Seed coat microsculpturing changes during seed development in diploid and amphidiploid Brassica species

BACKGROUND AND AIMS: Seed coat morphology is known to be an excellent character for taxonomic and evolutionary studies, thus understanding its structure and development has been an important goal for biologists. This research aimed to identify the developmental differences of seed coats between amphidiploids and their putative parents in Brassica. METHODS: Scanning electron microscope (SEM) studies were carried out on six species (12 accessions), three amphidiploids and their three diploid parents. KEY RESULTS: Twelve types of basic ornamentation patterns were recognized during the whole developmental process of the seed coat. Six types of seed coat patterns appeared in three accessions of Brassica rapa, five types in B. oleracea, B. nigra and B. carinata, seven types in B. napus, and eight types in B. juncea. There was less difference among seed coat patterns of the three accessions of B. rapa. The reticulate and blister types were two of the most common patterns during the development of seeds in the six species, the blister-pimple and the pimple-foveate patterns were characteristic of B. rapa, and the ruminate of B. oleracea and B. nigra. The development of seed coat pattern in amphidiploids varied complicatedly. Some accessions showed intermediate patterns between the two putative parents, while others resembled only one of the two parents. CONCLUSIONS: The variation in the patterns of seed coat development could be used to provide a new and more effective way to analyse the close relationship among amphidiploids and their ancestral parents.     

Chloroplast DNA diversity reveals the contribution of two wild species to the origin and evolution of diploid safflower (Carthamus tinctorius L.)

The identity of the wild progenitor of one of the most important oil crop species, Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, has been the subject of numerous studies at morphological, biochemical, cytogenetic, and biosystematic levels, but no definitive conclusions have been made. The nuclear, mitochondrial, and chloroplast genomes of the two botanical varieties of C. tinctorius, C. tinctorius var. tinctorius and C. tinctorius var. inermis, and two wild species, C. palaestinus and C. oxyacantha, were assayed at the nucleotide sequence level and by DNA markers. The nuclear and mitochondrial DNA assays were not helpful in conclusively identifying the diploid ancestor of C. tinctorius. The chloroplast DNA diversity, on the other hand, unambiguously provided new and novel evidence that C. palaestinus and C. oxyacantha contributed their plastomes to the evolution of C. tinctorius var. inermis and C. tinctorius var. tinctorius, respectively. This study, therefore, affirms a startling revelation of a rare event of two wild species contributing to the origin and evolution of safflower, a major world oilseed crop about whose genetics very little is known.     

Chloroplast and nuclear DNA variation in common wheat: insight into the origin and evolution of common wheat

To understand the origin and evolution of common wheat, chloroplast (ct) and nuclear DNA variations were studied in five hexaploid and three tetraploid wheat subspecies. Based on chloroplast simple sequence repeats at 24 loci, they were classified into two major plastogroups. Plastogroup I consisted of 11 plastotypes, including the major plastotype H10 that occurred at the highest frequency (59%) in common wheat. Plastogroup II consisted of five plastotypes and occurred in eight out of 27 accessions of T. aestivum ssp. spelta and one accession of ssp. aestivum. As for nuclear DNA variations, AFLP data using 10 primer sets revealed two major clades of a phylogenetic tree constructed by UPGMA (unweighted pair-group method with arithmetic mean), one consisting of common wheat and the other of emmer wheat. The clade of common wheat was further divided into two major and six minor subclades. One of the major subclades consisted only of non-free-threshing ssp. spelta accessions, which were grouped into two clusters, one consisting only of accessions with plastogroup I ctDNA and the other with both plastogroups I and II. T. aestivum ssp. macha, another non-free-threshing common wheat, formed the other cluster. Taken together, our data indicate the existence of at least two maternal lineages in common wheat and support the hypothesis that European spela wheat originated in Europe separately from other groups of common wheat.     

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium? array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.     

