Interspecific Backcross Population Research Articles

Genetic analysis and identification of QTLs associated with yield-enhancing traits in Oryza sativa IR64 × Oryza glaberrima interspecific backcross populations

Context Broadening the gene pool of staple food crops by incorporating desirable genes from wild/relative species is way to achieve higher genetic gains. The African rice species Oryza glaberrima Steud. is a source of genes for genetic improvement of Oryza sativa L. to various biotic and abiotic stresses. Aim The study was undertaken to identify genomic regions associated with yield and yield-related traits in interspecific backcross mapping populations derived from variety IR64 (O. sativa indica) × O. glaberrima. Methods The BC1F2 and BC1F2:3 populations were phenotyped for days to 50% flowering, plant height, number of tillers, number of productive tillers, panicle length, spikelet fertility and grain yield under irrigated conditions. The BC1F2 population was genotyped with 99 genome-wide polymorphic SSR markers. Quantitative trait loci (QTLs) associated with yield-enhancing traits were identified through single marker analysis. Key results Significant genetic variability was observed with moderate to high heritability in mapping populations. Genotyping revealed the presence of segregation distortion and skewed genome composition. Single marker analysis identified 20 QTLs linked to the studied traits. A QTL linked to grain yield, qGY-4.1, derived from O. glaberrima was found in both populations with high phenotypic variance. The chromosome region around marker RM510 was identified as a QTL hotspot and putative candidate genes were identified. Conclusions Our study demonstrates that O. glaberrima can serve as a valuable genetic resource for genetic improvement of yield-enhancing traits in O. sativa cultivars. Implications The identified QTLs regions can be employed in molecular breeding. The promising introgression lines identified can be used as pre-breeding lines in future breeding programs.

Genotyping-by-sequencing and multilocation evaluation of two interspecific backcross populations identify QTLs for yield-related traits in pigeonpea.

This study has identified single-nucleotide polymorphism (SNP) markers associated with nine yield-related traits in pigeonpea by using two backcross populations (BP) developed through interspecific crosses and evaluating them at two locations and 3years. In both the populations, markers have shown strong segregation distortion; therefore, a quantitative trait locus (QTL) mapping mixed model was used. A total of 86 QTLs explaining 12-21% phenotypic variation were detected in BP-1. On the other hand, 107 QTLs explaining 11-29% phenotypic variation were detected in BP-2. Although most QTLs were environment and trait specific, few stable and consistent QTLs were also detected. Interestingly, 11 QTLs in BP-2 were associated with more than one trait. Among these QTLs, eight QTLs associated with days to 50% flowering and days to 75% maturity were located on CcLG07. One SNP "S7_14185076" marker in BP-2 population has been found associated with four traits, namely days to 50% flowering, days to 75% maturity, primary branches per plant and secondary branches per plant with positive additive effect. Hence, the present study has not only identified QTLs for yield-related traits, but also discovered novel alleles from wild species, which can be used for improvement of traits through genomics-assisted breeding.

Combination of Acylglucose QTL reveals additive and epistatic genetic interactions and impacts insect oviposition and virus infection

Acylsugars are important insect defense compounds produced at high levels by glandular trichomes of the wild tomato, Solanum pennellii. Marker-assisted selection was used to select for plants containing the three AGQTL named AG3QTL, AG4QTL, and AG11QTL from self-pollinated populations derived from an interspecific backcross population of CU071026 x (CU071026 x S. pennellii LA716). High acylglucose-accumulating lines were selected from these populations that possess these three AGQTL and the fewest number of extraneous S. pennellii LA716 introgressions. Incorporation of these three acylglucose QTL in the presence of the five standard S. pennellii introgressions of CU071026 altered acylsugar level and sugar moiety, demonstrating epistatic interactions between the acylglucose QTL on both of these traits. Comparison of the lines generated from the two breeding techniques indicated the three acylglucose QTL are essential but not necessarily sufficient for the production of elevated levels of acylglucose acylsugars. Fine-mapping of AG3QTL, AG4QTL, and AG11QTL resulted in less than 1 Mbp intervals for the locations of AG4QTL and AG11QTL; proposals of the causal genes underlying these acylglucose QTL are discussed. Characterization of the fatty acid profile of lines selected out of the interspecific backcross populations revealed an increase in the proportion of acylsugar n-C10 fatty acid acyl chains, possibly governed by one or more of the three acylglucose QTL. Characterization of the acylsugar profile of acylglucose lines selected from the interspecific backcross populations also demonstrated interactions among the acylglucose QTL to further modulate the diversity of acylsugars accumulated. Evaluation of an acylglucose line and controls against the tomato insect pest Frankliniella occidentalis demonstrated that levels of resistance differed among these lines and that the acylsugars accumulated by the acylglucose line were effective at reducing both F. occidentalis oviposition and incidence of Tomato spotted wilt orthotospovirus. However, of some of the acylglucose lines and hybrids tested against Spodoptera exigua did not indicate differences for larval weight gain and survival.

