Alberto Spada

Evidence of a secondary grapevine domestication centre detected by SSR analysis

Abstract. The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

Development of male and female flower in Asparagus officinalis. Search for point of transition from hermaphroditic to unisexual developmental pathway

Asparagus officinalis is a dioecious plant. The flowers start to develop as hermaphrodites and later become unisexual. In female flowers the stamens degenerate, while in male flowers the ovary stops growing without degenerating. We have examined young asparagus flowers using SEM and optical microscopy in order to determine the exact moment of transition from hermaphroditic to unisexual development. We defined 13 stages of development, starting from flower primordia up to completely mature flowers and labelled them with numbers from -6 to 7. The first five stages are fully hermaphroditic: a difference between sexes becomes visible at stage — 1 when the style begins to develop in female flowers. Degeneration of stamens in female flowers starts somewhat later. At the stage of transition, some differences between sexes also appear in the bidimensional polypeptide pattern of flowers. RNase activity shows a distinct peak at this stage (in female flowers only), probably related to stamen degeneration.

The arrest of development of abortive reproductive organs in the unisexual flower of Vitis vinifera ssp. silvestris

During the first stages of development, flowers of most dioecious species are hermaphroditic, with their transition to unisexual flowers being the result of the developmental arrest of one set of reproductive organs. In this work, we describe the development of male and female flowers of the dioecious wild grape species Vitis vinifera ssp. silvestris through scanning electron microscopy analysis and cytological observations, focusing our attention on the transition from bisexual to unisexual development. We divide floral development of the wild grape into eight stages. Differences between male and female flowers appear first at stage 6, when the style and stigma start to differentiate in female but not in male flowers. Cytological analysis of the slowly growing abortive pistil of male flowers shows that megagametophyte formation is, surprisingly, not inhibited. Instead of pistil abortion in the male flower, sexual determination is accomplished through programmed death of external nucellus cells and some layers of integumentary cells. Sterility of male structures in female flowers follows a different pattern, with microspore abnormalities evident from the time of their release from the tetrad. Sterile microspores and pollen grains in female flowers display an abnormal round shape, lacking colpi and possessing uniformly thickened cell walls that impede germination.

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

Abstract An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM.

Morphological traits and molecular markers for classification of Echinochloa species from Italian rice fields

Abstract The relationship among 80 different Echinochloa accessions were studied in 2000–2002 by applying amplified fragment length polymorphism (AFLP) analysis and examining the main morphological traits of these plants. The plants were collected from 40 different areas in which the main rice-growing region of northern Italy was divided on the basis of uniform environmental and agronomical conditions. Echinochloa accessions were grouped in three different species (E. crus-galli, E. erecta, E. phyllopogon) according to Pignattis classification key and in four different species according to Carreteros taxonomy (E. crus-galli, E. hispidula, E. oryzicola, and E. oryzoides). The E. crus-galli accessions clustered as a specific group under both AFLP analysis and morphological traits analysis carried out according to Pignattis and Carreteros keys. AFLP analysis revealed a separate group containing two accessions with a green basal stem section. These were classified as E. crus-galli by Pignattis taxonomy and E. oryzoides according to Carreteros taxonomy. A red basal stem section, a trait not considered by either morphological classification, was the main morphological trait that characterizes all E. crus-galli accessions. All other accessions with green basal stem section clustered in a group that included E. erecta and E. phyllopogon according to Pignattis taxonomy and E. hispidula and E. oryzicola according to Carreteros taxonomy. All species were evenly distributed in the monitored rice area, except the two E. oryzoides accessions, which were found only in the southwestern zone. Nomenclature: Barnyardgrass, Echinochloa crus-galli (L.) Beauv. ECHCG; junglerice, E. colonum (L.) Link ECHCO; late watergrass, E. phyllopogon (Stapf) Vasc. ECHPH; gulf cockspur, E. crus-pavonis (H. B. K.) Schultes ECHCV; early watergrass, E. oryzoides (Ard.) Fritsch. ECHOR; E. hostii (Bieb.) Boros; E. erecta (Pollacci) Pign.; E. hispidula (Retz.) Nees ex Royle; E. oryzicola (Vasing.) Vasing; rice, Oryza sativa L.

RAPD analysis and flavonoid composition of Aconitum as an aid for taxonomic discrimination

Abstract The RAPD technique and phytochemical analysis, based on the investigation of flavonoid composition, were used to study Aconitum vulparia , A. paniculatum , A. napellus subsp. tauricum (from two different localities), and A. napellus subsp. neomontanum . Twenty primers were screened for the genetic analysis and 14 were selected providing 51 polymorphic bands. The phenogram based on UPGMA clustering of Jaccard coefficient revealed a clear division between yellowish and blue Aconitum plants and inside this second group A. paniculatum is clearly separated from all populations belonging to A. napellus group. Also the flavonoid profile well characterise all species and subspecies under study.

The MADS box gene AOM1 is expressed in reproductive meristems and flowers of the dioecious species Asparagus officinalis.

Abstract MADS box genes are implicated in different steps of plant development. Some of them are expressed in vegetative organs. Most of them, however, are expressed in flower tissues and are involved in different phases of flower development. Here we describe the isolation and characterization of an Asparagus officinalis MADS box gene, AOM1. The deduced AOM1 protein shows the highest degree of similarity with FBP2 of Petunia hybrida and AGL9 (SEP3), AGL2 (SEP1) and AGL4 (SEP2) of Arabidopsis thaliana. In situ hybridization analyses, however, show that the expression profile of AOM1 is different from that of these genes: AOM1 is expressed not only in flower organs but also in inflorescence and flower meristems. These data indicate a possible function of AOM1 during flower development as well as in earlier stages of the flowering process. Asparagus officinalis is a dioecious species which bears male and female flowers on different individuals. AOM1, which is expressed very early during the process of flowering and has a similar expression profile in male and female flowers, does not seems to be involved in asparagus sex differentiation.

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones.

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 1∶1 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.

Anisocotyly and meristem initiation in an unorthodox plant, Streptocarpus rexii (Gesneriaceae)

In common with most Old World Gesneriaceae; Streptocarpus Lindl. shows anisocotylous growth, i.e., the continuous growth of one cotyledon after germination. Linked to this phenomenon is an unorthodox behaviour of the shoot apical meristem (SAM) that determines the growth pattern of acaulescent species (subgenus Streptocarpus). In contrast caulescent species develop a conventional central post-embryonic SAM (mainly subgenus Streptocarpella). We used S. rexii Lindl. as a model to investigate anisocotyly and meristem initiation in Streptocarpus by using histological techniques and analyses of the expression pattern of the meristematic marker SrSTM1 during ontogeny. In contrast to Arabidopsis thaliana (L.) Heynh., S. rexii does not establish a SAM during embryogenesis, and the first evidence of a SAM-like structure occurs during post-embryonic development on the axis (the petiolode) between the two cotyledons. The expression pattern of SrSTM1 suggests a function in maintaining cell division activity in the cotyledons before becoming localized in the basal meristem, initially at the proximal ends of both cotyledons, later at the base of the continuously growing macrocotyledon, and the groove meristem on the petiolode. The latter is equivalent to a displaced SAM seemingly originating de novo under the influence of endogenous factors. Applied cytokinin retains SrSTM1expression in the small cotyledon, thus promoting isocotyly and re-establishment of a central post-embryonic SAM. Hormone-dependent delocalization of the process of meristem development could underlie anisocotyly and the unorthodox SAM formation in Streptocarpus.

AOM3 and AOM4: two MADS box genes expressed in reproductive structures of Asparagus officinalis

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.