Identification of molecular markers associated with sugar-related traits in a <Emphasis Type="Italic">Saccharum</Emphasis> interspecific cross

The cultivated sugarcane (Saccharum spp. hybrids, 2n = 100–130) is one crop for which interspecific hybridization involving wild germplasm has provided a major breakthrough in its improvement. Few clones were used in the initial hybridization event leading to a narrow genetic base for continued cultivar development. Molecular breeding would facilitate the identification and introgression of novel alleles/genes from the wild germplasm into cultivated sugarcane. We report the identification of molecular markers associated with sugar-related traits using an F₁ population derived from a cross between S. officinarum ‘Louisiana Striped’ × S. spontaneum ‘SES 147B’, the two major progenitor species of cultivated sugarcane. Genetic linkage maps of the S. officinarum and S. spontaneum parents were produced using the AFLP, SRAP and TRAP molecular marker techniques. The mapping population was evaluated for sugar-related traits namely, Brix (B) and pol (P) at the early (E) and late (L) plant growing season in the plant cane (04) and first ratoon (05) crops (04EB, 04LB, 04LP, 05EB and 05EP). For S. officinarum, combined across all the traits, a total of 30 putative QTLs was observed with LOD scores ranging from 2.51 to 7.48. The phenotypic variation (adj. R²) explained by all QTLs per trait ranged from 22.1% (04LP) to 48.4% (04EB). For S. spontaneum, a total of 11 putative QTLs was observed with LOD scores ranging from 2.62 to 4.70 and adj. R² ranging from 9.3% (04LP) to 43.0% (04LB). Nine digenic interactions (iQTL) were observed in S. officinarum whereas only three were observed in S. spontaneum. About half of the QTLs contributed by both progenitor species were associated with effects on the trait that was contrary to expectations based on the phenotype of the parent contributing the allele. Quantitative trait loci and their associated effects were consistent across crop-years and growing seasons with very few QTLs being unique to the early season. When the data were reanalyzed using the non-parametric discriminant analysis (DA) approach, significant marker-trait associations were detected for markers that were either identical to or in the vicinity of markers previously identified using the traditional QTL approach. Discriminant analysis also pointed to previously unidentified markers some of which remained unlinked on the map. These preliminary results suggest that DA could be used as a complementary approach to traditional QTL analysis in a crop like sugarcane for which saturated linkage maps are unavailable or difficult to obtain.     

Verification of the introgression of Erianthus arundinaceus germplasm into sugarcane using molecular markers

Erianthus arundinaceus has a number of important agronomic traits including good ratooning ability, tolerance to both drought and water logging, disease resistance and vigour and is of interest as a potential source of parental germplasm to sugarcane breeders. However, to date, attempts to produce fertile hybrids between sugarcane (Saccharum spp.) and E. arundinaceus have been unsuccessful. Microsatellite markers that generated genus‐specific markers and were highly polymorphic within sugarcane were identified. The microsatellite markers and 5S rDNA PCR were used to screen intergeneric (F1) clones from Saccharum officinarum×E. arundinaceus crosses, and two Saccharum backcross progeny (BC1) populations derived from crosses between selected F1 clones and sugarcane (Saccharum spp.), to identify genuine Saccharum spp. BC1 progeny. The 5S rDNA PCR marker and highly polymorphic microsatellites with Erianthus‐specific bands confirmed the F1 parent of the two putative BC1 populations was a S. officinarum×E. arundinaceus hybrid and allowed the identification of the genuine BC1 progeny from selfs of the F1 parent. This is the first verification of BC1 progeny from an F1 intergeneric hybrid x sugarcane (Saccharum spp.) clone with molecular markers and confirms the introgression of E. arundinaceus germplasm into sugarcane. It should now be possible to exploit genes of value from E. arundinaceus in sugarcane breeding programmes.     

