Comparative Analysis of SSR Markers Developed in Exon,Intron, and Intergenic Regions and Distributed in Regions Controlling Fruit Quality Traits in <Emphasis Type="Italic">Prunus</Emphasis> Species: Genetic Diversity and Association Studies

Simple sequence repeats (SSRs) are genome domains located in both coding and non-coding regions in eukaryotic genomes. Although SSRs are often characterized by low polymorphism, their DNA-flanking sequences could be a useful source of DNA markers, which could help in genetic studies and breeding because they are associated with genes that control traits of interest. In this study, 56 genotypes from different Prunus species were used, including peach, apricot, plum, and almond (already phenotyped for several agronomical traits, including self-compatibility, flowering and ripening time, fruit type, skin and flesh color, and shell hardness). These Prunus genotypes were molecularly characterized using 28 SSR markers developed in exons, introns, and intergenic regions. All these genes were located in specific regions where quantitative trait loci (QTLs) for certain fruit quality traits were also located, including flowering and ripening times and fruit flesh and skin color. A sum of 309 SSR alleles were identified in the whole panel of analyzed cultivars, with expected heterozygosity values of 0.61 (upstream SSRs), 0.17 (exonic SSRs), 0.65 (intronic SSRs), and 0.58 (downstream SSRs). These values prove the low level of polymorphism of the exonic (gene-coding regions) markers. Cluster and structural analysis based on SSR data clearly differentiated the genotypes according to either specie (for the four species) and pedigree (apricot) or geographic origin (Japanese plum). In addition, some SSR markers mainly developed in intergenic regions could be associated with genes that control traits of interest in breeding and could therefore help in marker-assisted breeding. These findings highlight the importance of using molecular markers able to discriminate between the functional roles of the gene allelic variants.     

DNA marking of some quantitative trait loci in the cultivated edible mushroom Pleurotus ostratus (Fr.) Kumm

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.     

DNA marking of some quantitative trait loci in the cultivated edible mushroom <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> (Fr.) Kumm.

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.     

Mapping quantitative trait loci for a common bean (Phaseolus vulgaris L.) ideotype.

Breeding a model plant that encompasses individual traits thought to enhance yield potential, known as ideotype breeding, has traditionally focused on phenotypic selection of plants with desirable morphological traits. Broadening this breeding method to the molecular level through the use of molecular markers would avoid the environmental interactions associated with phenotypic selection. A population of 110 F5 recombinant inbred lines (RILs), derived from the cross between WO3391 and 'OAC Speedvale', was used to develop a genetic linkage map consisting of 105 random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and sequence-tagged site (STS) markers. The map has a total length of 641 cM distributed across 8 linkage groups (LGs). Five of them were aligned on the core linkage map of bean. Twenty-one quantitative trait loci (QTLs) were identified over three environments for eight agronomic and architectural traits previously defined for a bean (Phaseolus vulgaris L.) ideotype. The QTLs were mapped to seven LGs with several regions containing QTLs for multiple traits. At least one QTL was located for each trait and a maximum of four were associated with lodging. Total explained phenotypic variance ranged from 10.6% for hypocotyl diameter to 45.4% for maturity. Some of the QTLs identified will be useful for early generation selection of tall, upright, high-yielding lines in a breeding program.     

Identification of Associated SSR Markers for Yield Component and Fiber Quality Traits Based on Frame Map and Upland Cotton Collections

Detecting QTLs (quantitative trait loci) that enhance cotton yield and fiber quality traits and accelerate breeding has been the focus of many cotton breeders. In the present study, 359 SSR (simple sequence repeat) markers were used for the association mapping of 241 Upland cotton collections. A total of 333 markers, representing 733 polymorphic loci, were detected. The average linkage disequilibrium (LD) decay distances were 8.58 cM (r² > 0.1) and 5.76 cM (r² > 0.2). 241 collections were arranged into two subgroups using STRUCTURE software. Mixed linear modeling (MLM) methods (with population structure (Q) and relative kinship matrix (K)) were applied to analyze four phenotypic datasets obtained from four environments (two different locations and two years). Forty-six markers associated with the number of bolls per plant (NB), boll weight (BW), lint percentage (LP), fiber length (FL), fiber strength (FS) and fiber micornaire value (FM) were repeatedly detected in at least two environments. Of 46 associated markers, 32 were identified as new association markers, and 14 had been previously reported in the literature. Nine association markers were near QTLs (at a distance of less than 1–2 LD decay on the reference map) that had been previously described. These results provide new useful markers for marker-assisted selection in breeding programs and new insights for understanding the genetic basis of Upland cotton yields and fiber quality traits at the whole-genome level.     

