Establishment of a New Cross of the Rice Blast Fungus Derived from Japanese Differential Strain Ina168 and Hermaphroditic Rice Pathogen Guy11

Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.     

AVR1-CO39 is a predominant locus governing the broad avirulence of Magnaporthe oryzae 2539 on cultivated rice (Oryza sativa L.)

Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.     

Genetic Analysis and Molecular Mapping of a Rolling Leaf Mutation Gene in Rice

A rice mutant with rolling leaf, namely γ-rl, was obtained from M2 progenies of a native indica rice stable strain Qinghuazhan （QHZ） from mutagenesis of dry seeds by γ-rays. Genetic analysis using the F2 population from a cross between this mutant and QHZ indicated the mutation was controlled by a single recessive gene. In order to map the locus for this mutation, another F2 population with 601 rolling leaf plants was constructed from a cross between y-rl and a japonica cultivar 02428. After primary mapping with SSR （simple sequence repeats） markers, the mutated locus was located at the short arm of chromosome 3, flanked by RM6829 and RM3126. A number of SSR, InDel （insertion/deletion） and SNP （single nucleotide polymorphism） markers within this region were further developed for fine mapping. Finally, two markers, SNP121679 and InDe1422395, were identified to be flanked to this locus with genetic distances of 0.08 cM and 0.17 cM respectively, and two SNP markers, SNP75346 and SNPl10263, were found to be co-segregated with this locus. These results suggested that this locus was distinguished from all loci for the rolling leaf mutation in rice reported so far, and thus renamed rl10（t）. By searching the rice genome database with closely linked markers using BLAST programs, an e-physical map covering rl10（t） locus spanning about a 50 kb region was constructed. Expression analysis of the genes predicted in this region showed that a gene encoding putative flavin-containing monooxygenase （FMO） was silenced in γ-rl, thus this is the most likely candidate responsible for the rolling leaf mutation.     

Genetic basis of hybrid breakdown in a Japonica/Indica cross of rice,Oryza sativa L

Reproductive barriers often arise in hybrid progeny between two varietal groups of Asian cultivated rice ( Oryza sativa L.), Japonica and Indica. Hybrid breakdown showing poor growth habit, and complete sterility was found in the backcrossed progeny derived from a cross between a Japonica variety, Asominori, and an Indica variety, IR24. We employed RFLP analysis in the segregating population to study the genetic basis underlying hybrid breakdown. It was found that the hybrid breakdown is caused by a set of two nuclear genes, which were symbolized as hwe1 and hwe2. The parental varieties, Asominori and IR24, carry hwe1(+) hwe1(+) hwe2hwe2 and hwe1hwe1hwe2(+) hwe2(+) genotypes, respectively, whereas the progenies that showed a weakness performance carry the double recessive genotype ( hwe1hwe1hwe2hwe2). Abnormality was not observed in the progenies that carry the other genotypes, indicating that a single dominant allele at either locus is necessary for normal growth. Based on linkage analysis with RFLP markers, the hwe1 locus was located between RFLP markers R1869 and S1437 on chromosome 12 and the hwe2 locus was located between R3192 and C1211 on chromosome 1. The genetic basis was reconfirmed using near-isogenic lines carrying the genes with reciprocal genetic backgrounds. The present study provides clear evidence, viewed by previous workers, that hybrid breakdown is attributed to complementary genes from both parents.     