Linkage mapping and genome analysis in a Saccharum interspecific cross using AFLP, SRAP and TRAP markers

Framework genetic linkage maps of two progenitor species of cultivated sugarcane, Saccharum officinarum ‘La Striped’ (2n = 80) and S. spontaneum ‘SES 147B’ (2n = 64) were constructed using amplified fragment length polymorphism (AFLP), sequence related amplified polymorphism (SRAP), and target region amplification polymorphism (TRAP) markers. The mapping population was comprised of 100 F₁ progeny derived from the interspecific cross. A total of 344 polymorphic markers were generated from the female (S. officinarum) parent, out of which 247 (72%) were single-dose (segregating in a 1:1 ratio) and 33 (9%) were double-dose (segregating in a 3.3:1 ratio) markers. Sixty-four (19%) markers deviated from Mendelian segregation ratios. In the S. spontaneum genome, out of a total of 306 markers, 221 (72%) were single-dose, 43 (14%) were double-dose, and 42 markers (14%) deviated from Mendelian segregation ratios. Linkage maps with Kosambi map distances were constructed using a LOD score ≥5.0 and a recombination threshold of 0.45. In Saccharum officinarum, 146 markers were linked to form 49 linkage groups (LG) spanning 1732 cM whereas, in S. spontaneum, 121 markers were linked to form 45 LG spanning 1491 cM. The estimated genome size of S. officinarum ‘La Striped’ was 2448 cM whereas that of S. spontaneum ‘SES 147B’ was 3232 cM. Based on the two maps, genome coverage was 69% in S. officinarum and 46% in S. spontaneum. The S. officinarum parent ‘La Striped’ behaved like an auto-allopolyploid whereas S. spontaneum ‘SES 147B’ behaved like a true autopolyploid. Although a large disparity exists between the two genomes, the existence of simple duplex markers, which are heterozygous in both parents and segregate 3:1 in the progeny, indicates that pairing and recombination can occur between the two genomes. The study also revealed that, compared with AFLP, the SRAP and TRAP markers appear less effective at generating a large number of genome-wide markers for linkage mapping in sugarcane. However, SRAP and TRAP markers can be useful for QTL mapping because of their ability to target gene-rich regions of the genome, which is a focus of our future research.     

Genetic relationships between attributes in sugarcane clones closely related to Saccharum spontaneum

Summary Saccharum spontaneum is being used in sugarcane breeding programs in attempt to improve characteristics such as ratooning ability and stress tolerance. A population of F₁ (Saccharum officinarum or commercial variety x S. spontaneum) and F₁ x F₁ sugarcane clones was evaluated for sugar yield and a range of yield components in a plant and two ratoon crops. The aim was to determine genetic correlations between attributes in clones with a large component of S. spontaneum, that could be used to help derive appropriate selection indices in such populations.There were close associations between the same attributes measured in different crop-years and the associations between different attributes were generally similar across crop-years. Stalk number and fibre content were positively correlated, as were stalk weight and CCS. The latter two attributes (which are low in S. spontaneum but high in S. officinarum) were negatively correlated with the former two (high in S. spontaneum, low in S. officinarum). Sugar yield was more closely associated with stalk weight and CCS than with stalk number but became more closely associated with stalk number with successive ratoon crops.CCS was positively correlated (r_g=0.55) with cane yield in the plant crop but showed a small negative correlation with cane yield (r_g=–0.20) in the second ratoon crop. CCS (measured in any crop) also had a negative correlation with cane yield in the ratoon crops expressed as a percentage of plant cane yield. This suggests that CCS is negatively correlated with levels of traits contributing to ratooning ability. Intensive selection among such populations for CCS without consideration of ratooning performance may reduce the frequency of favourable specific ratooning characteristics.Abbreviations CCS commercial cane sugar     

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.     

Genome size variation in three Saccharum species

Saccharum species are autopolyploids with ploidy level ranging from 5× to 16x, and are considered the most complex genomes among crop plants. In present study, the genome sizes of 28 Saccharum spontaneum accessions, 15 Saccharum officinarum accessions, 28 Saccharum robustum accessions, and 12 Saccharum hybrids spp. were analyzed using flow cytometry. The estimated genome sizes of S. officinarum accessions ranged from 7.50 to 8.55?Gb with an average size of 7.88?Gb. In S. robustum, the estimated genome sizes ranged from 7.65 to 11.78, reflecting the variation of ploidy level. In S. spontaneum, the estimated genome sizes varied widely, with a range from 3.36 to 12.64?Gb, also due to variation of ploidy level. The average monoploid genome size of S. officinarum was 985?Mb, and that of S. spontaneum was 843?Mb. The results also showed that genome sizes were correlated with chromosome numbers, and based which, that the unknown chromosome numbers of some accessions could be predicted. The estimated genome sizes of Saccharum germplasm also helped identify some mislabeled accessions and yielded information critical for sugarcane breeding and genome sequencing programs.     

