population was higher for plantsderived from leaf roll callus compared tobud propagated cane. Yield components weresimilar for plants derived from an apicalmeristem and bud propagation. Individualplant phenotypic variants resulting fromsomaclonal variation were not observed inany of the cultivars derived from eitherexplant source. In summary, genotype andexplant source affected persistent, uniformphenotypic variation resulting from tissueculture that changed some yield components. However, apical meristem culture wassuitable for production of seed-cane, assugarcane derived by meristem culture ofthree cultivars did not differsignificantly from the original germplasmfor any measured yield trait.     

Genetic characterization of 218 elite CIMMYT maize inbred lines using RFLP markers

Characterization of genetic diversity among maize inbred lines can facilitate organization of germplasm and improve efficiency of breeding programs. A set of 218 phenotypically diverse inbred maize lines developed at CIMMYT for hybrid production was characterized using 32 RFLP markers to: (1) analyze the genetic diversity present; (2) define potential heterotic groups based on clusters formed with marker data; and (3) identify the most representative testers for each potential heterotic group. Lines were clustered using five different genetic distance measurements to find consensus non-hierarchical clusters. Dendrograms were produced to study hierarchical classification within smaller groups of lines. A very high average allelic diversity was seen in this germplasm. Lines did not cluster based on phenotype, environmental adaptation, grain color or type, maturity, or heterotic response (as determined based on hybrid performance with testers), but lines related by pedigree usually did cluster together. Previously defined testers from opposite heterotic groups were not genetically differentiated, and did not represent well their heterotic group. Discrete clusters were difficult to find; thus, potential heterotic groups will be difficult to suggest using RFLP markers alone. However, suggestions on how to use molecular markers and cross performance information to refine heterotic groups and select representative testers are presented.     

Colchine-induced aneuploids from cell culture of sugarcane

Summary Sugarcane (Saccharum spp.) clones are amenable to gross chromosome manipulation due to their high polyploid nature (2n=100–120). This study was conducted to analyze the effects on plant morphology of altering chomosome number via callus culture. Callus cultures from clone H69-9092 were established, and plants were regenerated following colchicine treatment of cultured cells. Cytological analysis showed that variant somaclones were aneuploids with a wide range in chromosome numbers (2n=66–196). Some 22 visually distinct somaclones were planted in 1.35 m² plots with five replications to compare morphological and quality characteristics with H69-9092 at 8 months of growth. Extreme morphological variation was observed between somaclones, but coefficients of variation for quality factors-fibers %, refractometer solids %, pol %, and juice purity-and stomatal length were smaller than those for morphological traits associated with stalk volume and leaf area. Significant negative correlations were found between chromosome number and most morphological traits, e.g., stalk length (r=-0.58), number (r=-0.69), diameter (r=-0.54) and volume (r=-0.65); internode length (r=-0.57); and leaf area (r=-0.48). A positive correlation was found between chromosome number and stomatal length (r=-0.66). No significant correlations were found between chromosome number and quality factors. Aneuploids with higher than parental chromosome number had reduced growth. However, depression in growth was generally not observed in somaclones lower in chromosome number than the parent.Published with the approval of the Director as Paper No. 598 in the Journal Series of the Experiment Station, Hawaiian Sugar Planters' Association.     

Screening for and heritability of flood-tolerance in the Florida (CP) sugarcane breeding population

Summary An experiment screening for flood-tolerance and estimating the heritability of flood-tolerance was conducted on 160 sugarcane clones in the Canal Point, Florida, USA sugarcane breeding population. Clones were grown in two environments, a drained control and a continuous, five-month flood. The test ran for two crop years, plant-cane and first ratoon. A wide range of production was observed, with some clones dying out after the first year. Several clones had two-year mean cane production in flood that was 70% of their production in control. Heritabilities calculated by parent-offspring regression ranged from 0.29 to 0.51.     

Isolation,fusion and multiplication of sugarcane protoplasts and comparison of sexual and parasexual hybridization

Summary The problems associated with the isolation of leaf protoplasts in sugarcanes have been overcome by the choice of spindle tissue as source material. It is now possible to isolate, fuse, regenerate and multiply sugarcane protoplasts. However, the usefulness is limited due to the shortlived chromosomal stability during the callus stage. Nevertheless, preliminary results are promising and the method should prove feasible with refinement in technique. Two breeding systems for using parasexual hybridization are discussed.     