Genetic Mapping of Magnaporthe grisea Avirulence Gene Corresponding to Leaf and Panicle Blast Resistant QTLs in Jao Hom Nin Rice Cultivar

The avirulence characteristic of Magnaporthe grisea isolate TH16 corresponding to Jao Hom Nin (JHN) rice cultivar was studied by mapping population of 140 random ascospore progenies derived from the cross between B1-2 and TH16 isolates. Segregation analyses of the avirulence characteristic performing on JHN rice at the seedling and flowering stages were performed in this mapping population. We used the reference map of Guy11/2539 to choose microsatellite DNA markers for mapping the avirulence gene. The genetic map of this population was constructed from 39-microsatellite markers. The genetic map was spanned by covering seven chromosomes with an average distance of 11.9 cM per marker. In mapping population the distribution of pathogenic and non-pathogenic progenies on JHN rice were found to be fitted to 1 : 1 ratio for two of the rice stages, seedling and flowering stages. The Quantitative Trait Loci (QTL) analysis for avirulence genes corresponding to two rice stages were located at the same region on chromosome 2 between markers Pyms305 and Pyms435. The LOD score and percentage of phenotypic variance explained (PVE) on two rice stages were 5.01/16.69 and 6.73/20.26, respectively. These loci were designated as Avr-JHN(lb) and Avr-JHN(pb) corresponding to leaf and panicle blast characteristics. The findings of this study can be the initial step for positional cloning and identifying any function of avirulence genes corresponding to leaf and panicle blast characteristics.     

Analysis of genetic diversity and population structure of rice cultivars from Korea, China and Japan using SSR markers

A total of 29 simple sequence repeat (SSR) markers were used to analyze the genetic diversity of 150 accessions of cultivated rice (Oryza sativa L.) from Korea, China, and Japan. A total of 375 alleles were detected with an average of 12.9 per locus. The averaged values of gene diversity and polymorphism information content (PIC) for each SSR locus were 0.7001 and 0.6683, respectively. Alleles per locus in Korean rice were 8.8, whereas 8.1 and 7.2 alleles per locus were found in Chinese and Japanese rice, respectively. The mean gene diversity in Korean, Chinese, and Japanese rice was 0.6058, 0.6457, and 0.5174, respectively, whereas the mean PIC values for each SSR locus were 0.5759, 0.6138, and 0.4881, respectively. The genetic diversity of the Korean and Chinese cultivars was higher than that of the Japanese cultivars, and the genetic diversity ofjaponica was higher than that ofindica. The model-based structure analysis revealed the presence of three subpopulations, which was basically consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 22% in contrast to 78% for the within-population component. The overallFST value was 0.2180, indicating a moderate differentiation among groups. The results could be used for designing effective breeding programs aimed at broadening the genetic bases of commercially grown varieties.     

Genetic architecture underlying the evolutionary change of competitive ability in Asian cultivated and wild rice

Breeding of competitive cultivars has long been fraught with difficulty owing to limited knowledge of the genetic basis of competitive ability. In this study, we examined the diversity of competitive ability in Asian rice and the genetic basis of this variation. Cultivated strains and wild perennial strains have higher competitive ability than wild annual strains. Quantitative trait locus (QTL) analysis of competitive ability for three weed species was conducted in the cross between cultivated and wild annual strains, and three QTLs for general competitive ability (GCA) were identified. GCA-QTLs conferred higher competitive ability by the cultivated rice alleles and were co-located with QTLs for plant architecture and root growth, detected in the same mapping population. Furthermore, a significant change in GCA was achieved by accumulation and epistatic interaction of three QTLs. Further studies on the genetic control of competitive ability would facilitate the breeding of competitive cultivars in rice.     

Identification of flanking SSR markers for a major rice gall midge resistance gene <Emphasis Type="Italic">Gm1</Emphasis> and their validation

Host-plant resistance is the preferred strategy for management of Asian rice gall midge (Orseolia oryzae), a serious pest in many rice-growing countries. The deployment of molecular markers linked to gall midge resistance genes in breeding programmes can accelerate the development of resistant cultivars. In the present study, we have tagged and mapped a dominant gall midge resistance gene, Gm1, from the Oryza sativa cv. W1263 on chromosome 9, using SSR markers. A progeny-tested F₂ mapping population derived from the cross W1263/TN1 was used for analysis. To map the gene locus, initially a subset of the F₂ mapping population consisting of 20 homozygous resistant and susceptible lines each was screened with 63 parental polymorphic SSR markers. The SSR markers RM316, RM444 and RM219, located on chromosome 9, are linked to Gm1 at genetic distances of 8.0, 4.9 and 5.9 cM, respectively, and flank the gene locus. Further, gene/marker order was also determined. The utility of the co-segregating SSR markers was tested in a backcross population derived from the cross Swarna/W1263//Swarna, and allelic profiles of these markers were analysed in a set of donor rice genotypes possessing Gm1 and in a few gall midge-susceptible, elite rice varieties.     