Successful Introgression of Abiotic Stress Tolerance from Wild Tepary Bean to Common Bean

Common bean (Phaseolus vulgaris L.) production is limited due to abiotic stresses, including drought and subzero temperatures. Tepary bean (P. acutifolius Gray), a relative of common bean, has been reported to be tolerant to these stresses. Preliminary studies screening wild tepary accessions for cold tolerance demonstrated that W6 15578 is a potential donor of cold tolerance, so an interspecific backcross population derived from a cross between it and common bean (NY5‐161) was developed. A 3‐yr field study was conducted in Saskatoon, SK, to identify lines able to withstand subzero temperatures better than the common bean parent at the seedling stage. Introgression lines were also tested for response to terminal drought under field conditions in Isabela, PR, over 3 yr. Days to flowering and yield measurements, along with subzero temperature and drought stress tolerance data, are presented. Although the interspecific introgression lines were backcrossed twice to common bean to improve the fertility and increase the proportion of common bean genome, several introgression lines performed better than the common bean parent under both stress conditions. Future breeding objectives include backcrossing to tepary to try to recover additional abiotic stress tolerance genes, as well as using selected introgression lines as breeding material to develop common bean varieties with increased subzero temperature stress and drought stress tolerance. Interspecific introgression of portions of the tepary bean genome into common bean is a promising method for increasing abiotic stress tolerance in common bean.

Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima.

A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene. Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.

Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm

Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

Introgressing White Mold Resistance from Phaseolus coccineus PI 439534 to Common Pinto Bean

ABSTRACTWhite mold [caused by Sclerotinia sclerotiorum (Lib.) de Bary] is a devastating disease of common bean (Phaseolus vulgaris L.) in cool‐ to moderate‐temperature and wet‐production regions worldwide. Use of resistant cultivars is crucial for effective and economical white mold control. Partial resistance exists in cultivated and wild common bean and Phaseolus species of the secondary gene pool. The objectives were to (i) develop highly resistant breeding lines (BL) from a recurrent interspecific backcross of common pinto bean ‘UI 320’ with P. coccineus PI 439534, and (ii) compare their response with known sources of resistance. Five pinto BL derived from UI 320 × 2/PI 439534 interspecific backcross population, the two parents, nine known sources of resistance, and susceptible pinto ‘Othello’ were screened in the greenhouse. Sclerotinia sclerotiorum isolates ARS12D and ND710 were used at the University of Idaho, Kimberly, in 2012 and isolates CO467 and NY133 at Colorado State University, Fort Collins, in 2013. All five interspecific pinto BL (VC13‐1, VC13‐3, VC13‐4, VC13‐5, VC13‐6) in Idaho and three (VC13‐4, VC13‐5, VC13‐6) in Colorado exhibited significantly (P ≤ 0.05) higher levels of resistance than PI 439534. Their resistance was either similar to (in Colorado) or higher (in Idaho) than the highest levels available in other interspecific BL (92BG‐7, I9365‐31, VCW 54, VRW 32) derived from the secondary gene pool thus far, as well as medium‐ (USPT‐WM‐1) and small‐ (‘ICA Bunsi’) seeded Middle American and large‐seeded Andean dry (G 122) and green (NY 6020‐4) beans. The effectiveness of the five interspecific pinto BL for controlling white mold with and without fungicides and other disease management strategies should be performed. Genetics of resistance and tagging and mapping of new resistance genes and/or quantitative trait loci should be performed. Also, high levels of resistance from the five interspecific pinto BL should be pyramided across Phaseolus species and/or transferred into cultivars.

