DNAMarkMaker: streamlining ARMS and CAPS marker development from resequencing data with NGS short reads.

DNA markers serve as essential tools in breeding selection and genetic analysis. However, developing DNA markers can be time-consuming and labor-intensive due to the need to identify polymorphisms between cultivars/lines and to design suitable primers. To address these challenges, we have developed DNAMarkMaker, a tool designed to automate the process of primer design for Amplification Refractory Mutation System (ARMS) and Cleaved Amplified Polymorphic Sequences (CAPS) markers, utilizing resequencing data. One key feature of DNAMarkMaker is its user-friendly graphical user interface (GUI), ensuring its accessibility and ease of use, even for researchers not well-versed in bioinformatics. We confirmed DNAMarkMaker's applicability by developing DNA markers for rice, potato, and turnip-each representing distinct genome structures: homozygous diploid, heterozygous autotetraploid, and heterozygous diploid, respectively. DNAMarkMaker will contribute to the rapid and efficient development of DNA markers, accelerating breeding and genetic analysis in various crops.

Polyploid QTL-seq revealed multiple QTLs controlling steamed tuber texture and starch gelatinization temperature in sweetpotato.

Sweetpotato (Ipomoea batatas) includes diverse cultivars with flesh textures ranging from dry to moist. Moist-fleshed cultivars often contain starch with a lower gelatinization temperature (GT). To elucidate the genetic determinants of flesh texture and starch GT, we conducted a QTL analysis using F1 progenies obtained from a cross between dry-fleshed and moist-fleshed cultivars, 'Benikomachi' (BK) and 'Amahazuki' (AH), by using an updated polyploid QTL-seq pipeline. Flesh texture was assessed based on the wet area ratio (WAR) observed on the cut surface of steamed tubers, as progenies with dry and moist flesh exhibited low and high WAR values, respectively, demonstrating a strong correlation. Three QTLs were found to regulate the WAR. Notably, two AH-derived alleles at 4.30 Mb on Itr_chr05 and 21.01 Mb on Itr_chr07, along with a BK-derived allele at 2.89 Mb on Itr_chr15, were associated with increased WAR. Starch GT, which displayed no correlation with either flesh texture or WAR, was distinctly influenced by two QTLs: a GT-increasing BK-derived allele at 1.74 Mb on Itr_chr05 and a GT-decreasing AH-derived allele at 30.16 Mb on Itr_chr12. Consequently, we developed DNA markers linked to WAR, offering a promising avenue for the targeted breeding of sweetpotato with the desired flesh textures.

Detection and validation of QTLs for green stem disorder of soybean (Glycine max (L.) Merr.).

In mechanically harvested soybean, green stem disorder (GSD) is an undesirable trait that causes green-stained seeds, which are graded lower in Japan. To obtain DNA markers for reduced GSD, we conducted a quantitative trait locus (QTL) analysis for 2 years using F4 and F5 lines from a cross between 'Suzuotome' (less GSD) and 'Fukuyutaka' (more GSD). We validated the effect of a detected QTL for GSD by first identifying F4 or F5 plants in which one or more markers in the QTL region were heterozygous. The F5 or F6 progeny of each plant was used to form a pair consisting of two groups in which the QTL region was homozygous for either the 'Suzuotome' or 'Fukuyutaka' allele in a similar genetic background, and the two groups within each pair were compared for GSD. Over 3 years of testing, the 'Suzuotome' allele of a QTL on chromosome 6 was found to reduce the level of GSD. This novel QTL was mapped to the region around DNA marker W06_0130, and was not closely linked to QTLs for important agronomic traits including yield components. Using this marker, the low level of GSD from 'Suzuotome' could be conferred to 'Fukuyutaka' or other high-GSD cultivars.

PMEL p.Leu18del dilutes coat color of Kumamoto sub-breed of Japanese Brown cattle.

BACKGROUND: Coat color is important for registration and maintenance of livestock. Standard coat color of Kumamoto sub-breed of Japanese Brown cattle is solid brown, but individuals with diluted coat color have been observed recently. In this study, we attempted to identify polymorphism(s) responsible for coat color dilution by whole genome analysis. RESULTS: One of the diluted cattle possessed 7302 exonic polymorphisms which could affect genes' function. Among them, 14 polymorphisms in 10 coat color-related genes were assumed to be specific for the diluted cattle. Subsequent genotyping with three diluted cattle and 74 standard cattle elucidated that PMEL p.Leu18del was the causative polymorphism for coat color dilution in this sub-breed. Individuals with del/del type of this polymorphism showed diluted coat color, but coat color of heterozygotes were intermediate with various dilution rates. CONCLUSIONS: Coat color dilution of Kumamoto sub-breed was caused by PMEL p.Leu18del. The causative del allele has been detected in several genetically distant cattle breeds, suggesting that PMEL p.Leu18del can be used as a DNA marker to control cattle coat color.