Somaclonal variation in sugarcane through tissue culture and evaluation for quantitative and quality traits

Eight genotypes of Saccharum officinarum were crossed with Saccharum spontaneum and 14 genotypes of S. officinarum were crossed with Erianthus arundinaceus. A total of 39 hybrids were evolved. These 39 hybrids were raised in the field and used as donor clones for in vitro culture studies. Plantlets were regenerated from 1-month-old callus. The grown up plants were transplanted to well prepared field, to study the variations generated for the biometric as well as for biochemical characters. There were significant differences between the donor clones and their sub clones for all the character of interest. The somatic segregation was gradual and wider, showing a range of divergence from the mean towards the end of the scale. Fifty-one sub clones were selected with commercial potential which have 13% fibre, 200 cm stalk length, 10 cm internode length and pure obtainable cane sugar per cent of 10.     

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.     

A re-examination of the mechanisms of 2n gamete formation in potato and its implications for breeding

Summary The claim that first division restitution gametes are produced through the unique meiotic abnormality of parallel spindle formation at anaphase II of microsporogenesis in Solanum species appears to be not justified. An examination of the phenomenon of meiotic nuclear restitution in fifty-six diploid Solanum clones revealed that the parallel orientation of spindles either at metaphase or anaphae II does not necessarily lead to dyad or unreduced microspore formation. However, the fused spindle formation at metaphase II of microsporogenesis invariably leads to dyad and unreduced microspore formation. The 2n gametes produced through fused spindles may be genetically equivalent to first division restitution gametes, and therefore, they may be useful in potato breeding. In addition to fused spindle formation, at least three other types of meiotic abnormalities that could lead to 2n gamete formation were also observed. Although any one, or two, of these mechanisms were predominantly observed in certain clones, it was by no means certain that other mechanisms did not operate in these clones at the same time, or at a later stage. Because of the great modifying influence of environment on the process of meiosis, and meiotic nuclear restitution, it might be erroneous to conclude that one or the other meiotic nuclear restitution mechanism alone operates at a time in a given clone. In order to elucidate the genetic basis of 2n gamete formation in potato, it is desirable (i) to answer whether there are exclusive genetic mechanisms for the formation of first or second division restitution gametes; (ii) to establish reliable cytological criteria for their detection; and (iii) to determine the effect of environmental factors on the occurrence of different mechanisms.     

Distribution and frequency of Bru1, a major brown rust resistance gene,in the sugarcane world collection

Brown rust, caused by Puccinia melanocephala, is an important disease of sugarcane worldwide. Molecular markers for a major brown rust resistance gene, Bru1, were used to screen a total of 1,282 clones in the World Collection of Sugarcane and Related Grasses (WCSRG) to determine the distribution and frequency of the gene in Saccharum species and related genera. Bru1 was found across all species within the Saccharum complex, but the frequency varied among species. Bru1 was more prevalent in S. robustum clones (59.1%), whereas it occurred in low frequency and exhibited the highest level of variability as determined by the presence of one or both markers (18.8%) in clones of S. spontaneum. Bru1 frequency was highest in the two secondary cultivated species, S. barberi (79.3%) and S. sinense (71.8%). The frequency of Bru1 was 26.4% and 21.0% in S. officinarum and interspecific hybrid clones, respectively. Knowledge of the distribution and frequency of Bru1 in the WCSRG will complement efforts to characterize diversity in the Saccharum complex for the expected expanded use of marker‐assisted selection in the future.     

