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RAPD, RFLP, nuclear SSLP and chloroplast SSLP analyses were carried out to clarify the phylogenetic relationships among A-genome species of rice. In total, 12 cultivars of Oryza sativa (4 Japonica, 3 Javanica and 5 Indica), one cultivar of O. glaberrima, and 17 wild accessions (12 O. rufipogon, 2 O. glumaepatula, 1 O. longistaminata, 1 O. meridionalis and 1 O. barthii) were used. Their banding patterns were scored and compared to evaluate the similarity between accessions. Genetic differentiation within and between taxa was examined based on the average similarity indices. Except for chloroplast SSLP analysis, the average similarities were higher within O. sativa than within O. rufipogon, and O. sativa Indica had greater intrasubspecific variation than Japonica and Javanica. Comparisons between cultivated and wild species showed that O. sativa was closely related to O. rufipogon, while O. glaberrima was closely related to O. barthii. This indicated that two cultivated species, O. sativa and O. glaberrima, originated from O. rufipogon and O. barthii, respectively. Domestication of O. sativa seemed to be diphyletic, since strong similarity was observed between O. sativa Japonica-Javanica and O. rufipogon from China and between O. sativa Indica and O. rufipogon from tropical Asia. In addition, dendrograms for RAPD, RFLP, and nuclear and chloroplast SSLP analyses were constructed to reveal the overall genetic relationships among A-genome species. In all analyses, O. sativa and O. glaberrima formed groups with O. rufipogon and O. barthii, respectively. However, their manners of clustering with other wild species were not the same. The results of RAPD and RFLP analyses indicate that O. glumaepatula was relatively close to the groups of O. sativa and O. glaberrima whereas O. longistaminata and O. meridionalis were highly differentiated from other A-genome species. On the other hand, clear interspecific relationships were not obtained by nuclear or chloroplast SSLP analyses.

A genetic linkage map was developed with 86 F2 plants derived from an interspecific cross between azuki bean (Vigna angularis, 2n=2x=22) and rice bean (V. umbellata, 2n=2x=22). In total, 14 linkage groups, each containing more than 4 markers, were constructed with one phenotypic, 114 RFLP and 74 RAPD markers. The total map size was 1702 cM, and the average distance between markers was 9.7 cM. The loci showing significant deviation from the expected ratio clustered in several linkage groups. Most of the skewed loci were due to the predominance of rice bean alleles. The azuki-rice bean linkage map was compared with other available maps of Vigna species in subgenus Ceratotropis. Based on the lineage of the common mapped markers, 7 and 16 conserved linkage blocks were found in the interspecific map of azuki bean ×V. nakashimae and mungbean map, respectively. Although the present map is not fully saturated, it may facilitate gene tagging, QTL mapping and further useful gene transfer for azuki bean breeding.

Electrofusion was performed to produce somatic hybrids between Solanum tuberosum (2n=4x=48) and dihaploid S. acaule (2n=2x=24). Among the 18 regenerated plants obtained, 14 were found to be hexaploid hybrids based on RAPD analysis and cytological observation. In order to identify their cytoplasmic genome constitution, RFLP analysis of chloroplast and mitochondrial DNA was carried out. Six and eight hybrids were found to have S. tuberosum and S. acaule chloroplast genomes, respectively, suggesting that the chloroplast genomes of the somatic hybrids segregated randomly. In addition, two regenerated plants derived from the same callus had different chloroplast genomes, indicating that the segregation of the chloroplast genome occurred before shoot formation from callus. Concerning the mitochondrial genome constitution, 13 hybrids harbored the genome of S. tuberosum type and the others the S. acaule type genome. This mode of segregaion is obviously in favor of S. tuberosum mitochondrial genome. Regarding cytoplasmic genome segregation in somatic hybrids, the mitochondrial genome was not associated with the chloroplast genome. For the cytoplasmic genomes of both species, no hybrid was observed with recombinant or heterogeneous genome from both parents. In most of the somatic hybrids, some characters such as plant habit, shape of foliage and shape of tuber, were generally similar to those of S. tuberosum. The high level of resistance to potato virus X (PVX) detected in dihaploid S. acaule and somatic hybrids, suggests that the resistance to PVX is incorporated by fusion with S. acaule.