Identification of molecular markers associated with mite resistance in coconut (Cocos nucifera L.).

Coconut mite (Aceria guerreronis 'Keifer') has become a major threat to Indian coconut (Co?cos nucifera L.) cultivators and the processing industry. Chemical and biological control measures have proved to be costly, ineffective, and ecologically undesirable. Planting mite-resistant coconut cultivars is the most effective method of preventing yield loss and should form a major component of any integrated pest management stratagem. Coconut genotypes, and mite-resistant and -susceptible accessions were collected from different parts of South India. Thirty-two simple sequence repeat (SSR) and 7 RAPD primers were used for molecular analyses. In single-marker analysis, 9 SSR and 4 RAPD markers associated with mite resistance were identified. In stepwise multiple regression analysis of SSRs, a combination of 6 markers showed 100% association with mite infestation. Stepwise multiple regression analysis for RAPD data revealed that a combination of 3 markers accounted for 83.86% of mite resistance in the selected materials. Combined stepwise multiple regression analysis of RAPD and SSR data showed that a combination of 5 markers explained 100% of the association with mite resistance in coconut. Markers associated with mite resistance are important in coconut breeding programs and will facilitate the selection of mite-resistant plants at an early stage as well as mother plants for breeding programs.     

中俄茄子种质资源遗传多样性研究

利用25对SSR分子标记和24个表型性状对105份中俄茄子材料进行遗传多样性分析。表型变异分析结果表明:24个表型性状在中俄材料间均表现出了不同程度的多样性,但是同一性状在中俄材料中多样性不同;主成分分析可将24个表型性状概括为果形因子、颜色因子、果实外观因子、叶片形态因子、果萼刺和花药条纹6个指标,其中果实特征占主要成分;利用UPGMA法进行聚类,遗传相似系数在0.4~0.8之间,平均值0.6。25对多态性SSR标记,扩增出122个条带,含有等位基因82个,其中有效等位数24.8个,PIC值为0.3~0.7。分子聚类的遗传相似系数在0.5~1之间,平均值是0.7。表型聚类和分子标记聚类的结果相似,105份茄子种质资源间的类群划分与地理来源之间没有直接关系,但与茄子的果实性状有一定的相关性。     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Genetic diversity, linkage disequilibrium, and association mapping analyses of peach (Prunus persica) landraces in China

The genetic diversity, population structure, and linkage disequilibrium (LD) of peaches are greatly important in genome-wide association mapping. In the current study, 104 peach landrace accessions from six Chinese geographical regions were evaluated for fruit and phenological period. The accessions were genotyped with 53 genome-wide simple sequence repeat (SSR) markers. All SSR markers were highly polymorphic across the accessions, and a total of 340 alleles were detected, including 59 private alleles. Of the six regions studied, the northern part of China as well as the middle and lower reaches of the Changjiang River were found to be the most highly diverse genetically. Based on population structure analysis, the peaches were divided into five groups, which well agreed with the geographical distribution. Of the SSR pairs in these accessions, 18.07% (P?<?0.05) were in LD. The mean r ² value for all intrachromosomal loci pairs was 0.0149, and LD decayed at 6.01?cM. The general linear model was used to calculate the genome-wide marker-trait associations of 10 complex traits. The traits include flesh color around the stone, red pigment in the flesh, flesh texture, flesh adhesion, flesh firmness, fruit weight, chilling requirement, flowering time, ripening time, and fruit development period. These traits were estimated by analyzing the 104 landraces. Many of the associated markers were located in regions where quantitative trait loci (QTLs) were previously identified. Peach association mapping is an effective approach for identifying QTLs and may be an alternative to QTL mapping based on crosses between different lines.     

