Comparative genetic maps of foxtail millet (Setaria italica) and rice (Oryza sativa)

 A foxtail millet-rice comparative genetic map was constructed using mapped rice RFLP markers and wheat genomic and cDNA clones with known map position in rice. About 74% and 37% of the cDNA and genomic clones, respectively, were transferable to foxtail millet, confirming that conservation at the DNA level is greatest in genic regions. A high degree of conserved colinearity was observed between the two genomes. Five entire foxtail millet chromosomes appear to be colinear with five entire rice chromosomes. The remaining four foxtail millet linkage groups each show colinearity with segments of two rice chromosomes. The rearrangements of rice chromosomes 3 and 10 to form foxtail millet chromosome IX, and 7 and 9 to form chromosome II are very similar to those required to form maize chromosomes 1 and 7 and sorghum linkage groups C and B, indicating Setaria’s clear taxonomic position within the subfamily of the Panicoideae. Received: 18 December 1996 / Accepted: 4 August 1997     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

A genome-wide analysis of wide compatibility in rice and the precise location of the S5 locus in the molecular map

 The discovery of wide-compatibility varieties (WCVs) that are able to produce normal fertility hybrids when crossed both to indica and japonica rice has enabled the fertility barrier between indica and japonica subspecies to be broken and provided the possibility of developing inter-subspecific hybrids in rice breeding programs. However, a considerable variation in the fertility level of hybrids from the same WCV crossed to different varieties has often been observed. One hypothesis for this variable fertility is that additional genes are involved in hybrid fertility besides the wide-compatibility gene (WCG). To assess such a possibility, we performed a genome-wide analysis by assaying a large population from a three-way cross ‘02428’/‘Nanjing 11’//‘Balilla’ using a total of 171 RFLP probes detecting 191 polymorphic loci distributed throughout the entire rice linkage map. Our analysis recovered 3 loci conferring significant effects on hybrid fertility. The major locus on chromosome 6 coincided in chromosomal location with the previously identified S ₅ locus, and the 2 minor loci that mapped to chromosomes 2 and 12, respectively, were apparently distinct from all previously reported hybrid sterility genes. Interaction between the indica and japonica alleles at each of the loci caused a reduction in hybrid fertility. The joint effect of the 2 minor loci could lead to partial sterility even in the presence of the WCG. The location of the S ₅ locus on the molecular marker linkage map was determined to be approximately 1.0 cM from the RFLP locus R2349. This tight linkage will be useful for marker-aided transfer of the WCG in hybrid rice breeding and for map-based cloning. Received: 5 February 1997 / Accepted: 4 April 1997     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F₃ families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F₂ plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region. Received: 15 December 1999 / Accepted: 18 February 2000     

MADS box genes expressed in developing inflorescences of rice and sorghum

With the aim of elucidating the complex genetic system controlling flower morphogenesis in cereals, we have characterized two rice and two sorghum MADS box genes isolated from cDNA libraries made from developing inflorescences. The rice clones OsMADS24 and OsMADS45, which share high homology with the Arabidopsis AGL2 and AGL4 MADS box genes, are expressed in the floral meristem, in all the primordia, and in mature floral organs. High expression levels have also been found in developing kernels. The sorghum clone SbMADS1 is also homologous to AGL2 and AGL4: expression analysis and mapping data suggest that it is the ortholog of OsMADS24. The pattern of expression of SbMADS2, the other sorghum MADS box gene, suggests that it may play a role as a meristem identity gene, as does AP1 in Arabidopsis, to which it shows considerable homology. The four genes have been mapped on a rice RFLP genetic map: the results are discussed in terms of synteny among cereals. Received: 25 April 1996 / Accepted: 29 August 1996     

Molecular cloning, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice ( Oryza sativa L.)