Identification of major quantitative trait loci qSBR11-1 for sheath blight resistance in rice

Sheath blight caused by Rhizoctonia solani Kühn is one of the important diseases of rice, resulting in heavy yield loss in rice every year. No rice line resistant to sheath blight has been identified till date. However, in some rice lines a high degree of resistance to R. solani has been observed. An indica rice line, Tetep, is a well documented source of durable and broad spectrum resistance to rice blast as well as quantitative resistance to sheath blight. The present study identified genetic loci for quantitative resistance to sheath blight in rice line Tetep. A mapping population consisting of 127 recombinant inbred lines derived from a cross between rice cultivars HP2216 (susceptible) and Tetep (resistant to sheath blight) was evaluated for sheath blight resistance and other agronomic traits for 4 years across three locations. Based on sheath blight phenotypes and genetic map with 126 evenly distributed molecular markers, a quantitative trait loci (QTLs) contributing to sheath blight resistance was identified on long arm of chromosome 11. Two QTL mapping approaches i.e., single marker analysis and composite interval mapping in multi environments were used to identify QTLs for sheath blight resistance and agronomical traits. The QTL qSBR11-1 for sheath blight resistance was identified between the marker interval RM1233 (26.45 Mb) to sbq33 (28.35 Mb) on chromosome 11. This region was further narrowed down to marker interval K39516 to sbq33 (~0.85 Mb) and a total of 154 genes were predicted including 11 tandem repeats of chitinase genes which may be responsible for sheath blight resistance in rice line Tetep. A set of 96 varieties and a F₂ population were used for validation of markers linked to the QTL region. The results indicate that there is very high genetic variation among varieties at this locus, which can serve as a starting point for allele mining of sheath blight resistance.     

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.     

Genetic diversity and phylogeny of Japanese sake-brewing rice as revealed by AFLP and nuclear and chloroplast SSR markers

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.     

Identification of <Emphasis Type="Italic">qSOR1</Emphasis>, a major rice QTL involved in soil-surface rooting in paddy fields

Specific Indonesian lowland rice (Oryza sativa L.) cultivars elongate thick primary roots on the soil surface of paddy fields. To clarify the genetic factors controlling soil-surface rooting, we performed quantitative trait locus (QTL) analyses using 124 recombinant inbred lines (RILs) derived from a cross between Gemdjah Beton, an Indonesian lowland rice cultivar with soil-surface roots, and Sasanishiki, a Japanese lowland rice cultivar without soil-surface roots. These cultivars and the RILs were tested for soil-surface rooting in a paddy field. We identified four regions of chromosomes 3, 4, 6, and 7 that were associated with soil-surface rooting in the field. Among them, one major QTL was located on the long arm of chromosome 7. This QTL explained 32.5–53.6% of the total phenotypic variance across three field evaluations. To perform fine mapping of this QTL, we measured the basal root growth angle of crown roots at the seedling stage in seven BC₂F₃ recombinant lines grown in small cups in a greenhouse. The QTL was mapped between markers RM21941 and RM21976, which delimit an 812-kb interval in the reference cultivar Nipponbare. We have designated this QTL qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1).     