Chloroplast DNA variation and evolution in the genus Lens Mill

 Chloroplast DNA (cpDNA) restriction site diversity was assessed by 21 enzyme/probe combinations in 30 accessions of six Lens species, including the recently recognized L. lamottei and L. tomentosus. A total of 118 fragments were scored and 26 restriction site mutations were identified. The cpDNA restriction pattern supports circumscribing L. lamottei and L. tomentosus as independent species. The value of the data for reconstructing phylogeny in the genus is discussed. The cpDNA of all 13 accessions of the lentil’s wild progenitor, L. culinaris subsp. orientalis, differed from that of the single lentil cultivars used in this study. This diversity indicates that other populations of this subspecies from Turkey and Syria examined by Mayer and Soltis (1994) are potentially the founder members of lentil. Examination of L. lamottei×L. nigricans hybrids between accessions having different restriction patterns showed paternal plastid inheritance in L. nigricans. Received: 2 July 1996 / Accepted: 19 July 1996     

Chloroplast DNA indicates a single origin of the allotetraploid Arabidopsis suecica

DNA sequencing was performed on up to 12 chloroplast DNA regions [giving a total of 4288 base pairs (bp) in length] from the allopolyploid Arabidopsis suecica (48 accessions) and its two parental species, A. thaliana (25 accessions) and A. arenosa (seven accessions). Arabidopsis suecica was identical to A. thaliana at all 93 sites where A. thaliana and A. arenosa differed, thus showing that A. thaliana is the maternal parent of A. suecica. Under the assumption that A. thaliana and A. arenosa separated 5 million years ago, we estimated a substitution rate of 2.9 x 10(-9) per site per year in noncoding single copy sequence. Within A. thaliana we found 12 substitution (single bp) and eight insertion/deletion (indel) polymorphisms, separating the 25 accessions into 15 haplotypes. Eight of the A. thaliana accessions from central Sweden formed one cluster, which was separated from a cluster consisting of central European and extreme southern Swedish accessions. This latter cluster also included the A. suecica accessions, which were all identical except for one 5 bp indel. We interpret this low level of variation as a strong indication that A. suecica effectively has a single origin, which we dated at 20 000 years ago or more.     

Nicotiana chloroplast genome III. Chloroplast DNA evolution

Summary Nicotiana chloroplast genomes exhibit a high degree of diversity and a general similarity as revealed by restriction enzyme analysis. This property can be measured accurately by restriction enzymes which generate over 20 fragments. However, the restriction enzymes which generate a small number (about 10) of fragments are extremely useful not only in constructing the restriction maps but also in establishing the sequence of ct-DNA evolution. By using a single enzyme, Sma I, a elimination and sequential gain of its recognition sites during the course of ct-DNA evolution is clearly demonstrated. Thus, a sequence of ct-DNA evolution for many Nicotiana species is formulated. The observed changes are all clustered in one region to form a hot spot in the circular molecule of ct-DNA. The mechanisms involved for such alterations are mostly point mutations but inversion and deficiency are also indicated. Since there is a close correlation between the ct-DNA evolution and speciation in Nicotiana a high degree of cooperation and coordination betwen organellar and nuclear genomes is evident.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.     

Chloroplast DNA variation within and among five Plantago species

Chloroplast DNA variation was studied within and among five Plantago species. We determined polymorphism by surveying 86% of the chloroplast genome using 9 or 11 restriction enzymes for intra or interspecific variation, respectively. All five species were polymorphic for both site and length mutations. The outcrossing P. lanceolata has six chloroplast haplotypes of which some were found in the Netherlands as well as in the United States. The highly selfing P. major had five haplotypes and Dutch and United States populations were differentiated from each other in cpDNA. Plantago species are highly differentiated from each other. Of 252 restriction-sites, 52% were variable among species. Most of this variation was localized in part of the large single copy region. We derived a molecular phylogeny of the five species from restriction-site variability using PAUP 3.0. P. coronopus and P. maritima form a group as do P. major and P. media. P. lanceolata is more distantly related to the other four species. In a consensus tree both P. major haplotypes and both P. media haplotypes were connected to one node.     