Stability and potential use of RAPD markers in a sugarcane genealogy

Summary A complete ancestral history of the recently developed and closely related South African commercial sugarcane varieties N11 and NCo376, which differ markedly in their response to sugarcane mosaic virus (SCMV), was elucidated from archival records. The genealogy spans seven generations, starting with early intraspecific crosses between varieties of Saccharum officinarum and interspecific crosses between S. officinarum and either S. spontaneum or S. barberi. In total, the genealogy comprises 38 different varieties. Genomic DNA samples from N11 and NCo376 respectively were screened for polymorphisms using the PCR-RAPD technique. Ten polymorphic fragments ranging in molecular size from 317 to 1263bp were identified from a total of 1159 loci amplified with 100 random decamer primers. Two of the 10 polymorphic fragments were shown to be consistently present in N11 (resistant) and absent in NCo376 (susceptible), while 8 showed the reverse occurrence. The primers producing the polymorphisms were used to screen genomic DNA samples from all 19 varieties representing the genealogy. Results have indicated that (1) specific PCR-RAPD generated polymorphic fragments can indeed be identified across the seven generations; (2) certain fragments are sufficiently definitive to be used as markers to trace parentage; (3) the validity of documented crosses and/or the authenticity of germplasm material may be questioned using this technique, and (4) there is the potential to subject the markers to linkage analysis once a full and accurate assessment of the SCMV resistance phenotype is obtained.     

RFLP analysis of genetic variation in species of section Arachis, genus Arachis (Leguminosae)

Four A-genome species of the genus Arachis (A. cardenasii, A. correntina, A. duranensis, A. kempff-mercadoi), three B genomes species (A. batizocoi, A. ipaënsis and A. magna),the AABB allotetraploid A. hypogaea (cultivated peanut) and introgression lines resulting from a cross between A. hypogaea and A. cardenasii were analyzed by RFLP. The A genome species (cytologically characterized by the presence of a small chromosome pair ‘A’) were closely similar to each other and shared a large number of restriction fragments. In contrast, the B genome species differed more from one another and shared few fragments. The results of this study indicate that the absence of the small chromosome pair is not a good criterion for grouping species of section Arachis as B genome species, since their genome might be quite distinct from the B genome of A. hypogaea.The lowest genetic variation was detected within accessions of A. duranensis (17 accessions), followed by A. batizocoi (4 accessions) and A. cardenasii (9 plants of accession GKP 10017).The high level of genetic variation found in A. cardenasii might indicate that not all accessions of wild species of Arachis are autogamous, as reported for A. hypogaea.     

Relationships among American and Spanish populations of maize

Summary Two experiments were carried out with two objectives. First, to establish the phenetic relationships among the maize (Zea mays L.) landraces from Galicia (Northwestern Spain) maintained at the Misión Biológica de Galicia. Second, to assess the resemblance between a collection of Spanish populations (including the landraces from Galicia) and a set of US Corn Belt varieties. For the first objective 73 varieties from Galicia, along with 9 hybrid checks, were grown in 9×9 simple lattices at two locations for two years. For the second objective 131 populations from the US Corn Belt and Spain, along with 9 hybrid checks, were grown for three years in unreplicated experiments. Cluster analyses were carried out with the first principal components that accounted for a significant amount of the total variation. Four groups were found among the landraces from Galicia. The populations from Spain and America were classified as belonging to nine main groups. The replicated experiment was more accurate than the unreplicated one. However, it is concluded that an unreplicated test grown in several environments is accurate enough to detect the main groups, although some inaccuracies should be expected.     

The genetic structure of cocoa populations (Theobroma cacao. L.) revealed by RFLP analysis

The genetic structure of 175 genotypes of Theobroma cacao L. was investigated using 27 RFLP/cDNA loci. The number of alleles per locus was never higher than four and a high genetic diversity was found. Criollo genotypes appeared differentiated from Forastero genotypes. A deficiency of heterozygotes was found in all populations and some alleles were fixed in some populations. Within population gene diversity was high. When four morphological groups were considered, Upper Amazon Forastero was the most polymorphic and diverse population. Almost all the alleles of the whole species could be found within this group. Observed heterozygosity was the highest within Criollo and Trinitario populations but a certain proportion of homozygous genotypes was present in all groups. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identifying the alien chromosomes in wheat – Leymus multicaulis derivatives using GISH and RFLP techniques

Genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) were used to identify the Leymus multicaulis (XXNN, 2n = 28) chromosomes in wheat-L. muliticaulis derivatives. Fifteen lines containing L. multicaulis alien chromosomes or chromosomal fragments were identified. All alien chromosomes or fragments in these 15 lines were from the X genome and none were from the N genome. Eleven L. multicaulis disomic addition lines and four translocation-addition lines were identified with chromosome rearrangements among homoeologous groups 2, 3, 6 and 7. Only homoeologous group 1 lacked rearrangements in addition or translocation chromosomes. The results revealed that translocation in non-homoeologous chromosomes widely exists in the Triticeae and therefore it is necessary to identify the alien chromosomes (segments) in a wheat background using these combined techniques. During the course of the work, probe PSR112, was found to detect X genome addition lines involving L. multicaulischromosomes. This may prove to be a valuable probe for the identification of alien chromosomes in a wheat background. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of a set of 14 c-DNA probes from wheat to detect restriction fragment length polymorphism (RFLP) in barley

Summary A set of 14 probes from wheat cDNA clones was used to search for restriction fragment length polymorphism (RFLP) in six barley lines. The degree of polymorphism among the lines varied greatly between probes and between the various restriction enzymes. Two probes revealed a high degree of polymorphism in all probe/enzyme combinations. Seven of 14 probes did not reveal RFLP. The average level of polymorphism based on all 840 pairwise comparisons was 14.0%, which is higher than has been reported in wheat, but lower than in maize, rice, potato and lettuce. Most of the probes that detected RFLP correspond to sites on the long arms of wheat chromosomes.     

Genetic diversity and phylogenetic relationships among the annual Cicer species as revealed by isozyme polymorphism

Summary There are few estimates of genetic variability within and among populations of the nine annual Cicer species and for the wild species this information is based on few accessions. The present study was undertaken to examine genetic variation within and between annual Cicer species. One hundred and thirty-nine accessions of nine annual Cicer species were used for electrophoretic analysis at ICARDA. High levels of polymorphism in all eight wild annual Cicer species was found. This is in contrast to earlier research which had shown high polymorphism only in C. reticulatum. Cicer reticulatum had the highest proportion of polymorphic loci. However, for the cultigen, among 14 loci assayed, only two were polymorphic, ADH and PGD2. The nine species formed four phylogenetic groups based on the neighbor-joining method. The first group comprised C. arietinum, C. Reticulatum and C. echinospermum, the second C. bijugum, C. judaicum and C. pinnatifidum, the third C. chorassanicum and C. yamashitae; and the fourth group consisted of one species, C. cuneatum. The phylogenetic tree developed from the neighbor-joining technique illustrated that C. reticulatum is the probable progenitor of C. arietinum and that C. echinospermum split off from a common ancestor at an earlier stage in the evolutionary history of Cicer. Genetic diversity data showed that the greatest diversity was within C. reticulatum and the lowest with the cultigen, C. arientinum. With the exception of C. reticulatum, genetic diversity increased with genetic distance from the cultigen. Little geographic variation in genetic diversity was found.     

Detection of linkage between RFLP markers and genes affecting anthocyanin pigmentation in maize (Zea mays L.)

Summary Linkages between molecular markers and genes involved in the expression of agronomical traits have already been described in all of the major crops. In most cases, the genetic model underlying the Quantitative Traits Loci (QTL) is discussed. Here, Restriction Fragment Length Polymorphisms (RFLPs) and Mapmaker-QTL have been used to pinpoint seven regions of the genome significantly correlated with four pigmentation qualitative traits of maize (Zea mays L.). Two of these, located on chromosomes 2 and 10, explain most of the variation of these traits. The R and B gene loci known to be involved in the regulation of the anthocyanin pathway map to the same regions and we suggest that these loci could be the candidate genes involved in the correlations detected with RFLPs. This type of result is in accordance with the hypothesis of the candidate gene which supposes that, if we have a very high density map of randomly-selected cDNA clones, it should theoretically be possible to associate a cloned genic sequence with a phenotypic trait where correlations are found.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

Tissue and cell culture as AIDS to sugarcane breeding. I. Creation of genetic variation through callus culture