High density molecular map of semidwarfing gene, sd-1, was constructed using 178 F2 plants derived from the cross between the semidwarf nearisogenic line and its recurrent parent, japonica cultivar “Shiokari”. The total size of the map including nine molecular markers (five RAPD and four RFLP markers) and sd-1 was 6.4 cM. The most closely linked RAPD markers in both sides of sd-1 were OPVI0500 and OPAE141000, and the distance between them was 1.7 cM. Since sd-1 is a recessive gene, these closely linked RAPD markers will be very powerful to monitor the gene for large scale selection in breeding program.

Genes involved in the transition from wild to cultivated crop species should be of great agronomic importance. Population genomic approaches utilizing genome resequencing data have been recently applied for this purpose, although it only reports a large list of candidate genes with no biological information. Here, by resequencing more than 30 genomes altogether of wild rice Oryza rufipogon and cultivated rice O. sativa, we identified a number of regions with clear footprints of selection during the domestication process. We then focused on identifying candidate domestication genes in these regions by utilizing the wealth of QTL information in rice. We were able to identify a number of interesting candidates such as transcription factors that should control key domestication traits such as shattering, awn length, and seed dormancy. Other candidates include those that might have been related to the improvement of grain quality and those that might have been involved in the local adaptation to dry conditions and colder environments. Our study shows that population genomic approaches and QTL mapping information can be used together to identify genes that might be of agronomic importance.

A genetic linkage map of azuki bean (Vigna angularis) was constructed with molecular and morphological markers using an F2 population of an interspecific cross between azuki bean and its wild relative, V. nakashimae. In total, 132 markers (108 RAPD, 19 RFLP and five morphological markers) were mapped in 14 linkage groups covering 1250 cM; ten remained unlinked. The clusters of markers showing distorted segregation were found in linkage groups 2, 8 and 12. By comparing the azuki linkage map with those of mungbean and cowpea, using 20 RFLP common markers, some sets of the markers were found to belong to the same linkage groups of the respective maps, indicating that these linkage blocks are conserved among the three Vigna species. This map provides a tool for markerassisted selection and for studies of genome organization in Vigna species.

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.

In order to clarify the phylogenetic relationships in A-genome species of rice, RAPD analysis was carried out using 29 accessions (8 Oryza sativa, 6 O. glaberrima, 13 O. perennis, and 2 O. breviligulata). Their total DNA was used as template for PCR with 14 decamer primers. The amplified products were electrophoresed in agarose gel, and the banding patterns were observed. Average number of total amplified bands for each accession using 14 primers was 84.6. By comparing the banding patterns, the proportion of common bands between 29 accessions was calculated and a dendrogram showing genetic relationships was constructed. Most of the accessions formed clusters corresponding to their taxonomic grouping. The Oceanian and African forms of O. perennis were substantially different from all other groups. The Asian form of O. perennis, however, formed a complex with O. sativa while O. breviligulata was clustered with O. glaberrima. This indicates that O. sativa and O. glaberrima have probably originated from the Asian form of O. perennis and from O. breviligulata, respectively.

Reduction in seed shattering was an important phenotypic change during cereal domestication. Here we show that a simple morphological change in rice panicle shape, controlled by the SPR3 locus, has a large impact on seed-shedding and pollinating behaviors. In the wild genetic background of rice, we found that plants with a cultivated-like type of closed panicle had significantly reduced seed shedding through seed retention. In addition, the long awns in closed panicles disturbed the free exposure of anthers and stigmas on the flowering spikelets, resulting in a significant reduction of the outcrossing rate. We localized the SPR3 locus to a 9.3-kb genomic region, and our complementation tests suggest that this region regulates the liguleless gene (OsLG1). Sequencing analysis identified reduced nucleotide diversity and a selective sweep at the SPR3 locus in cultivated rice. Our results suggest that a closed panicle was a selected trait during rice domestication.

Studies were conducted to determine farmers’ perception on Striga control options and their potential for adoption in two Extension Planning Areas (EPAs) in Central Malawi. Individual interviews were conducted in Mpingu (Lilongwe District) and Mponela (Dowa District) EPAs in 2010 in a sample of 247 respondents. The study revealed that crop production was the main source of livelihood for most households. Farmers identified Striga as a constraint to maize production and attributed its increasing incidence to insufficient funds to purchase inputs, soil fertility and grazing animals. On Striga control mechanisms, manure application was perceived to be the best by farmers, followed by crop rotation, fertilizer application and hand pulling. Even though Striga infestation is increasing in farmers fields, they have not adopted the control options. The low adoption of the options has been justified as “too risky” as farmers do not trust them. Emphasis should be laid on undertaking on-farm trials and development of technologies should involve farmers if they are to gain wide acceptability.