Relationships among ancestral species of sugarcane revealed with RFLP using single copy maize nuclear probes

Summary DNA restriction fragment length polymorphism (RFLP) analysis was performed on 50 wild and old cultivated sugarcane accessions. Ninety-four maize low copy nuclear DNA sequences of known chromosomal position were screened for hybridization to digested sugarcane genomic DNA blots. Seventy-five (80%) gave very strong hybridization signals and usually yielded many bands and detected profuse polymorphism. Twenty-nine probes and 36 probe/enzyme combinations were selected on the basis of the scorability of the banding profiles. A total of 1110 fragments were separately identified among the 50 genotypes. Multivariate analyses of the data allowed the separation of the three basic species, Saccharum spontaneum, S. robustum and S. officinarum, showed that S. spontaneum had structure which could be related to the geographic origin of the clones and supported current hypotheses on the origin of secondary species S. barberi and S. sinense. The use of more probes did not improve the resolution between the various species examined but identified a few key polymorphisms which were not accounted for by current phylogenetic hypotheses and can guide future analyses. RFLPs in sugarcane will be useful essentially for depicting the genomic constitution of modern varieties of interspecific origin.     

Matromorphy in Brassica oleracea L. VII. Research on products of microsporogenesis and gametogenesis from prickle pollinated plants

Summary A research has been carried out on the occurrence of 2n gametes in Brassica oleracea and on their possible way of origination. After microsporogenesis it appeared that unreduced PMC's, dyads and triads occurred. Giant pollen grains with two (diploid) and four (haploid) generative nuclei, respectively, were found, resulting from a deviating cytokinesis and karyokinesis. Diploid heterozygous matromorphic embryos may develop from such unreduced gametes formed after macrosporogenesis.     

Problems in breeding and cytology of sugar cane

In this article a survey is given on sugar cane breeding, as it was performed in Java during a period of about fifty years. When in the eighties of the nineteenth century sugar cane was heavily affected by the sereh disease it was Soltwedel, the first director of the Sugar Experiment Station Midden Java, who tried to obtain sereh resistant cane varieties by species hybridization, until his early death intervened. The first species hybrids were obtained in 1893 by Wakker, who crossed noble sugar cane, Saccharum officinarum, with Kassoer, considered by him as a wild species. In later years it appeared from morphological investigations by Jeswiet (1916) and from cytological investigations by Bremer (1921) that Kassoer is to be considered as a spontaneous hybrid between S. officinarum and S. spontaneum, the wild glagah. In 1895 Kobus imported the Indian sugar cane Chunnee in Java. Chunnee, not belonging to S. officinarum, was crossed with noble sugar cane (for the first time) in 1897. From this cross many clones were obtained which appeared to be resistant against the sereh disease but were highly susceptible to mosaic. The continued crossing between noble sugar cane and Kassoer, however, was very successful. Many clones were obtained, which as commercial varieties, showed a high degree of resistance against sereh disease and mosaic and moreover gave a much higher sugar production than the susceptible varieties of noble sugar cane.The following terms are introduced: first, second and third nobilisation of the wild S. spontaneum. Kassoer cane itself is a product of the first nobilisation, the direct cross between noble sugar cane and wild cane. The back cross between Kassoer and noble sugar cane is called the second nobilisation. When cane varieties belonging to the second nobilisation are crossed again with noble sugar cane, the third nobilisation of S. spontaneum is said to take place. The well-known sugar cane variety 2878 P.O.J. belongs to the third nobilisation.In all clones investigated Saccharum officinarum has 2n=80 chromosomes. Within S. spontaneum types occur which in chromosome number vary from 2n=48 to 2n=128. The Java glagah has 2n=112 chromosomes.Clones of the first glagah nobilisation S. officinarum (n=40) x S. spontaneum (n=56) did not have 2n=40+56 chromosomes, but 2n=40+40+56=136 chromosomes.In relation to the taxonomy of Saccharum many other cytological details are given. Within S. officinarum the basic chromosome number x=10. About S. spontaneum opinions are divided. The author supposes that x=6,8 and 10 are basic numbers of S. spontaneum.     