Association analysis of yield and fibre quality characters in interspecific population of cotton (Gossypium spp.)

Seed cotton yield and fibre quality are controlled by polygenes and highly influenced by the environment. Hence, selection merely based on yield is not effective. In order to enhance the yield potential of the cotton varieties, an understanding of the relationship among different characters is of more importance. Knowledge on the direct contribution of different characters to yield would be highly important for formulating a selection programme. Correlation and path coefficient analysis were carried out using 62 progenies of an interspecific backcross population (Gossypium hirsutum L. var. KC 3 x Gossypium barbadense L. var. Suvin) x KC 3. Genotypic correlation studies revealed that number of bolls per plant, boll weight, number of sympodia per plant, number of seeds per boll, seed index, lint index, days to first flowering, and plant height have a significant positive association with seed cotton yield per plant. Path coefficient analysis showed that number of bolls per plant contributed maximum direct effect to seed cotton yield per plant followed by boll weight and lint index. The highest positive indirect effect on seed cotton yield per plant was also observed through number of bolls per plant. This indicated that seed cotton yield per plant was highly influenced by number of bolls per plant directly and indirectly. Thus, a greater emphasis should be given to number of bolls per plant, boll weight, and lint index for increasing seed cotton yield per plant.

Chromosomal regions associated with dollar spot resistance in colonial bentgrass

With 1 figure and 4 tables Abstract Interspecific hybridization between colonial bentgrass and creeping bentgrasses may be a useful approach for introducing resistance to Sclerotinia homoeocarpa, the causal agent of dollar spot disease, into creeping bentgrass. An interspecific backcross population of 271 genotypes was evaluated for dollar spot in a 2-year field study, and 11% of the genotypes were resistant to dollar spot. In the subset of the backcross population used for genetic linkage mapping, some resistant plants were identified, but no quantitative trait locus (QTL) was detected. A model of qualitative 3-gene recessive epistasis for dollar spot resistance in colonial bentgrass was proposed, and the limitations of QTL analysis to detect genomic regions conferring multigenic qualitative traits were examined. In the simulations, monogenic traits and 2-gene recessive epistatic traits were readily detectable by QTL analysis, whereas for 3-gene recessive epistasis, a QTL was not detectable. As an alternative to QTL analysis, screening of resistant plants from the backcross population for genomic regions held in common suggests that linkage groups 2A1 and 3A1 are possibly genomic locations of dollar spot resistance in colonial bentgrass.

Development and Evaluation of Primary Introgression Lines of Rice by Advanced Backcross QTL Strategy for Gaozhou Wild Rice (<i>O. rufipogon</i>)

Rice ( Oryza sativa L.) is the major cereal crop in China. The modern rice cultivars have narrow genetic base for most of the available traits, wild relatives of rice species contain trait-enhancing genes. The goal of this study was to identify introgression lines that can significantly increase yield in cultivated rice by yield-related traits from an inter-specific advanced backcross population (AB-population) between O.statia×O.rufipogon. AB-population ( O. statia ' Yuexiangzhan'× O. rufipogon ‘G52-9’) has evaluated using statistical methods for 17 agronomic traits pertaining to yield and yield-related components. Our studies indicated that AB-population had higher values for most traits analyzed in comparison to the recurrent parent, Yuexiangzhan. According to the statistical results, about 33.20% of the BC3F1 lines showed better than Yuexiangzhan in some traits. Grain shape and grain weight have selected for QTL mapping as an illustration of AB-QTL analysis. A total of 23 QTLs were identified for grain length (GL), grain width (GW), Grain length/grain width (LW), and grain weight (GWt). Phenotypic variation of each QTL ranged from 3.77%-28.67%. The grain weight of the recurrent ‘Yuexiangzhan’ was improved 15.10%, 28.67%, 26.29%, 24.28% and 12.90% by five main QTLs of GWt (from Gaozhou wild rice). This study confirms that wild progenitor species present potential donor sources for some complex traits and the advanced backcross population provides an efficient source of gene pool for future breeding programs. Therefore, Gaozhou wild rice is an efficient source of gene pool and can further utilize.

