The grapevine fleshless berry mutation. A unique genotype to investigate differences between fleshy and nonfleshy fruit

In flowering plants, fruit morphogenesis is a distinct process following fertilization resulting in the formation of a specialized organ associated with seeds. Despite large variations in types and shapes among species, fleshy fruits share common characteristics to promote seed dispersal by animals such as organ growth and metabolite accumulation to attract animal feeding. The molecular biology of fruit ripening has received considerable attention, but little is known about the determinism of early fruit morphogenesis and why some fruits are fleshy while others lack flesh. We have identified in grapevine (Vitis vinifera) a mutation we have named fleshless berry (flb) that reduces by 20 times the weight of the pericarp at ripening without any effect on fertility or seed size and number. The flb mutation strongly impaired division and differentiation of the most vacuolated cells in the inner mesocarp. The timing of ripening was not altered by the mutation although the accumulation of malic acid in the green stage was noticeably reduced while sucrose content (instead of hexoses) increased during ripening. The mutation segregates as a single dominant locus. These results indicate that the Flb- mutant is suitable material to advance our understanding of the genetic and developmental processes involved in the differentiation of an ovary into a fruit.     

The grape microvine – a model system for rapid forward and reverse genetics of grapevines

A grapevine model system is described that is suitable for rapid forward and reverse genetic studies in small controlled environments. It is based on the Vvgai1 mutant allele that confers a dwarf stature, short generation cycles and continuous flowering (‘microvine’). Black and white berry microvine genotypes were developed that can be transformed by Agrobacterium tumefaciens. Near‐homozygous lines were created for efficient bi‐allelic single nucleotide polymorphism (SNP) marker mapping and mutagenesis studies. A genetic mapping strategy based on picovine‐derived microvine progeny populations was used to rapidly phenotype and map the flower sex and fleshless berry loci and identify a new lethal recessive locus, Vvlrl1. The microvine provides a unique model system for rapid genetic studies of grapevine by changing the perennial long life cycle of the plant to one with features and advantages similar to an annual plant.     

Molecular mapping of the fasciation mutation in soybean, Glycine max (Leguminosae)

The spontaneous fasciation mutation generates novel developmental diversity in cultivated soybean, Glycine max (L.) Merrill. An increased apical dominance in the mutant inhibits axillary buds, causes a branchless phenotype, and restricts reproduction to shoot apices. The fasciation mutation is encoded by a recessive (f) allele at a single locus. The mutation, despite its importance in soybean development, has no locus assignment on previously reported molecular maps of soybean. A population of 70 F(2) progeny was derived from a cross between 'Clark 63' and the fasciation mutant. More than 700 molecular markers (amplified restriction fragment length polymorphisms [AFLPs], random amplified polymorphic DNAs [RAPDs], restriction fragment length polymorphisms [RFLPs], and simple sequence repeats [SSRs]) were used in mapping of the fasciation phenotype. Twenty linkage groups (LGs) corresponding to the public soybean molecular map are represented on the Clark 63 × fasciation mutant molecular map that spans 3050 centimorgans (cM). The f locus was mapped on LG D1b+W and linked with two AFLPs and four SSR markers (Satt005, Satt141, Satt600, and Satt703). No linkage was found between the f locus and several cDNA polymorphic loci between the wild type and the mutant. The known map position of the f locus and demonstration of the mutant phenotype from early postembryonic throughout reproductive stages provide an excellent resource for investigations of molecular mechanisms affecting soybean ontogeny.     

Genetic identification of a female partial-sterile mutant in soybean.