Genetic strategies to determine the mode of 2n egg formation in diploid potatoes

Summary In this study, nineteen diploid potato clones (Solanum spp. 2n=2x=24) were identified as 2n egg producers on the basis of fruit set in 2x–4x crosses. The segregation of three genes mapped close to the centromere, Got-1 (1.1 cM), Pgm-2 (2.0 cM), and Sdh-1 (8.3 cM), were analyzed in the tetraploid offspring in these 2x–4x crosses to discriminate between First Division Restitution (FDR) and Second Division Restitution (SDR) modes of 2n egg formation. The co-dominant nature of these markers lead to more precise estimates of the recombinational frequencies as a result of completely classifying the segregating progenies. 2x–4x data revealed a predominance of SDR mechanisms occurring in 20 of the 21 families analyzed. With a SDR mode established, half-tetrad analysis (HTA) of four distal loci, 6-Pgdh-3, Mdh-1, Pgi-1, and Aps-1, revealed two SDR segregation patterns in some of the families. One pattern fit the expectations for the distal arm position. The gene-centromere map distances based upon SDR modes in the families following this pattern, were generally close to 4x–2x (FDR) estimates suggesting similar recombination rates between micro- and mega-sporogenesis. Heterozygosity transmission, on average, was 39.1%. In the other segregation pattern, in which the diploid parents were derived from S. chacoense PI 230580, higher than expected homozygosity levels were found in the female 2n gametophyte populations. A post-meiotic doubling of the reduced megaspore, which generates homozygous 2n eggs, is suggested to operate in three families. The common genetic background of the diploid clones suggested a heritable nature of this mechanism. Pooled data from these three deviant families calculated that 1.8% of the heterozygosity was transmitted to the tetraploid progeny.It is concluded that utilization of seven enzyme-coding loci, with previously established gene-centromere map distances, in 2x–4x crosses improved half-tetrad analysis (HTA) as a means to determine the mode of 2n gamete formation in megasporogenesis and megagametogenesis of diploid Solanum species.     

Pedigree,marker recruitment,and genetic diversity of modern sugarcane cultivars in China and the United States

Sugarcane (Saccharum spp) is an important crop for both sugar and biofuel production. However, the sugarcane breeding process has resulted in modern sugarcane cultivars with a narrow genetic basis. To broaden the genetic basis and promote international collaborations in sugarcane cultivar development, we documented the peidgrees of representative sugarcane culativars widely used in China and the United States of America (USA), recruited more than six thousand simple sequence repeat (SSR) markers for sugarcane, and assessed the genetic diversity and relationships beween representative sugarcane cultivars and their potential ancestry accessions. The SSR gentoyping results indicated that both the USA and Chiniese cultivars had low genetic diversity, specifically the Chinese cultivars. The USA sugarcane cultivars experienced high presure of selection for sugar content as they had the closest relationship with S. officinarum, followed by Chinese cultivars, S. robustum, and S. spontaneum. The sugarcane accessions assessed could be divided into five and four groups through cluster and principal component analysis, respectively. S. spontaneum as a potential ancestor contributing to the stress tolerance of sugarcane cultivars was grouped into distinct clusters, and S. officinarum was grouped with sugarcane cultivars in both countries. S. robustum did not seem to contribute to the sugarcane cultivar development in China, but may have contributed to the USA cultivar development. This study not only provided a collection of easy to use SSR markers, but also detailed genetic diversity and relationship among the cultivars in the two counties, which will be referable to promote international collaboration and broaden the genetic basis of sugarcane cultivars.     

Evidence for second division restitution as the basis for 2n + n maternal chromosome transmission in a sugarcane cross

Chromosome doubling in megaspores of sugarcane was first reported over 70 years ago and was seen primarily in interspecific crosses between Saccharum officinarum and Saccharum spontaneum. Since then, there has been robust debate on the mechanism(s) responsible for this doubling with hypotheses ranging from second division restitution (SDR), post-meiotic restitution to fusion of the two innermost cells of the megaspore tetrad. In the present study, we sought to answer this question using molecular marker data from a cross which demonstrated 2n + n chromosome transmission between an S. officinarum clone (IJ76-514; 2n transmission) and a commercial hybrid (Q165 ; n transmission). Molecular marker data from four other bi-parental crosses were used to form a “base-line” for expected segregation ratios (n + n transmission). Comparison of segregation ratios showed that 2n transmission from the S. officinarum clone could be identified by the increased segregation ratios, a characteristic of both SDR and tetrad cell fusion. Simulation was then undertaken based on current understandings of sugarcane meiosis, to determine if the observed segregation ratios matched those simulated for SDR/tetrad cell fusion. Analysis of the simulation confirmed that most and perhaps all progeny appear to have been formed from a mechanism akin to SDR/tetrad cell fusion. The genetic consequences of this are discussed.     