Association analysis and population structure of flowering-related traits in super sweet corn inbred lines with simple sequence repeat markers

This study assessed the genetic and phenotypic variation of 90 super sweet corn inbred lines and performed association analyses of six agronomical traits using 100 simple sequence repeats (SSR), ultimately detecting 590 alleles, with an average of 5.90 alleles per locus. The average genetic diversity and Polymorphism information content values were 0.54 and 0.50, respectively. Using population structure analysis, inbred lines were divided into three major groups and one admixed group. Association analysis was performed with a general linear model using a Q-matrix (Q GLM) and a mixed linear model using Q and K-matrices (Q + K MLM). Q GLM found 33 marker-trait associations involving 20 SSR markers that were associated with six agronomic traits. Q + K MLM identified four marker-trait associations involving three markers that were associated with traits of days of tasseling (DT) and days of silking (DS). Q GLM and Q + K MLM detected four significant marker-trait associations (SMTAs), with a level of significance of P < 0.01. In overlapping SMTAs, phi051 was associated with DT, umc1708 was associated with DS, and umc2341 was associated with two traits: DT and DS. The detection of loci associated with traits in this study may provide greater opportunities to improve quality by marker-assisted selection (MAS). Finally, these results will be helpful for breeders in choosing parental lines for crossing combinations as well as markers for using MAS in super sweet corn breeding programs in Korea.     

Genome-wide association mapping of yield components and drought tolerance-related traits in cotton

Drought causes serious yield losses in cotton production throughout the world. Association mapping allows identification and localization of the genes controlling drought-related traits which will be helpful in cotton breeding. In the present study, genetic diversity analysis and association mapping of yield and drought traits were performed on a panel of 99 upland cotton genotypes using 177 SSR (simple sequence repeat) markers. Yield parameters and drought tolerance-related traits were evaluated for two seasons under two watering regimes: water-stressed and well-watered. The traits included seed cotton yield (SCY), lint yield (LY), lint percentage (LP), water-use efficiency (WUE), yield potential (YP), yield reduction (YR), yield index (YI), drought sensitivity index (DSI), stress tolerance index (STI), harmonic mean (HM), and geometric mean productivity (GMP). The genotypes with the least change in seed cotton yield under drought stress were Zeta 2, Delcerro, Nazilli 87, and DAK 66/3 which were also the most water-use efficient cultivars. The average genetic diversity of the panel was 0.38. The linkage disequilibrium decayed relatively rapidly at 20–30 cM (r²?≥?0.5). We identified 30 different SSR markers associated with the traits. Fifteen and 23 SSR markers were linked to the traits under well-watered and water-stress conditions, respectively. To our knowledge, most of these quantitative yield and drought tolerance-associated loci were newly identified. The genetic diversity and association mapping results should facilitate the development of drought-tolerant cotton lines with high yield in molecular breeding programs.     

Development and mapping of Oryza glumaepatula-derived microsatellite markers in the interspecific cross Oryza glumaepatula x O. sativa

Wild germplasm of domesticated crops is a source of genetic variation little utilized in breeding programs. Interspecific crosses can potentially uncover novel gene combinations that can be important for quantitative trait analysis. The combined use of wide crosses and genetic maps of chromosomal regions associated with quantitative traits can be used to broaden the genetic basis of rice breeding programs. Oryza glumaepatula is a diploid (AA genome) wild rice species native from South and Central America. A genetic map was constructed with 162 PCR-based markers (155 microsatellite and 7 STS markers) using a backcross population derived from the cross O. glumaepatula, accession RS-16 from the Brazilian Amazon Region x O. sativa BG-90-2, an elite rice inbred line. The map included 47 new SSR markers developed from an O. glumaepatula genomic library enriched for AG/TC sequences. All SSR markers were able to amplify the O. sativa genome, indicating a high degree of SSR flanking region conservation between O. glumaepatula and O. sativa species. The map covered 1500.4 cM, with an average of one marker every 10 cM. Despite some chromosomes being more densely mapped, the overall coverage was similar to other maps developed for rice. The advantage to construct a SSR-based map is to permit the combination of the speed of the PCR reaction, and the codominant nature of the SSR marker, facilitating the QTL analysis and marker assisted selection for rice breeding programs.     