Drought resistance in an interspecific backcross population of rice ( Oryza spp.) derived from the cross WAB56-104 ( O. sativa) × CG14 ( O. glaberrima)

The ability to identify drought-resistant lines in all seasons would accelerate variety introduction. A total of 202 backcross-inbred lines of rice were subjected to drought during the 2006 and 2007 dry seasons at AfricaRice in Benin. Two irrigation regimes were applied: fully irrigated to maturity and 21 days of drought from 45 days after sowing. Plants were harvested at maturity. Increased canopy temperature under drought as compared to fully irrigated condition was observed. Delays were observed in plant flowering and maturity, with drought susceptibility index reaching 26.8 for flowering. Plant leaves were greener (2.9% increase) under drought than when fully irrigated. Drought negatively affected tiller number, plant height, number of leaves, leaf width and grain yield (16.9%, 13.7%, 6.7%, 14.1% and 26.7% respective reductions). Highly significant ( P ≤ 0.01) correlations were observed between traits measured under fully irrigated and drought conditions ( r between 0.52 and 0.92), except for leaf greenness (SPAD), leaf width and canopy temperature ( r = 0.42 ns, −0.03 ns and −0.30**, respectively). The study identified canopy temperature, SPAD, plant height and leaf number as possible traits that best correlated with grain yield. The performance of these traits under drought was a function of the rice line.

Comparative linkage mapping of Oryza glumaepatula and Oryza sativa interspecific crosses based on microsatellite and expressed sequence tag markers

Molecular linkage maps representing the rice genome have been an important tool for breeding programs because they allow the elucidation of polygenic traits and are an efficient tool for monitoring wild introgressions in interspecific crosses. Common markers among rice genetic maps are important in defining the homology of chromosomes and the synteny between genomic target regions. We used 148 markers (expressed sequence tags, microsatellites and single nucleotide polymorphisms) to construct a molecular linkage map based on co-dominant markers for an interspecific backcross population using a wild rice (Oryza glumaepatula) from Brazil and performed a comparative analysis with other interspecific maps. The comparative analysis revealed a Spearman correlation index of 0.86 for marker order conservation to a previous map constructed for an interspecific cross using the same wild parent. Approximately 90% of markers common to other interspecific maps kept the same order. These results indicate that it will be possible to generate a unique genetic map using the wild donor and that it may be a helpful tool for breeding programs because plants derived from different interspecific populations can be rapidly scanned using markers associated with useful wild traits.

Genetics of flower size and nectar volume in Petunia pollination syndromes

The two related Petunia species, P. axillaris and P. integrifolia, are sympatric at various locations in South America but do not hybridise. Divergent pollinator preferences are believed to be in part responsible for their reproductive isolation. The volume of nectar produced and several components of flower morphology might contribute to pollinator-dependant reproductive isolation. In this study, we aimed to identify the genetic changes underlying the quantitative differences observed between these two Petunia species in flower size and nectar volume. We mapped quantitative trait loci (QTL) responsible for the different phenotypes of P. axillaris and P. integrifolia in an inter-specific backcross population. QTL of small to moderate effect control the differences in flower size and volume of nectar. In addition, we observed strong suppression of meiotic recombination in Petunia, even between closely related species, which precluded a fine resolution of QTL mapping. Thus, our data suggest that flower size and nectar volume are highly polygenic. They are likely to have evolved gradually through pollinator-mediated adaptation or reinforcement, and are not likely to have been primary factors in early steps of pollinator isolation of P. axillaris and P. integrifolia.

A major QTL introgressed from wild Lycopersicon hirsutum confers chilling tolerance to cultivated tomato (Lycopersicon esculentum)