By using differential display PCR (DD-PCR) technique, two salt-inducible and one salt-repressed cDNA fragments were isolated from rice. The three cDNA fragments were characterized respectively as partial sequence of rice S-adenosylmethionine decarboxylase (SAMDC) gene, a new member of translation elongation factor 1A gene (namedREF1 A), and a novel gene whose function is unknown (namedSRG1). The full-length cDNA of SAMDC gene (namedSAMDC1) was further isolated by RT-PCR approach and the deduced polypeptide was found to be homologous to SAMDC proteins of other plants, yeast and buman. Northern hybridization revealed that expression of SAMDCl and REFlA was induced, while SRGl was dramatically repressed, by salinity stress. Southern blot analysis demonstrated that SAMDCl and SRGl were present as a single copy gene in rice genome, whereas riceREF1 A gene was organized as a gene family. TheREF1 A,SAMDC1, andSRG1 genes were located on chromosome 3,4, and 6 respectively by RFLP mapping approach using ZYQ8/JX17 DH population and RFLP linkage maps. Project supported by the National “863” High-Technology Program.     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998     

Direct amplification of minisatellite-region DNA with VNTR core sequences in the genus Oryza

 A polymerase chain reaction (PCR) application, involving the directed amplification of minisatellite-region DNA (DAMD) with several minisatellite core sequences as primers, was used to detect genetic variation in 17 species of the genus Oryza and several rice cultivars (O. sativa L.). The electrophoretic analysis of DAMD-PCR products showed high levels of variation between different species and little variation between different cultivars of O. sativa. Polymorphisms were also found between accessions within a species, and between individual plants within an accession of several wild species. The DAMD-PCR yielded genome-specific banding patterns for the species studied. Several DAMD-PCR-generated DNA fragments were cloned and characterized. One clone was capable of detecting multiple fragments and revealed individual-specific hybridization banding patterns using genomic DNA from wild species as well as rice cultivars. A second clone detected only a single polymorphic locus, while a third clone expressed a strong genome specificity by Southern analysis. The results demonstrated that DAMD-PCR is potentially useful for species and genome identification in Oryza. The DAMD-PCR technique also allows for the isolation of informative molecular probes to be utilized in DNA fingerprinting and genome identification in rice. Received: 1 October 1996 / Accepted: 25 April 1997     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

Construction of an RFLP linkage map for cultivated sunflower

 An RFLP linkage map was constructed for cultivated sunflower Helianthus annuus L., based on 271 loci detected by 232 cDNA probes. Ninety-three F₂ plants of a cross between inbred lines RHA 271 and HA 234 were used as the mapping population. These genetic markers plus a fertility restoration gene, Rf ₁, defined 20 linkage groups, covering 1164 cM of the sunflower genome. Of the 71 loci 202 had codominant genotypic segregation, with the rest showing dominant segregation. Thirty-two of the 232 probes gave multiple locus segregation. There were 39 clusters of tightly linked markers with 0 cM distance among loci. This map has an average marker-to-marker distance of 4.6 cM, with 11 markerless regions exceeding 20 cM. Received: 17 June 1997 / Accepted: 19 June 1997     

Identification of random amplified polymorphic DNA (RAPD) markers linked to the v locus in barley, Hordeum vulgare L.

 Recombinant backcross lines of barley were produced from a cross between Kanto Nakate Gold (KNG; two-rowed) and Azumamugi (AZ; six-rowed) after backcrosses of F₁ plants with AZ as the recurrent parent. Each of these lines had an introgressed segment from chromosome 2 of KNG. Two recombinant backcross lines, L1 and M3-13, were used for an initial screening of polymorphism. After screening a total of 888 oligonucleotides as arbitrary primers, we identified eight random amplified polymorphic DNAs (RAPDs) between backcross lines and AZ. Among the RAPD fragments, CMNA-38₇₀₀ was linked to the v locus with a recombination frequency of zero, while OPJ-09₈₅₀ and OPP-02₇₀₀ were linked to the v locus at a map distance of 1.4 cM. Thus, the three RAPD markers were clustered around the v locus since the lengths of introgressed chromosomal segments in the L1 and M3-13 lines were no less than 38 cM. The other five RAPD fragments that we identified were not linked to the v locus. Received: 14 January 1997 / Accepted: 14 February 1997     