The Asian wild rice, Oryza rufipogon, has partial outcrossing behavior and shows high levels of genetic variation. To estimate an accurate outcrossing rate of annual form of O. rufipogon, two backcross lines (Lines R1 and R2) between Oryza sativa Nipponbare and O. rufipogon W630 were examined under field conditions. A chromosome survey confirmed that these lines had wild chromosomal segments at more than 92% of marker loci. As for the traits of glume, stamen and pistil, Line R1 showed similar floral morphology as that of O. rufipogon W630, whereas Line R2 had larger glumes. In 2005, 2006 and 2008, a total of 22 backcross plants were planted in the middle of wild rice plots. The successive progenies of each plant were examined using microsatellite markers that could clearly detect self-pollination and outcrossing. The outcrossing rates of Line R1 plants ranged from 4.04% to 25.50% with an average of 10.20%. This indicates that cross-pollination of wild rice is a chance event affected by many environmental factors. The outcrossing rates of Line R2 plants also varied, however, no significant difference was observed between the averages of Lines R1 and R2, suggesting that the glume sizes are not critical for outcrossing ability.

To decrease the age at first calving in Holsteins, the effects of average daily body weight gain (ADG) and crude protein (CP) level until first insemination on growth performance and milk production were examined. The MM group had a target ADG of 0.75 kg and received a diet with a CP level of 14%. The HM and HH groups had a target ADG of 1 kg; both these groups received a diet with CP levels 14% and 16%, respectively. The ADG in the HM and HH groups was 1.1 kg, whereas in the MM group it was 0.97 kg (P0.01). The HM and HH groups showed no differences in withers height at body weight 350 kg. The ages at first calving in MM, HM and HH groups were 23.1, 21.0 and 21.8 months, respectively. The HM and HH groups had lower milk yield at day 305 than the MM group (P0.01). These results suggest that growth performance until first insemination should be maintained at an ADG of 0.97 kg or less with a CP level of approximately 14%, to shorten time until first insemination and prevent the decrease of milk yield.

It is known that the common cultivated rice (Oryza sativa) was domesticated from Asian wild rice, O. rufipogon. Among the morphological differences between them, loss of seed shattering is one of the striking characters specific for the cultivated forms. In order to understand the genetic control on shattering habit, QTL analysis was carried out using BC(2)F(1) backcross population between O. sativa cv. Nipponbare (a recurrent parent) and O. rufipogon acc. W630 (a donor parent). As a result, two strong QTLs were detected on chromosomes 1 and 4, and they were found to be identical to the two major seed-shattering loci, qSH1 and sh4, respectively. The allelic interaction at these loci was further examined using two sets of backcross populations having reciprocal genetic backgrounds, cultivated and wild. In the genetic background of cultivated rice, the wild qSH1 allele has stronger effect on seed shattering than that of sh4. In addition, the wild alleles at both qSH1 and sh4 loci showed semi-dominant effects. On the other hand, in the genetic background of wild rice, non-shattering effects of Nipponbare alleles at both loci were examined to inspect rice domestication from a viewpoint of seed shattering. It was serendipitous that the backcross plants individually having Nipponbare homozygous alleles at either shattering locus (qSH1 or sh4) shed all the seeds. This fact strongly indicates that the non-shattering behavior was not obtained by a single mutation in the genetic background of wild rice. Probably, some other minor genes are still associated with the formation or activation of abscission layer, which enhance the seed shattering.