We report here the genetic identification of a female partial-sterile mutant derived from soybean mutant L67-3483. L67-3483, which originated from the cultivar Clark after X-ray irradiation, is male and female fertile. All F1 plants in reciprocal pollinations of L67-3483 with 'Clark', 'Minsoy', or 'BSR 101' were female partial sterile. Partial sterility is expressed in the heterozygous condition at a single locus and upon self-pollination this locus exhibits a 1:1 segregation pattern. This locus is located on the terminus of the soybean molecular linkage group D1b+W, between simple sequence repeat (SSR) markers Satt157 and Satt266, and is linked to each by 5.3 and 1.2 cM, respectively. This gene is transmitted through both female and male gametes and there was no segregation distortion of SSR markers linked to this gene. We concluded that this female partial-sterile gene is a new mutation class, and differs from the previously reported mutation classes in soybean, i.e., sporophytic mutation, gametophytic female-specific mutation, and general gametophytic mutation. Restriction of recombination around the mutant gene suggested that this gene is located near or within (a) small inversion(s) or adjacent to (a) chromosomal deletion(s).     

Genetic analysis and molecular mapping of a pale flower allele at the W4 locus in soybean.

In soybean (Glycine max (L.) Merr.), the w4-mutable line that harbors the w4-m allele was identified in 1983. It was proposed that this line contained an autonomous transposable element at the W4 locus, which is a major locus controlling the biosynthesis of anthocyanin. The w4-m allele can revert to the W4 allele that produces the wild-type phenotype, or sometimes to other alleles that produce intermediate phenotypes. Mutant plants that produce pale flowers were identified among the progeny of a single germinal revertant event from the w4-mutable line. Through genetic analysis, we established that the pale-flower mutation was conditioned by a new allele (w4-p) at the W4 locus. The w4-p allele is dominant to the w4 allele but recessive to the W4 allele, and the w1 allele has an epistatic effect on the w4-p allele. The pale-mutant line (w4-pw4-p) was designated as Genetic Type Collection number T369. An F2 mapping population derived from the cross of Minsoy (W4W4) x T369 (w4-pw4-p) was used to map the W4/w4-p locus, using simple sequence repeat (SSR) markers. The W4 locus was located at one end of molecular linkage group D2, 2.3 cM from the SSR marker Satt386 and close to the nearby telomere.     

Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation

Potassium accumulation is essential for grapevine (Vitis vinifera L.) growth and development, but excessive levels in berries at harvest may reduce wine quality particularly for red wines. In addition to decreasing the free acid levels, potassium also combines with tartaric acid to form largely insoluble potassium bitartrate. This precipitates during winemaking and storage, resulting in an increase in wine pH that is associated with negative impacts on wine colour, flavour, and microbiological stability. For these reasons, a better understanding of potassium transport and accumulation within the vine and berries is important for producing fruit with improved winemaking characteristics. Here two genes encoding KUP/KT/HAK-type potassium transporters that are expressed in grape berries are described. Their function as potassium transporters was demonstrated by complementation of an Escherichia coli mutant. The two transporters are expressed most highly in the berry skin during the first phase of berry development (pre-veraison), with similar patterns in two grapevine varieties. The timing and location of expression of these transporters are consistent with an involvement in potassium accumulation in grape berries.     

QTL analysis for fruit yield components in table grapes (Vitis vinifera)

A segregation population of 184 genotypes derived from a pseudo-testcross of table grapes (Vitis vinifera), together with 203 AFLP and 110 SSR markers was used to detect quantitative trait loci (QTLs) for fruit yield components. Diffferent QTLs, a low percentage of phenotypic variance explained by the QTLs detected and QTL instability over years were detected for each fruit yield component. These results confirm the complex genetic architecture of the yield components in grapevine due to the perennial nature of this species, which has to adapt to yearly variations in climate. Phenotypic correlation analyses between fruit yield components were also performed. The negative correlation between berry weight and the number of berries per cluster seems to have an indirect negative effect on cluster weight, as revealed by the path coefficient analysis; however, this negative correlation was not supported at the molecular level because no coincident QTLs were observed between these traits. Nonetheless, the possibility to select seedless genotypes with large berries without affecting cluster weight needs to be substantiated in future experiments because factors such as sample size and heritability might influence QTL identification in table grapes.     