A weak allele of OsNRAMP5 confers moderate cadmium uptake while avoiding manganese deficiency in rice.

Decreasing cadmium (Cd) concentrations in rice grains can effectively reduce potential risks to human health because rice is the major contributor to Cd intake in many diets. Among several genes involved in rice Cd accumulation, the loss of function of OsNRAMP5 is known to be effective in reducing grain concentration by inhibiting root uptake. However, disruption of this gene simultaneously decreases manganese (Mn) uptake because OsNRAMP5 is a major Mn transporter. With the aim of improving Mn uptake in OsNRAMP5 mutants while still restricting the grain Cd concentration below the upper limit of international standards, we identified a novel OsNRAMP5 allele encoding a protein in which glutamine (Q) at position 337 was replaced by lysine (K). The mutant carrying the OsNRAMP5-Q337K allele showed intermediate Cd and Mn accumulation between that of the wild-type and OsNRAMP5-knockout lines, and exhibited more resistance to Mn deficiency than the knockout lines. Different amino acid substitutions at position Q337 significantly affected the Cd and Mn transport activity in yeast cells, indicating that it is one of the crucial sites for OsNRAMP5 function. Our results suggest that the OsNRAMP5-Q337K allele might be useful for reducing grain Cd concentrations without causing severe Mn deficiency in rice cultivars through DNA marker-assisted breeding.

Start Codon Targeted (SCoT) markers for the assessment of genetic diversity in yeast isolated from Turkish sourdough.

Molecular markers are valuable tools for assessing the genetic variation in yeast. Here, we investigated the utility of SCoT markers for the genetic characterization of yeast strains at inter and intraspecies levels. A total of 345 endogenous yeast strains were isolated from 65 Type I sourdough samples collected from six different regions of Turkey. The seven SCoT primers produced 221 bands, of which 95.47% were polymorphic. Each primer could successfully differentiate species, supported by PIC and RP values. The ITS sequencing of isolates selected from the UPGMA dendrogram revealed that Saccharomyces cerevisiae predominated the microflora, followed by Kazachstania servazzii, K. humilis, Wickerhamomyces anomalus, Torulaspora delbrueckii, and Pichia kudriavzevii, respectively. The AMOVA revealed a high genetic variation between (49%) and within populations (51%) for S. cerevisiae. The high gene flow observed among S. cerevisiae populations suggests that it may have contributed to the geographical evolution of S. cerevisiae via the transportation of the sourdough samples. The different geographical origins were most likely to group separately on the UPGMA and PCoA. Saccharomyces cerevisiae strains from more distant populations generally displayed more significant genetic variation. SCoT markers can successfully be used alone or with the other existing DNA markers for DNA fingerprinting and analyzing the genetic variation between and within species.

Prevalence of causative agents of transmissive tick-borned ricketsious in the Crimean peninsula.

The paper presents the results of a study of the prevalence of Ixodid ticks - potential carriers of tick-borne rickettsiosis pathogens. Ectoparasites were collected in various natural and climatic zones of the Crimean Peninsula within the year 2016-2018. As a result of screening with the help of real-time PCR analysis (PCR-RT), a genetic marker (a section of the gltA gene) of the rickettsia group of tick-borne spotted fever was detected in ticks. The most common DNA marker of rickettsia was found in ticks in the eastern regions of the steppe zone - 50,6 %, in the north-western part of the steppe zone this value was 12,0 %. The least amount of rickettsia target DNA was detected in ticks collected in the mountain forest and south bank zones - 4,5 %. As a result of sequencing of positive DNA samples from fragments of the gltA, ompA, ompB, and sca4 genes, the species composition of rickettsias was established. The DNA of 8 species of rickettsia was identified: Circulation of three R. conorii, R. massiliae, R. sibirica subsp. mongolotimonae, R. slovaca, R. aeschlimannii, R. monacensis, R. helvetica, R. raoultii. R. massiliae, R. slovaca, and R. helvetica were established in the Crimean Peninsula for the first time. The peculiarities of the geographical distribution of the identified rickettsia species were determined, which was due to the spread of mites-carriers of pathogens. The revealed diversity of rickettsia species and their vectors, due to the isolation of the areas of the main feeding animals and the established routes of migratory birds, suggests the circulation of other rickettsia species on the territory of the Crimean Peninsula. The obtained results suggest that the diseases of tick-borne rickettsiosis in the Crimean Peninsula can be caused not only by R. conorii, as previously thought, but also by other types of rickettsii.

RNA-Seq-based DNA marker analysis of the genetics and molecular evolution of Triticeae species.