Restriction endonuclease analysis of chloroplast DNA was carried out to clarify the chloroplast genome differentiation between Asian varieties of Oryza sativa. Based on the restriction fragment patterns obtained with six endonucleases, i.e., EcoRI, HindIII, PstI, PvuII, SmaI, and XhoI, chloroplast genomes of 68 local varieties from 15 Asian countries could be classified into five types (types 1, 3, 10, 11, and 12). Among these types, four length mutations and two base substitutions were found; these changes were located on the PstI physical map of rice chloroplast DNA. In a dendrogram showing genetic relationships among five chloroplast genomes, they are mainly divided into two groups, which we have named as the Japonica (types 1, 11, and 12) and Indica (types 3 and 10) chloroplast genome groups. Both groups are distributed widely in Asian countries. The Japonica isozyme group classified by J.C. Glaszmann (1985. Rice Genetics. International Rice Research Institute, Manila, Philippines, pp. 83–90) carries only the Japonica chloroplast genome, whereas the Indica isozyme group contains both Japonica and Indica chloroplast genomes.Key words: Oryza sativa, Asian varieties, chloroplast DNA, restriction endonuclease analysis, chloroplast genome differentiation.

A simple procedure for the detection of rice RFLPs with non-radioactive probes is described. Rice single-copy DNA was labeled with non-radioactive digoxigenin-d UTP. When digested total DNA was hybridized with the non-radioactive labeled DNA probes, RFLPs for rice single-copy DNA could be successfully detected.

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.

Polymorphism over approximately 26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (pi = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (pi = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to approximately 50 kb in O. sativa ssp. indica.

This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism.

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.

Japanese rice ( Oryza sativa L.) cultivars that are strictly used for the brewing of sake (Japanese rice wine) represent a unique and traditional group. These cultivars are characterized by common traits such as large grain size with low protein content and a large, central white-core structure. To understand the genetic diversity and phylogenetic characteristics of sake-brewing rice, we performed amplified fragment length polymorphism and simple sequence repeat analyses, using 95 cultivars of local and modern sake-brewing rice together with 76 cultivars of local and modern cooking rice. Our analysis of both nuclear and chloroplast genome polymorphisms showed that the genetic diversity in sake-brewing rice cultivars was much smaller than the diversity found in cooking rice cultivars. Interestingly, the genetic diversity within the modern sake-brewing cultivars was about twofold higher than the diversity within the local sake-brewing cultivars, which was in contrast to the cooking cultivars. This is most likely due to introgression of the modern cooking cultivars into the modern sake-brewing cultivars through breeding practices. Cluster analysis and chloroplast haplotype analysis suggested that the local sake-brewing cultivars originated monophyletically in the western regions of Japan. Analysis of variance tests showed that several markers were significantly associated with sake-brewing traits, particularly with the large white-core structure.

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10 000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trait locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.

Striga spp. is considered to be the greatest biological constraint to food production in sub-Saharan Africa, a more serious problem than insects, birds and plant diseases. They are among the most specialized root-parasitic plants inflicting serious injury to their host depriving them water, minerals and photosynthate. The greatest diversity of Striga spp. occurs in grassland. However, Striga hermonthica mainly occurs in farmland infecting grasses. The parasite devastating effect is accomplished prior to its emergence from the soil. It may cause yield losses in cereals ranging from 15% under favourable conditions to 100% where several stress factors are involved, thereby affecting the livelihood of millions of resource-poor farmers. Piecemeal approach to address one aspect of Striga problem at a time has been a setback in technology transfer to producers. Future Striga control programs should not be conducted separately, but should rather be conducted in an integrated approach that combines research talents of various institutions. This will facilitate collaborative research and achieve qualitative interaction between stakeholders, which can easily produce reliable technologies that are practical and available to farmers. Striga being a pervasive pest, time is of essence in controlling it. There is an urgent need for the establishment of policies to promote, implement, and ensure a long-term sustainable Striga control program.

Relationships between chloroplast genomes (=ctDNAs) from diploid, A-genome species of rice (Oryza spp.) were established using length differences in restriction fragments of ctDNAs

Restriction endonuclease analysis of chloroplast (ct) DNA variation in two cultivated rice species, Oryza sativa and O. glaberrima, using three endonucleases, EcoRI, HindIII and PstI, indicated the differentiation of their chloroplast genome into three types. O. sativa differed from O. glaberrimain its EcoRI restriction pattern, whereas both O. sativa ecospecies Japonica and ecosp. Javanica were differentiated from ecosp. Indica by their HindIII and PstI restriction patterns. In contrast, no ctDNA variation was revealed in O, sativa ecosp. Japonica, Javanica, or Indica, or O. glaberrima (11, 4, 7 and 8 accessions, respectively). These findings demonstrated, for the first time, the existence of chloroplast genome diversity among cultivated rice species and ecospecies.