Association mapping of six yield-related traits in rapeseed (Brassica napus L.)

Yield is one of the most important traits for rapeseed (Brassica napus L.) breeding, but its genetic basis remains largely ambiguous. Association mapping has provided a robust approach to understand the genetic basis of complex agronomic traits in crops. In this study, a panel of 192 inbred lines of B. napus from all over the world was genotyped using 451 single-locus microsatellite markers and 740 amplified fragment length polymorphism markers. Six yield-related traits of these inbred lines were investigated in three consecutive years with three replications, and genome-wide association studies were conducted for these six traits. Using the model controlling both population structure and relative kinship (Q + K), a total of 43 associations (P < 0.001) were detected using the means of the six yield-related traits across 3 years, with two to fourteen markers associated with individual traits. Among these, 18 markers were repeatedly detected in at least 2 years, and 12 markers were located within or close to QTLs identified in previous studies. Six markers commonly associated with correlated traits. Conditional association analysis indicated that five of the associations between markers and correlated traits are caused by one QTL with pleiotropic effects, and the remaining association is caused by linked but independent QTLs. The combination of favorable alleles of multiple associated markers significantly enhances trait performance, illustrating a great potential of utilization of the associations in rapeseed breeding programs.     

Evaluation of the best linear unbiased prediction method for breeding values of fruit-quality traits in citrus

Fruit-quality trait improvement is an important objective in citrus breeding; however, fruit breeding programs often accumulate highly unbalanced phenotypic records, which are a serious obstacle in comparing and selecting genotypes. The best linear unbiased prediction (BLUP) method can be used to overcome these difficulties, but few fruit breeding programs have adopted the method, and to our knowledge, the method has not yet been used to predict breeding values of traits based on pedigree information and genetic correlations between traits in citrus. Accordingly, we used the BLUP method to predict the breeding values of nine fruit-quality traits (fruit weight, fruit skin color, fruit surface texture, peelability, flesh color, pulp firmness, segment firmness, sugar content, and acid content) utilizing phenotypic records collected over several years as part of the citrus breeding program conducted at the Kuchinotsu branch of the National Institute of Fruit Tree Science in Japan. Although the accumulated phenotypic records were highly unbalanced, the BLUP method was able to predict the breeding values of all 2122 genotypes (111 parental cultivars and 2011 F₁ offspring from 126 pair-cross families), as well as estimates of several genetic parameters, including narrow-sense heritability and phenotypic and genotypic correlations. These findings demonstrate the utility of the BLUP method in citrus crossbreeding and provide predicted breeding values, which can be used to select superior genotypes in the Kuchinotsu Citrus Breeding Program and related genetic selection endeavors.     

Population structure and marker–trait associations for pomological traits in peach and nectarine cultivars

Marker–trait associations based on populations from controlled crosses have been established in peach using markers mapped on the peach consensus map. In this study, we explored the utility of unstructured populations for association mapping to determine useful marker–trait associations in peach/nectarine cultivars. We used 94 peach cultivars representing local Spanish and modern cultivars from international breeding programs that are maintained at the Experimental Station of Aula Dei, Spain. This collection was characterized for pomological traits and was screened with 40 SSR markers that span the peach genome. Population structure analysis using STRUCTURE software identified two subpopulations, the local and modern cultivars, with admixture within both groups. The local Spanish cultivars were somewhat less diverse than modern cultivars. Marker–trait associations were determined in TASSEL with and without modelling coefficient of membership (Q) values as covariates. The results showed significant associations with pomological traits. We chose three markers on LG4 because of their proximity to the endoPG locus (freestone–melting flesh) that strongly affects pomological traits. Two genotypes of BPPCT015 marker showed significant associations with harvest date, flavonoids and sorbitol. Also, two genotypes of CPPCT028 showed associations with harvest date, total phenolics, RAC, and total sugars. Finally, two genotypes of endoPG1 showed associations with flesh firmness and total sugars. The analysis of linkage disequilibrium (LD) revealed a high level of LD up to 20 cM, and decay at farther distances. Therefore, association mapping could be a powerful tool for identifying marker–trait associations and would be useful for marker-assisted selection in peach breeding.     