Many plants of tropical or subtropical origin, such as tomato, suffer damage under chilling temperatures (under 10 degrees C but above 0 degrees C). An earlier study identified several quantitative trait loci (QTLs) for shoot turgor maintenance (stm) under root chilling in an interspecific backcross population derived from crossing chilling-susceptible cultivated tomato (Lycopersicon esculentum) and chilling-tolerant wild L. hirsutum. The QTL with the greatest phenotypic effect on stm was located in a 28 cM region on chromosome 9 (designated stm 9), and enhanced chilling-tolerance was conferred by the presence of the Lycopersicon hirsutum allele at this QTL. Here, near-isogenic lines (NILs) were used to verify the effect of stm 9, and recombinant sub-NILs were used to fine map its position. Replicated experiments were performed with NILs and sub-NILs in a refrigerated hydroponic tank in the greenhouse. Sub-NIL data was analyzed using least square means separations, marker-genotype mean t-tests, and composite interval mapping. A dominant QTL controlling shoot turgor maintenance under root chilling was confirmed on chromosome 9 using both NILs and sub-NILs. Furthermore, sub-NILs permitted localization of stm 9 to a 2.7 cM interval within the original 28 cM QTL region. If the presence of the L. hirsutum allele at stm 9 also confers chilling-tolerance in L. esculentum plants grown under field conditions, it has the potential to expand the geographic areas in which cultivated tomato can be grown for commercial production.

Coordinated genetic regulation of growth and lignin revealed by quantitative trait locus analysis of cDNA microarray data in an interspecific backcross of eucalyptus.

Phenotypic, genotypic, and transcript level (microarray) data from an interspecific backcross population of Eucalyptus grandis and Eucalyptus globulus were integrated to dissect the genetic and metabolic network underlying growth variation. Transcript abundance, measured for 2,608 genes in the differentiating xylem of a 91 (E. grandis x E. globulus) x E. grandis backcross progeny was correlated with diameter variation, revealing coordinated down-regulation of genes encoding enzymes of the lignin biosynthesis and associated methylation pathways in fast growing individuals. Lignin analysis of wood samples confirmed the content and quality predicted by the transcript levels measured on the microarrays. Quantitative trait locus (QTL) analysis of transcript levels of lignin-related genes showed that their mRNA abundance is regulated by two genetic loci, demonstrating coordinated genetic control over lignin biosynthesis. These two loci colocalize with QTLs for growth, suggesting that the same genomic regions are regulating growth, and lignin content and composition in the progeny. Genetic mapping of the lignin genes revealed that most of the key biosynthetic genes do not colocalize with growth and transcript level QTLs, with the exception of the locus encoding the enzyme S-adenosylmethionine synthase. This study illustrates the power of integrating quantitative analysis of gene expression data and genetic map information to discover genetic and metabolic networks regulating complex biological traits. (Sequence data for this article have been deposited with the EMBL/GenBank data libraries under accession numbers CB 967505 to CB 968059; CD 667988 to CD 670002; CD 670004; CD 670097; CD 670101 to CD 670112; and CD 670114 to CD 670137.)

Detection of QTLs associated with shoot wilting and root ammonium uptake under chilling temperatures in an interspecific backcross population from Lycopersicon esculentum ×L. hirsutum

The genetic basis for shoot wilting and root ammonium uptake under chilling temperatures was examined in an interspecific backcross (BC1) population derived from Lycopersicon esculentum Mill. cv T5 and wild Lycopersicon hirsutum f. typicum accession LA1778. The chilling sensitivity of shoot wilting and ammonium uptake was evaluated in four replicated cuttings from each of 196 BC1 plants. Wilting was evaluated at two different times: 2 hours (wilting 2 h) and 6 hours (wilting 6 h recovery) after root exposure to 4°C. The BC1 plants were genotyped with 89 polymorphic RFLP markers, and composite interval mapping was used to detect quantitative trait loci (QTLs). Three QTLs, one each on chromosomes 5, 6 and 9, were detected for wilting 2 h. The presence of a L. hirsutum (H) allele at the QTL on chromosomes 5 and 9 decreased wilting, while the H allele at the QTL on chromosome 6 increased wilting. To analyze plant recovery from wilting at 6 h, subsets of the BC1 population were selected, based on phenotype and genotype, because not all plants wilted at 2 h. The phenotype subset (wilting 6 h-PS) included plants that wilted to a greater degree at 2 h, and the genotype subsets included plants carrying specific allelic compositions at the QTL for wilting 2 h on chromosomes 5 (wilting 6 h-GS-ch5), 6 (wilting 6 h-GS-ch6), and 9 (wilting 6 h-GS-ch9). On chromosome 6, a QTL was located that was associated with three subsets (wilting 6 h-PS, wilting 6 h-GS-ch5 and wilting 6 h-GS-ch9), while on chromosome 7 a QTL was detected with two subsets (wilting 6 h-PS and wilting 6 h-GS-ch5). Three additional QTLs were detected within a single subset: chromosome 1 (wilting 6 h-GS-ch6), chromosome 11 (wilting 6 h-GS-ch5) and chromosome 12 (wilting 6 h-GS-ch9). The presence of the H allele at the QTL on chromosomes 7 and 12 had a positive effect, enhancing recovery from wilting, while the H allele at the other QTL had a negative effect. Three traits were used to evaluate the chilling sensitivity of root ammonium uptake: ammonium uptake before a chilling episode, ammonium uptake after the chilling episode, and the relative inhibition of uptake (difference in uptake rates before and after chilling divided by the rate before chilling). One QTL was detected on chromosome 3 for the rate before chilling and one on chromosome 6 for the relative inhibition of ammonium uptake. Our results demonstrate that shoot wilting and ammonium uptake under chilling are controlled by multiple QTLs.