Occurrence, inheritance and use of reproductive mutants in alfalfa improvement

The widespread occurrence of 2ngametes (i.e. gametes with the somatic chromosome number) in the Medicago sativa-coerulea-falcata complex supports the concept that gene flow from diploid to tetraploid species occurs continuously in nature and plays a key role in alfalfa evolution. Breeders realized early that gene transfer between ploidy levels via 2n pollen and 2n eggs would have had potential use in cultivated alfalfa improvement. Cytological investigations provided insights into the types of meiotic abnormalities responsible for the production of 2n gametes. Alterations were defined as genetically equivalent to first (FDR) or second division restitution(SDR) mechanisms. For breeding purposes,data have proven that 2n gametes of the FDR type are more advantageous than those obtained by SDR for transferring parental heterozygosity and retaining epistatic interactions. The use of diploid meiotic mutants that produce 2ngametes is now recognized as one of the most effective methods available for exploiting heterosis and introgressing wild germplasm traits into cultivated tetraploid alfalfa via unilateral (USP) and bilateral sexual polyploidization (BSP) schemes. Both2n egg and 2n pollen producers could be used for direct gene transfer from wild diploid relatives into cultivated alfalfa by means of 2x-4x and4x-2x crosses. Although data have shown that forage yield improvement can be achieved when plants are sexually tetraploidized, problems related to reduced plant fertility and seed production remain largely unexplained. Apomixis has the potential of cloning plants through seed and thus provides a unique opportunity for developing superior tetraploid cultivars with permanently fixed heterosis and epistatic effects. A main goal in alfalfa breeding could be the introduction of functional apomixis (i.e. Apomeiosis and parthenogenesis) in cultivated alfalfa stocks. In the future, the efficiency of alfalfa breeding programs based on the use of reproductive mutants could be improved by direct selection at the genotype level using RFLPs and PCR-based markers. Suitable DNA markers and detailed linkage maps of alfalfa mutants should help to discover apomictic mutants and address basic genetic issues such as the extent of genomicre combination in polyploid hybrids and the effect of sexual polyploidization on heterosis. Molecular markers have recently been used in alfalfa for studying the inheritance of 2n gamete formation and identifying polymorphisms associated to genes involved in meiotic abnormalities. Molecular tagging of 2n egg and 2n pollen formation not only should explain the genetic control and regulation of these traits, but may also be an essential step towards marker-assisted selection of 2n gamete producers and implementation of USP and BSP breeding schemes. Future perspectives include strategies for the map-based cloning of genomic DNA markers,and screening of EST mini-libraries related to flowers at different developmental stages from meiotic mutants and wild-type scan lead to the identification of mRNAs and thus of candidate genes that control 2n gamete formation in alfalfa. This revised version was published online in July 2006 with corrections to the Cover Date.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Transmission of resistance to common scab from the diploid to the tetraploid level via 4x-2x crosses in potatoes

Summary Diploid potato clones selected for their reaction to common scab and their ability to produce 2n male gametes were used in a series of crosses to a susceptible tetraploid female parent (cv. Shepody). In addition, two tetraploid clones were also selected for their reaction to common scab and crossed with Shepody as a female parent. Results indicated that resistance to common scab can be effectively transmitted from the diploid to the tetraploid level via 4x-2x crosses. Diploid parents producing 2n pollen via either first division or second division restitution can be used to transmit scab resistance. A relatively small proportion of resistant individuals could be recovered from susceptible x susceptible crosses in both 4x-2x and 4x-4x combinations. The data support a previously developed hypothesis that scab resistance is relatively simply inherited.