White grapes arose through the mutation of two similar and adjacent regulatory genes

Most of the thousands of grapevine cultivars (Vitis vinifera L.) can be divided into two groups, red and white, based on the presence or absence of anthocyanin in the berry skin, which has been found from genetic experiments to be controlled by a single locus. A regulatory gene, VvMYBA1, which could activate anthocyanin biosynthesis in a transient assay, was recently shown not to be transcribed in white berries due to the presence of a retrotransposon in the promoter. We have found that the berry colour locus comprises two very similar genes, VvMYBA1 and VvMYBA2, located on a single bacterial artificial chromosome. Either gene can regulate colour in the grape berry. The white berry allele of VvMYBA2 is inactivated by two non-conservative mutations, one leads to an amino acid substitution and the other to a frame shift resulting in a smaller protein. Transient assays showed that either mutation removed the ability of the regulator to switch on anthocyanin biosynthesis. VvMYBA2 sequence analyses, together with marker information, confirmed that 55 white cultivars all contain the white berry allele, but not red berry alleles. These results suggest that all extant white cultivars of grape vines have a common origin. We conclude that rare mutational events occurring in two adjacent genes were essential for the genesis of the white grapes used to produce the white wines and white table grapes we enjoy today.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait ‘low brood-viability’ resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

Characterisation of the virescent locus controlling a recessive phenotype in apple rootstocks (Malus pumila Mill.)

Certain progenies of Malling apple rootstocks (Malus pumila) have been reported to segregate for a virescent trait: leaves are chlorotic at germination or bud break but turn green as the season progresses. The M432 rootstock mapping progeny, from which a linkage map has recently been elaborated with 323 simple sequence repeat (SSR) markers and 3,069 single nucleotide polymorphism (SNP) markers, also segregates for this phenotype. In this investigation, 188 seedlings were scored and, on the basis of a 3:1 segregation, virescence was attributed to the recessive gene (vir) for which the two parents, M.27 and M.116, are heterozygous. At least seven of 28 Malling rootstocks are heterozygous for this apparently deleterious trait. With the published marker data the gene was mapped to linkage group 12, tightly flanked by the SSR CH01g12 and the SNP marker 475880474, and was located in a physical interval of 2.36 Mb on the Golden Delicious genome sequence. A PCR-based marker was developed from the SNP and along with the SSR was scored in a set of Malus rootstock accessions. The screening of this collection demonstrated that those accessions known to be heterozygous at the vir locus all carried the 152 allele of the SSR and the G allele of the SNP, whilst a virescent accession was homozygous for the alleles. The results we present here could help predict the genotype of apple rootstocks at the vir locus, assist in the fine mapping of the vir locus to identify potential candidate genes for the trait and also aid rootstock breeding.     

Characterization and genetic analysis of a lettuce (Lactuca sativa L.) mutant,weary, that exhibits reduced gravitropic response in hypocotyls and inflorescence stems

A lettuce (Lactuca sativa L.) mutant that exhibits a procumbent growth habit was identified and characterized. In two wild type (WT) genetic backgrounds, segregation patterns revealed that the mutant phenotype was controlled by a recessive allele at a single locus, which was designated weary. Hypocotyls and inflorescence stems of plants homozygous for the weary allele exhibited reduced gravitropic responses compared with WT plants, but roots exhibited normal gravitropism. Microscopic analysis revealed differences in the radial distribution of amyloplasts in hypocotyl and inflorescence stem cells of weary and WT plants. Amyloplasts occurred in a single layer of endodermal cells in WT hypocotyls and inflorescence stems. By contrast, amyloplasts were observed in several layers of cortical cells in weary hypocotyls, and weary inflorescence stem cells lacked amyloplasts entirely. These results are consistent with the proposed role of sedimenting amyloplasts in shoot gravitropism of higher plants. The phenotype associated with the weary mutant is similar to that described for the Arabidopsis mutant sgr1/scr, which is defective in radial patterning and gravitropism.     

Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus

Bud sports are infrequent changes in phenotype affecting shoots of woody perennials but the molecular basis of these mutations has rarely been identified. In this report, we show that the bronze-coloured berries of the Malian cultivar, a documented bud sport of the wine grape Cabernet Sauvignon (Vitis vinifera L.), lack anthocyanins in the subepidermal cells compared to the red/black berried Cabernet Sauvignon in which both the epidermis and several subepidermal cell layers contain anthocyanin. The Malian phenotype is correlated with an alteration in the genome indicated by a reduction of hybridisation signal using a MYBA probe. In Shalistin, a white-berried bud sport of Malian, the red allele at the berry colour locus appears to have been deleted completely. These data suggest that Malian could be a L1/L2 periclinal chimera, which gave rise to Shalistin by an invasion of epidermal cells (L1) by the mutated subepidermal cells (L2). The red grape Pinot Noir has given rise to a number of pale coloured sports, although the provenance of the extant sports is not known. We show that a clone of Pinot Blanc (white-berried) does not have a deletion of the red allele of the same dimensions as that in Shalistin, though a small deletion is a likely explanation for the altered phenotype. However, the mechanism of deletion of the red allele of the berry colour locus is a possible means by which other red to white clonal mutations of grapevines have occurred.     

Inheritance of spontaneous mutant homostyles in Turnera subulata x krapovickasii and in autotetraploid T. scabra (Turneraceae)

To explore the genetic architecture of distyly in Turnera spp., we determined the inheritance and compatibility behaviour of two spontaneous homostyled mutants. A long-homostyled mutant shoot arose on an otherwise short-styled plant that was an artificial hybrid (Turnera subulata x T. krapovickasii) between two diploid distylous species. The mutation appears to be an allele, SH, of the distyly locus with the dominance relationships, S>SH>s, where S confers the short-styled phenotype, SH confers homostyly in SHSH and SHs genotypes, and ss genotypes are long-styled. Aberrant segregation ratios were observed among some crosses and might be the result of pollen competition. Compatibility relationships are consistent with the hypothesis that a gene complex determines distyly. Infrequently, revertant short-styled flowers have appeared on cuttings of the T. subulata x T. krapovickasii mutant and on occasion, short-styled progeny have appeared in crosses where none were expected. A second mutant homostyle was discovered in autotetraploid T. scabra. The mutation is inherited as above, however, tetrasomic inheritance occurs at the locus. This homostyled mutant carries two copies of the SH allele and has the duplex genotype SHSHss. Compatibility relationships were as observed above. The occurrence of homostyled mutants is consistent with the hypothesis that a linked gene complex underlies the inheritance of distyly in Turnera but we cannot discount the hypothesis that an allelic series is responsible.     

SSR markers for Quercus suber tree identification and embryo analysis.

Three Quercus simple sequence repeat (SSR) markers were amplified by polymerase chain reaction (PCR) from nuclear DNA extracts of trees and in vitro-induced haploid embryos from anther cultures of Quercus suber L. These markers were sufficiently polymorphic to identify 10 of 12 trees located in two Spanish natural areas. The same loci have been analyzed in anther-derived haploid embryos showing the parental tree allele segregation. All the alleles were present in the haploid progeny. The presence of diverse alleles in embryos derived from the same anther demonstrated that they were induced on multiple microspores or pollen grains and they were not clonally propagated. Also, diploid cultures and mixtures of haploid-diploid tissues were obtained. The origin of such cultures, either somatic or gametic, was elucidated by SSR markers. All the embryos showed only one allele, corroborating a haploid origin. Allelic composition of the haploid progeny permitted parental identification among all analyzed trees.     

A methyltransferase essential for the methoxypyrazine‐derived flavour of wine

Methoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F₂ progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3‐isobutyl‐2‐methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.