Development of microsatellite markers and construction of genetic map in rice blast pathogen Magnaporthe grisea

Magnaporthe grisea is the most destructive fungal pathogen of rice and a model organism for studying plant-pathogen interaction. Molecular markers and genetic maps are useful tools for genetic studies. In this study, based on the released genome sequence data of M. grisea, we investigated 446 simple sequence repeat (SSR) loci and developed 313 SSR markers, which showed polymorphisms among nine isolates from rice (including a laboratory strain 2539). The number of alleles of each marker ranged 2-9 with an average of 3.3. The polymorphic information content (PIC) of each marker ranged 0.20-0.89 with an average of 0.53. Using a population derived from a cross between isolates Guy11 and 2539, we constructed a genetic map of M. grisea consisting of 176 SSR markers. The map covers a total length of 1247 cM, equivalent to a physical length of about 35.0 Mb or 93% of the genome, with an average distance of 7.1cM between adjacent markers. A web-based database of the SSR markers and the genetic map was established (http://ibi.zju.edu.cn/pgl/MGM/index.html).     

Genetic diversity among cultivars, landraces and wild relatives of rice as revealed by microsatellite markers

Genetic diversity among 35 rice accessions, which included 19 landraces, 9 cultivars and 7 wild relatives, was investigated by using microsatellite (SSR) markers distributed across the rice genome. The mean number of alleles per locus was 4.86, showing 95.2% polymorphism and an average polymorphism information content of 0.707. Cluster analysis based on microsatellite allelic diversity clearly demarcated the landraces, cultivars and wild relatives into different groups. The allelic richness computed for the clusters indicated that genetic diversity was the highest among wild relatives (0.436), followed by landraces (0.356), and the lowest for cultivars. Allelic variability among the SSR markers was high enough to categorize cultivars, landraces and wild relatives of the rice germplasm, and to catalogue the genetic variability observed for future use. The results also suggested the necessity to introgress genes from landraces and wild relatives into cultivars, for cultivar improvement.     

Identification of a Cross Between Two Compatible Isolates of Magnaporthe grisea (Hebert) Barr and Genetic Analysis of Avirulence/Virulence of Rice

A cross was successful between two field isolates of Magnaporthe grisea Guy 11 and CD 128, both pathogenic to rice. One ascospore isolated in this cross could be backcrossed to the most fertile parental isolate Guy 11. Backcrosses to CD 128 were not compatible. All progenies were inoculated to seven rice varieties in order to analyze the genetic basis of avirulence. Avirulence to Kusabue was found to be controlled by one gene. Two other controls of avirulence to rice were shown: a) two independent genes involved in avirulence to Pi-n°4, both required by the isolates for virulence expression, b) two independent genes, each one leading to virulence on the rice varieties K 1 and Aichi asahi. Implications of these results to Flor's gene-for-gene hypothesis are discussed.     

Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.     

New evidence for the synteny of rice chromosome 1 and barley chromosome 3H from rice expressed sequence tags.

To provide improved access to the wealth of resources and genomic information that is presently being developed for rice a set of 88 rice expressed sequence tags (ESTs) previously mapped on rice chromosome I in the cross 'Nipponbare' x 'Kasalath' was used for comparative mapping in a cross of the barley cultivars 'Igri' and 'Franka'. As expected. most (89%) of the clones gave distinct banding patterns in barley of which about one-third was polymorphic between 'Igri' and 'Franka'. These polymorphisms were mapped, and most of these (56%) confirmed that rice chromosome 1 and barley chromosome 3H are syntenous. All single-copy markers identified conserved collinear positions, while markers with multiple copies did so in a few cases only. The markers that were not fitting in the collinear order were distributed randomly across the barley genome. The comparative maps of barley chromosome 3H and rice chromosome 1 comprise in total 26 common markers covering more than 95% of the genetic length of both chromosomes. A 30-fold reduction of recombination is seen around the barley centromere, and synteny may be interrupted in this region. However, the good overall synteny on a mesoscale (1-10 cM) justifies the use of rice as a platform for map-based cloning in barley.     

Molecular mapping of quantitative trait loci in japonica rice.