Grapevine Phenological Quantitative Trait SSR Genotyping Using High-Throughput HRM-PCR Analysis

Discrimination among grapevine varieties based on quantitative traits, such as flowering, veraison and ripening dates is crucial for variety selection in the context of climate change and in breeding programs. These traits are under complex genetic control for which 6 linked SSR loci (VVS2, VVIn16, VMC7G3, VrZAG29, VMC5G7, and VVIB23) have been identified. Using these markers in HRM-PCR analysis, we assessed genetic diversity among a large collection of 192 grapevine varieties. The grapevine germplasm used encompasses the majority of Greek vineyard with 181 varieties, 3 prominent foreign varieties and 11 varieties of Palestinian origin. The SSR markers used were highly polymorphic, displaying unique melting curves for unusually higher number of samples than generally observed in SSR analysis. This prompted us to examine sequence composition for selected samples and found that variation present as SNPs in the flanking sequences of SSR motifs was responsible for the observed polymorphism. Hence, HRM-PCR proved to be a tool of higher analytical power to distinguish genotypes surpassing the discrimination power of conventional gel-based SSR analysis. The study provides a better understanding of genetic variation of SSR marker loci associated to phenological traits in grapevine varieties, signifying an analytical methodology that may be of higher discrimination power in detection of polymorphism for utilization in breeding programs.     

Linkage Map Construction and Quantitative Trait Loci Analysis for Bolting Based on a Double Haploid Population of Brassica rapa

Early bolting of Chinese cabbage （Brassica rapa L.） during spring cultivation often has detrimental effects on the yield and quality of the harvested products. Breeding late bolting varieties is a major objective of Chinese cabbage breeding programs. In order to analyze the genetic basis of bolting traits, a genetic map of B. rapa was constructed based on amplified fragment-length poiymorphism （AFLP）, sequence-related amplified poiymorphism （SRAP）, simple sequence repeat （SSR）, random amplification of polymorphic DNA （RAPD）, and isozyme markers. Marker analysis was carried out on 81 double haploid （DH） lines obtained by microspore culture from F1 progeny of two homozygous parents： B. rapa L. ssp. pekinensis （BY） （an extra-early bolting Chinese cabbage line） and B. rapa L. ssp. rapifera （MM） （an extra-late bolting European turnip line）. A total of 326 markers including 130 AFLPs, 123 SRAPs, 16 SSRs, 43 RAPDs and 14 isozymes were used to construct a linkage map with 10 linkage groups covering 882 cM with an average distance of 2.71 cM between loci. The bolting trait of each DH line was evaluated by the bolting index under controlled conditions. Quantitative trait loci （QTL） analysis was conducted using multiple QTL model mapping with MapQTL5.0 software. Eight QTLs controlling bolting resistance were identified. These QTLs, accounting for 14.1% to 25.2% of the phenotypic variation with positive additive effects, were distributed into three linkage groups. These results provide useful information for molecular marker-assisted selection of late bolting traits in Chinese cabbage breeding programs.     