Summary Four-hundred and seventeen plants from 8 sugarcane varieties were selected among the 4600 plants that had been generated from calluses of shoot-apices, rolled young leaves and young inflorescences of 58 varieties. They were planted in the field (Field Test-II stage) in December, 1971 and examined for morphology, sugar content and chromosome number in the summer of 1972 and thereafter. The callus derivatives of the 8 varieties differed from their donors in the frequency of morphological changes: F146 (1.8%), N:Co310 (2.4%), F161 (4.8%), F164 (6.7%), F162 (7.4%), 56–2080 (15.8%), F170 (27.6%), and F156 (34.0%). Auricle length showed the greatest frequency of difference (8.6%); dewlap shape ranked next (6.5%); followed by hair group (6.2%); attitude of top leaves (1.9%). The sucrose content of callus derivative was increased over their donors by 2% to 12%. Chromosome number (2n) varied from 86 to 126 in the F156 derivatives in contrast with the donor's 114 whereas those of F164 varied from 88 to 108 as compared with the donor's 108. The derivatives of F146 generally centred around the original chromosome number of 110 and it is considered to be the most genetically stable cultivar. The cause of the occurrence of chromosome mosaicism in sugarcane is discussed. There was no apparent correlation between morphological modifications and changes in chromosome number.     

Cytogenetic analyses of sugarcane relatives (Andropogoneae: Saccharinae)

Summary Meiosis was studied in 31 wild Saccharum relatives, including Erianthus (8 clones), Miscanthus (5 clones), Narenga prophyrocoma (1 clone), S. robustum (3 clones), and S. spontaneum (14 clones). Chromosome number for 18 clones confirmed published counts or was typical of the particular species. Chromosome number for seven clones (Djatiroto 2n=58, Molokai 5099 2n=80, SES 84/58 2n=58, SES 114 2n=64, SES 260 2n=64, Taiwan 100 2n=112, and US 57-11-2 2n=60) differed from published counts (2n=112, 86-100, 64, 60, 60, 96, and 30, respectively). Counts were obtained for the first time for six clones (Local escape 2n=96, Nepal 2n=72, NG 77-77 2n=108–112, NG 77-199 2n=166, US 57-60-2 2n=20, and US 68-1-4 2n=38). Bivalent chromosome pairing predominated in all clones. Meiotic irregularity (numeric aberrations, univalents, multivalents, and telophase II micronuclei) tended to be associated with taxonomic grouping and level of polyploidy. Clones in Erianthus, Miscanthus, and Narenga were apparent euploids (2n=20–60) and tended to have fewer meiotic irregularities than Saccharum clones. Differences in level of meiotic stability among taxonomic groups may reflect error in chromosome association and synapsis associated with high chromosome number.     

Identification of DNA probes that reveal polymorphisms among closely related Phaseolus vulgaris lines

Summary Analyses of genetic diversity within populations could be of great benefit to plant genetic resources conservation. In order to identify genetic markers that are variable within populations, the genome of Phaseolus vulgaris was screened with several DNA sequences in order to identify hypervariable sequences. Polymorphisms were observed between Middle American and Andean cultivars using the protein III tandem repeat of the M13 phase and the 33.15 human minisatellite. Extensive differences were observed when the DNA of two divergent lines—BAT93 and Jalo EEP558, of Middle American and Andean origin, respectively—were digested with HinfI, TaqI, HaeIII and hybridized with the 33.15 human minisatellite. Similarly, numerous polymorphisms were observed when the M13 protein III tandem repeat region was hybridized with TaqI digests of these cultivars. Polymorphism was also detected among sister lines of two F₆ backcross materials involving Middle American and Andean lines when genomic DNA was digested with TaqI and hybridized with M13 tandem repeat region. In addition, polymorphism was observed among Porrillo cultivars that resulted from selection within a single landrace population. Whereas only one isozyme difference had been observed previously among the Porrillo cultivars, eleven restriction fragments detected by the M13 protein III tandem repeat sequence differentiated these cultivars. Ribosomal DNA also hybridized to several polymorphic bands on TaqI and EcoRI genomic Southern blots of the F₆ backcross material. Only one polymorphism was observed with EcoRI-digested genomic DNA of BAT93 and Jalo EEP558 was hybridized with microsatellite (GACA)₄. This probe might be useful in ascertaining relationships at the species and subspecies level, and as a marker in mapping studies. Our results show that both the human 33.15 minisatellite and M13 should be useful probes to detect within-population variability in common bean.