Rice (Oryza sativa L.) molecular maps have previously been constructed using interspecific crosses or crosses between the two major subspecies: indica and japonica. For japonica breeding programs, however, it would be more suitable to use intrasubspecific crosses. A linkage map of 129 random amplified polymorphic DNA (RAPD) and 18 restriction fragment length polymorphism (RFLP) markers was developed using 118 F2 plants derived from a cross between two japonica cultivars with high and low seedling vigor, Italica Livorno (IL) and Labelle (LBL), respectively. The map spanned 980.5 cM (Kosambi function) with markers on all 12 rice chromosomes and an average distance of 7.6 cM between markers. Codominant (RFLP) and coupling phase linkages (among RAPDs) accounted for 79% of total map length and 71% of all intervals. This map contained a greater percentage of markers on chromosome 10, the least marked of the 12 rice chromosomes, than other rice molecular maps, but had relatively fewer markers on chromosomes 1 and 2. We used this map to detect quantitative trait loci (QTL) for four seedling vigor related traits scored on 113 F3 families in a growth chamber slantboard test at 18 degrees C. Two coleoptile, five root, and five mesocotyl length QTLs, each accounting for 9-50% of the phenotypic variation, were identified by interval analysis. Single-point analysis confirmed interval mapping results and detected additional markers significantly influencing each trait. About two-thirds of alleles positive for the putative QTLs were from the high-vigor parent, IL. One RAPD marker (OPAD13720) was associated with a IL allele that accounted for 18.5% of the phenotypic variation for shoot length, the most important determinant of seedling vigor in water-seeded rice. Results indicate that RAPDs are useful for map development and QTL mapping in rice populations with narrow genetic base, such as those derived from crosses among japonica cultivars. Other potential uses of the map are discussed. Key words : QTL mapping, RAPD, RFLP, seedling vigor, japonica, Oryza sativa.     

Genetic analysis of rice CMS-WA fertility restoration based on QTL mapping

 The inheritance of fertility restoration of rice cytoplasmic male sterility of the wild abortive type was studied by means of QTL mapping. The two segregating populations examined showed high frequencies of highly sterile and highly fertile progenies, but a low frequency of partially sterile and partially fertile progenies. The distributions suggested that fertility restoration was mainly controlled by major genes. Based on a linkage map constructed with 57 RFLP and 61 AFLP markers on a B₁F₁ population, composite interval mapping (CIM) revealed that the fertility was restored by the additive effects of two restorer loci located on chromosome 10. One QTL, tightly linked to RFLP marker C1361 in the middle of the long arm of chromosome 10, explained 71.5% of the phenotypic variance. The second QTL was located between RFLP markers R2309 and RG257 on the short arm and explained 27.3% of the phenotypic variance. Similar results were obtained using the simple interval mapping (SIM) methods. Recived: 8 January 1998/Accepted: 22 April 1998     

Association mapping of yield and its components in rice cultivars

To make advances in rice breeding it is important to understand the relatedness and ancestry of introduced rice accessions, and identify SSR markers associated with agronomically important phenotypic traits, for example yield. Ninety-two rice germplasm accessions recently introduced from seven geographic regions of Africa, Asia, and Latin America, and eleven US cultivars, included as checks, were evaluated for yield and kernel characteristics, and genotyped with 123 SSR markers. The SSR markers were highly polymorphic across all accessions. Population structure analysis identified eight main clusters for the accessions which corresponded to the major geographic regions, indicating agreement between genetic and predefined populations. Linkage disequilibrium (LD) patterns and distributions are of fundamental importance for genome-wide mapping association. LD between linked markers decreased with distance and with a substantial drop in LD decay values between 20 and 30 cM, suggesting it should be possible to achieve resolution down to the 25 cM level. For the 103 cultivars, the complex traits yield, kernel width, kernel length, kernel width/length ratio, and 1000-kernel weight, were estimated by analysis of variety trial data. The mixed linear model method was used to disclose marker-trait associations. Many of the associated markers were located in regions where QTL had previously been identified. In conclusion, association mapping in rice is a viable alternative to QTL mapping based on crosses between different lines.