Identification and mapping of fruit rot resistance QTL in American cranberry using GBS

Sustainability of the cranberry industry is threatened by widespread and increasing losses due to fruit rot in the field as well as increasing restrictions on fungicide inputs. Breeding for resistance offers a partial solution but is challenging because fruit rot is caused by a complex of pathogenic fungi that can vary by location and from year to year. We identified four genetically diverse germplasm accessions that exhibit broad-spectrum fruit rot resistance under field conditions. Three of these accessions were used in biparental crosses to develop four populations segregating for resistance. Genotyping by sequencing was used to generate single-nucleotide polymorphism (SNP) markers for development of high-density genetic maps and quantitative trait locus (QTL) analyses. Nineteen QTL associated with fruit rot resistance, distributed on nine linkage groups, were discovered in our populations. Three of these QTL matched previously reported fruit rot resistance QTL. Four newly reported QTL found on linkage group 8 (Vm8), which explain between 21 and 33% of the phenotypic variance for fruit rot, are of particular interest to our breeding program. The populations described herein were also phenotyped for other horticulturally important traits, and QTL associated with yield and berry weight were identified. These QTL provide markers for candidate gene discovery and for future breeding efforts to enhance and pyramid disease resistance and other traits into elite horticultural backgrounds.     

Diversity Assessment of Turkish Maize Landraces Based on Fluorescent Labelled SSR Markers

Landraces of maize represent a valuable genetic resource for breeding and genetic studies. Since 1970, landraces have been collected from all over Turkey, but the genetic diversity represented in this collection is still largely unknown. In this study, a sample of 98 landraces sampled from 45 provinces of Turkey was assessed genotypically at 28 simple sequence repeat (SSR) loci and phenotypically for 19 morphological traits. The landraces varied significantly for all the latter traits. A total of 172 SSR alleles were detected, giving a mean of 6.21 alleles per locus. The genetic distance between pairs of landraces ranged from 0.18 to 0.63, with a mean of 0.35. Positive and negative correlation exists among different morphological and agronomic traits. Positive association among different traits showed that improvement of one character may simultaneously improve the other desired trait. Based on UPGMA dendrogram and Neighbor-Net (NNET) analyses from both morphological traits and SSR data, respectively, it is obvious that maize landraces from the same geographical region were often placed in different clusters, indicating that grouping based on genetic parameters was not closely related to the geographic origin. The wide diversity present in Turkish maize landraces could be used as genetic resource in designing maize breeding program for developing new cultivars adapted to different geographic and climatic conditions, and may also contribute to worldwide breeding programs.     

RAPD markers for the identification of yield traits in tomatoes under heat stress via bulked segregant analysis

Tomato production is limited to a large extent by climates with high temperatures. Yield-related traits in tomatoes are generally thought to be quantitative inherited traits that are significantly affected by variation in environmental factors. Breeding for heat tolerance is restricted due to the complexity of the traits. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers linked to heat-tolerance traits in tomatoes under heat stress. Forty-three F(7) recombinant inbred lines (RILs) derived from a wild cross between CL 5915 (heat-tolerant) and L4422 (heat-sensitive) were obtained and scored for flower number, fruit number, fruit set, fruit weight and yield under screen house conditions during the hot (summer) season of 2003. The distributions of average fruit weight, fruit number, fruit set and yield in the F(7) population were strongly skewed towards heat susceptibility, characteristic of L4422. Significant positive correlation was observed between fruit weight and yield, and between fruit number, fruit set and yield. However, the increase in yield and fruit set by selecting for large flower number may be fairly minor due to non-significant correlation between these traits. Selecting for flower number may not be a useful tool for improving yield under heat stress. A total of 200 RAPD primers were screened, among which 14 were identified as associated with heat-tolerance using bulk segregant analysis (BSA) based on the F(7) population grown in a heat-stressed environment. Some RAPD markers were unique to one specific trait, and others were linked to two traits. All markers for heat tolerance related traits had positive gene effects as a result of the contribution of the CL5915 gene that bolstered these traits. One hundred F(2) plants derived from the same parents (CL5915xL4422) were grown in the same location during the summer of 2004 to test for the stability and reliability of the 14 markers identified. Selection for the desired heat-tolerance genotypes corresponded well with targeting heat tolerance traits using the selected heat tolerance RAPD markers identified. Marker-assisted selection (MAS) for heat tolerance may be efficiently conducted by selecting individuals that contain high fruit number, high fruit weight, and high yield markers (P06, X01, D06 and D11), which would thus facilitate conventional breeding using CL5915 as a donor parent.