Identification of a maize linkage group related to apomixis in Brachiaria

 A bulked segregant analysis using RFLPs and RAPDs was carried out to identify molecular markers co-segregating with apomixis in a Brachiaria F₁ population. The test population used was a cross between sexual B. ruziziensis R44 and the aposporous apomictic Brachiaria brizantha cv Marandu. The Brachiaria genome was systematically scanned using 61 cDNA and genomic maize clones detecting 65 loci located at 40 cM, on average, one from each other in the maize genome. The finding of a clone that presented a polymorphic band co-segregating with apomixis (umc147) led to the identification of another marker within the same area (umc72). The clones belong to a duplicated linkage group that maps to the distal part of maize chromosome-1 long arm and chromosome-5 short arm. RAPD analysis using 184 primers from Operon sets yielded one more marker (OPC4) significantly linked to the trait mapping the same locus. OPC4 had been previously reported as a potential marker for apospory in Pennisetum. A map of the region was constructed using additional clones that belong to the same maize linkage group. Since that was the only genomic region that presented an apomixis-linked polymorphism our observations support the existence of a single locus directing apospory in Brachiaria. Received: 9 September 1996 / Accepted: 20 September 1996     

Integrated map of the Schizosaccharomyces pombe genome

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments. Received: 21 November 1996; in revised form: 3 March 1997 / Accepted: 27 March 1997     

Construction and characterization of a bacterial artificial chromosome library of apple

 A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus×domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of EcoRI to EcoRI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome. Received: 19 February 1998 / Accepted: 30 April 1998     

A RFLP-based linkage map of mustard [Brassica juncea (L.) Czern. and Coss.]

 A genetic linkage map of Brassica juncea was constructed based on restriction fragment length polymorphism (RFLP) detected by anonymous cDNA markers from B. napus, using a segregating F₁-derived doubled haploid (DH) progeny from a cross between a canola-quality mustard line (J90-4317) and a high-oil-content mustard line (J90-2733). The RFLP probes consisted of 229 cDNA probes from B. napus and a B. napus tandem repeat sequence, RDA2. The map consisted of 343 marker loci arranged in 18 major linkage groups plus five small segments with two to five marker loci, covering a total map distance of 2073 cM. Twenty-four percent of the markers were dominant in nature. Sixty-two percent of the marker loci were duplicated, and the majority were involved in inter-linkage group duplications, illustrating that complex duplications and subsequent rearrangements occurred after allopolyploidy. Deviation from the Mendelian segregation ratio for a DH population was observed for 27% of the markers. Two-thirds of these markers with a skewed segregation were clustered in 6 linkage groups and two unassigned segments. The overall average marker interval of the B. juncea map reported here was 6.6 cM, which would provide a marker density satisfactory for efficient use of the map in breeding applications, such as tagging of important agronomic traits and marker-assisted selection. Received: 14 May 1996 / Accepted: 11 October 1996     

RFLP linkage map of the Ethiopian cereal tef [Eragrostis tef (Zucc) Trotter]

Tef [Eragrostis tef (Zucc)Trotter] is one of the most important cereal crops in Ethiopia. It is an allotetraploid species with a genome size of 720 Mbp. In this paper we report results of genetic linkage-map construction for E. tef using tef and heterologous cDNA probes for the first time. One hundred and sixteen F₈ recombinant inbred lines (RILs) from the cross E. tef cv Kaye Murri×Eragrostis pilosa (accession30–5) were used for mapping. Parental lines were digested with nine restriction enzymes and screened using 159 tef cDNA and 162 heterologous probes including the grass genome anchor probes. The polymorphism level between parental lines was 66.9%. One hundred and thirty nine polymorphic probes were hybridized against 116 RILs. Both the tef and the heterologous probes hybridized well against tef genomic DNA. The linkage map defined 1,489 cM of the tef genome comprising 149 marker loci distributed among 20 linkage groups. The average interval between markers was 9.99 cM. A fraction (14.8%) of the markers deviated significantly from the expected segregation. Such a genetic linkage map is useful for tagging economically useful genes in tef because a wide range of agronomically important traits is segregating within this population. This would enable the use of a marker assisted breeding strategy which, in turn, will enhance breeding efficiency. Alignment of the tef RFLP map with the rice RFLP map indicates that a number of syntenic chromosomal fragments exist between tef and rice in which the gene order was for the most part collinear. The comparative mapping information should enable tef scientists to take advantage of whatever genetic progress is made on the cereal model species rice. Received: 9 June 2000 / Accepted: 31 August 2000     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.