Radionuclide-induced evolution of DNA and the origin of life

Summary Artesian groundwaters of high radionuclide concentration are ubiquitous and may have provided the large, sustained energy sources that were required to drive the multistage process of DNA and primordial cell evolution. The rapid, early development of the genetic code as well as its degeneracy can be attributed to exceptionally high radiation-induced mutation rates in this unique environment. The ability of double-strand DNA to direct enzymatic repair of radiation damage to single strands contributed importantly to its selective evolution. It is postulated that the polymerization of nucleotides took place at elevated temperatures within -particle tracks of high ion and free-radical density, followed by rapid quenching to ambient conditions. It also is evident that radiation resistance and ploidy were important selection factors in cellular evolution.     

Chloroplast DNA microsatellite analysis supports a polyphyletic origin for barley

Five barley chloroplast DNA microsatellites (cpSSRs) were used to study genetic relationships among a set of 186 barley accessions—34 Hordeum vulgare ssp. spontaneum (HS accessions) from Morocco, Ethiopia, Cyprus, Crete, Libya, Iraq, Iran, Turkey, Afghanistan and Israel, 122 H. vulgare ssp. vulgare landraces (HV landraces) from Spain, Bolivia (old Spanish introductions), Morocco, Libya and Ethiopia and 20 modern European spring barleys (HV cultivars). All loci were polymorphic in the material studied, with the number of alleles per locus ranging from two to three. Fifteen multi-locus haplotypes were observed, 11 in HS accessions and seven in HV landraces and cultivars. Of the seven haplotypes found in the HV lines, three were shared with the HS accessions, and four were unique. Cluster analysis revealed two main groups, one consisting of HS accessions from Ethiopia and the HV landraces from Spain, Bolivia (old Spanish), Morocco and Ethiopia, whereas the other larger group contained all of the other accessions studied. Based on these grouping and the existence of haplotypes found in the HV landraces and cultivars but not in the HS wild barley, a polyphyletic origin is proposed for barley, with further centres of origin in Ethiopia and the Western Mediterranean.     

Relationships between growth and gas exchange characteristics in some salt-tolerant amphidiploid Brassica species in relation to their diploid parents

Relationships between growth and different gas exchange characteristics of two amphidiploid salt tolerant species, Brassica napus, and B. carinata with respect to their salt sensitive parents, B. oleracea, and B. nigra were investigated. Twenty three-day old plants of these four species along with those of another amphidiploid moderately salt tolerant B. juncea (developed by hybridization of diploids, B. campestris and B. nigra), and a diploid moderately salt tolerant, B. campestris, were subjected for 28 days to salinized sand culture containing 0, 100 or 200 mol NaCl m(-3) in Hoagland's nutrient solution. The species B. napus and B. carinata produced significantly greater shoot fresh and dry matters than their parents under saline conditions. A close association was found between growth, and assimilation rate for all species differing in degree of salt tolerance. Stomatal conductance (g(s)) was reduced due to salt stress in all species but this variable had no significant correlation with assimilation rate (A). However, the amphidiploid salt tolerant species, B. napus and B. carinata had significantly greater photosynthetic rate, water use efficiency (A/E), intrinsic water use efficiency (A/g(s)) than those of their diploid parents. In conclusion, high salt tolerance of the two amphidiploid species, B. napus and B. carinata was associated with a high assimilation rate, water use efficiency and intrinsic water use efficiency but there was little association of the tolerance of these species with stomatal conductance, leaf water potential or transpiration rate (E).     

Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome.In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms.Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products.In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.     

Chloroplast DNA evolution in potato (Solanum tuberosum L.)