Development of a microsatellite framework map providing genome-wide coverage in rice (Oryza sativa L.)

Ninety-four newly developed microsatellite markers were integrated into existing RFLP framework maps of four rice populations, including two doubled haploid, a recombinant inbred, and an interspecific backcross population. These simple sequence repeats (SSR) were predominantly poly(GA) motifs, targetted because of their abundance in rice. They were isolated from a previously described sheared library and a newly constructed enzyme-digested library. Differences in the average length of poly(GA) tracts were observed for clones isolated from the two libraries. The length of GA motifs averaged 21 repeat units for clones isolated from the Tsp-509-digested library, while motifs averaged 17 units for clones from the sheared library. There was no evidence of clustering of microsatellite markers near centromeres or telomeres. Mapping of the 94 newly developed markers as well as of 27 previously reported microsatellites provided genome-wide coverage of the 12 chromosomes, with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16–20 cM.

Genomic localization of tomato genes that control a hypersensitive reaction to Xanthomonas campestris pv. vesicatoria (Doidge) dye.

Xanthomonas campestris pv. vesicatoria causes bacterial spot, one of the most serious diseases of tomatoes. The lycopersicon esculentum accession 'Hawaii 7998' is the only reliable source of resistance to race 1 strains of the pathogen. This resistance is associated with a hypersensitive reaction controlled by multiple nondominant genes. The inoculated area becomes fully necrotic 24 hr after inoculation in 'Hawaii 7998,' whereas full necrosis is observed 5 and 4 days after inoculation in the susceptible species L. pennellii (LA 716) and their F1, respectively. An interspecific backcross population, using 'Hawaii 7998' as the recurrent parent, was analyzed to determine the linkage relationships between the resistance genes and 135 molecular marker loci. The range of responses of the BC1 population included those of the parents. Linkage to a hypersensitive response factor was assessed by comparing the rates of necrosis development between homozygous and heterozygous plants at 8 hr-intervals. Three factors that affect the hypersensitive response of 'Hawaii 7998' were detected. One factor is on the short arm of chromosome I, another on the long arm of chromosome I, and a third on the long arm of chromosome 5. These factors appeared to act independently and to have additive effects.

Saturated molecular map of the rice genome based on an interspecific backcross population.

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F2 population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.

ANALYSIS OF RFLP LOCI IN WALNUTS: INHERITANCE, INTRA- AND INTERSPECIFIC VARIATION, AND CULTIVAR IDENTIFICATION.

RFLP analysis was employed to study the inheritance of and genetic diversity identified by cloned walnut genomic probes. An interspecific backcross population of (J. hindsii × J. regia) × J. regia was used to determine the inheritance of thirty low copy number RFLP cloned probes. Of these probes, approximately 20% correspond to single copy loci, 40% correspond to single major loci with multiple minor loci, and 40% correspond to two major loci. Twenty of these probes were used to analyze variability within and between 13 walnut species (Juglans spp.). Substantial genetic variation was identified within many wild walnut species, while limited variation was identified within butternut (J. cinerea) and the widely cultivated English walnut (J. regia). Extensive polymorphism was found between walnut species, allowing a phylogenetic relationship of walnuts based upon RFLP markers to be developed. Identification of clonally propagated walnut cultivars by RFLP typing was readily performed in black walnut (J. nigra) accessions, was more difficult in English walnut accessions, and rarely possible in butternut accessions.