Summary A deletion specific to chloroplast (ct) DNA of potato (Solanum tuberosum ssp. tuberosum) was determined by comparative sequence analysis. The deletion was 241 bp in size, and was not flanked by direct repeats. Five small, open reading frames were found in the corresponding regions of ctDNAs from wild potato (S. tuberosum ssp. andigena) and tomato (Lycopersicon esculentum). Comparison of the sequences of 1.35-kbp HaeIII ctDNA fragments from potato, tomato, and tobacco (Nicotiana tabacum) revealed the following: the locations of the 5 ends of both rubisco large subunit (rbcL) and ATPase beta subunit (atp) mRNAs were probably the same as those of spinach (Spinacia oleracea); the promoter regions of the two genes were highly conserved among the four species; and the 5 untranslated regions diverged at high rates. A phylogenetic tree for the three potato cultivars, one tomato cultivar, and one tobacco cultivar has been constructed by the maximum parsimony method from DNA sequence data, demonstrating that the rate of nucleotide substitution in potato ctDNA is much slower than that in tomato ctDNA. This fact might be due to the differences in the method of propagation between the two crops.     

Chloroplast DNA and isozyme evidence on the evolution ofSenecio vulgaris (Asteraceae)

Chloroplast DNA (cpDNA) and isozyme variation were analyzed over a range of populations of two infraspecific taxa of the tetraploidSenecio vulgaris. The isozyme data were supportive of the hypothesis that the weedy and cosmopolitanS. vulgaris var.vulgaris is an evolutionary derivative ofS. vulgaris subsp.denticulatus from the coasts of W Europe and montane altitudes in S Spain and Sicily. The two taxa exhibited a very high genetic identity with subsp.denticulatus containing slightly more isozyme diversity than was found in var.vulgaris. — Three cpDNA haplotypes (A, B, C) already known from other Mediterranean diploid species ofSenecio were resolved in var.vulgaris, and an additional fourth haplotype (E) was found in subsp.denticulatus. Two alternative hypotheses were chosen to account for the origin and maintenance of the observed cpDNA composition ofS. vulgaris. It either reflects (1) the retention of an ancestral polymorphism which stems from the recurrent and polytopic formation of ancestral tetraploid lineages; or (2)S. vulgaris originally was characterized by haplotype E, and haplotypes A, B and C were acquired through repeated introgressive hybridization with related diploid species. The finding that very low levels of nuclear (isozyme) diversity were present in both taxa ofS. vulgaris examined supports the second of these two hypotheses; however, more detailed analysis of nuclear genetic diversity is required before a firm conclusion can be reached on this matter.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Chloroplast DNA phylogeography of the arctic-montane species Saxifraga hirculus (Saxifragaceae)

The genetic structure of populations of an arctic-montane herb, Saxifraga hirculus (Saxifragaceae), was analysed by means of chloroplast restriction fragment-length polymorphism. Sampled populations were distributed across Europe and North America (Alaska and Colorado). There was no evidence for geographically structured genetically divergent lineages, and although no haplotypes were shared between North America and Europe, the haplotypes from different continents were intermixed on a minimum spanning tree. European populations were much more highly differentiated and had much lower levels of haplotype diversity than their Alaskan counterparts. Centres of haplotype diversity were concentrated in those Alaskan populations located outside the limits of the last (Wisconsin) glaciation, suggesting that they may have acted as refugia during the Pleistocene. It was not possible to identify putative migration routes or corresponding refugia in the European genepool. One British population, from the Pentland Hills, was genetically very distant from all the others, for reasons that are as yet unknown.     

Inter- and intra-genomic homology of the Brassica genomes: implications for their origin and evolution

In order to determine the homologous regions shared by the cultivated Brassica genomes, linkage maps of the diploid cultivated B. rapa (A genome, n = 10), B. nigra (B genome, n = 8) and B. oleracea (C genome, n = 9), were compared. We found intergenomic conserved regions but with extensitve reordering among the genomes. Eighteen linkage groups from all three species could be associated on the basis of homologous segments based on at least three common markers. Intragenomic homologous conservation was also observed for some of the chromosomes of the A, B and C genomes. A possible chromosome phylogenetic pathway based on an ancestral genome of at least five, and no more than seven chromosomes, was drawn from the chromosomal inter-relationships observed. These results demonstrate that extensive duplication and rearrangement have been involved in the formation of the Brassica genomes from a smaller ancestral genome.