The release of high-quality chromosome-level genome sequences of members of the Triticeae tribe has greatly facilitated genetic and genomic analyses of important crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Due to the large diploid genome size of Triticeae plants (ca. 5 Gbp), transcript analysis is an important method for identifying genetic and genomic differences among Triticeae species. In this review, we summarize our results of RNA-Seq analyses of diploid wheat accessions belonging to the genera Aegilops and Triticum. We also describe studies of the molecular relationships among these accessions and provide insight into the evolution of common hexaploid wheat. DNA markers based on polymorphisms within species can be used to map loci of interest. Even though the genome sequence of diploid Aegilops tauschii, the D-genome donor of common wheat, has been released, the diploid barley genome continues to provide key information about the physical structures of diploid wheat genomes. We describe how a series of RNA-Seq analyses of wheat relatives has helped uncover the structural and evolutionary features of genomic and genetic systems in wild and cultivated Triticeae species.

Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

Molecular approaches for improving oil palm for oil.

The oil palm, originating from Africa, is the most productive oil crop species. Palm oil is an important source of edible oil. Its current global plantation area is over 23 million ha. The theoretical oil yield potential of the oil palm is 18.2 tons/ha/year. However, current average oil yield is only 3.8 tons/ha/year. In the past 100 years, conventional breeding and improvement of field management played important roles in increasing oil yield. However, conventional breeding for trait improvement was limited by its very long (10-20 years) phenotypic selection cycle, although it improved oil yield by ~10-20% per generation. Molecular breeding using novel molecular technologies will accelerate genetic improvement and may reduce the need to deforest and to use arable land for expanding oil palm plantations, which in turn makes palm oil more sustainable. Here, we comprehensively synthesize information from relevant literature of the technologies, achievements, and challenges of molecular approaches, including tissue culture, haploid breeding, mutation breeding, marker-assisted selection (MAS), genomic selection (GS), and genome editing (GE). We propose the characteristics of ideal palms and suggest a road map to breed ideal palms for sustainable palm oil.

Improvement of diagnostic markers for resistance to Globodera pallida and application for selection of resistant germplasms in potato breeding.

Occurrence of pale potato cyst nematode, Globodera pallida (Stone) Behrens, was first recorded in Japan in 2015. Among several control measures, cultivation of resistant potato (Solanum tuberosum L.) varieties is the most effective in cost and environmental impact. As no G. pallida-resistant varieties have yet been developed in Japan, great emphasis is being placed on screening of germplasm possessing the resistance and development of the resistant varieties. In this study, we first improved previously reported DNA markers linked to the G. pallida resistance loci (GpaIVs adg and Gpa5) and then used these to screen more than 1,000 germplasms to select several candidate germplasms with resistance. We performed inoculation testing on the selected candidates and identified several resistant germplasms to the Japanese G. pallida population. Furthermore, we developed a simultaneous detection method combining three DNA markers linked to G. pallida and Globodera rostochiensis (Wollenweber) Behrens resistance loci. We validated the ability of C237-I marker to select resistant allele of GpaIVs adg and predict the presence of resistance in a Japanese breeding population. Resistant germplasms identified in this study could potentially be used to develop G. pallida-resistant varieties. The marker evaluation methods developed in this study will contribute to the efficient development of resistant varieties.

Linkage analysis of rice bacterial blight resistance gene xa20 in XM6, a mutant line from IR24.

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is an important disease constraining rice (Oryza sativa L.) production worldwide. The XM6 line was induced by N-methyl-N-nitrosourea from IR24, an Indica cultivar that is susceptible to Philippine and Japanese Xoo races. XM6 was confirmed to carry a recessive gene named xa20, resistant to six Philippine and five Japanese Xoo races. The chromosomal gene location was found using 10 plants with the shortest lesion length in an F2 population consisting of 298 plants from a susceptible Japonica variety Koshihikari × XM6. Analysis using PCR-based DNA markers covering the whole rice genome indicated the gene as located on the distal region of the long arm of chromosome 3. The IKC3 line carries IR24 genetic background with Koshihikari fragment on chromosome 3 where a resistance gene was thought to be located. The F2 population from IKC3 × XM6 clearly showed a bimodal distribution separating resistant and susceptible plants. Further linkage analysis conducted using this F2 population revealed that xa20 is located within the 0.8 cM region flanked by DNA markers KIC3-33.88 (33.0 Mb) and KIC3-34.06 (33.2 Mb). This study yields important findings for resistance breeding and for the genetic mechanism of Xoo resistance.

Development of PCR-based DNA marker for detection of white carrot contamination caused by Y2 locus.

In carrot (Daucus carota L.), the taproot colors orange, yellow and white are determined mostly by the Y, Y2, and Or loci. One of the most severe issues in carrot seed production is contamination by wild white carrot. To evaluate the contamination ratio, easily detectable DNA markers for white carrot are desired. To develop PCR-based DNA markers for the Y2 locus, we have re-sequenced two orange-colored carrot cultivars at our company (Fujii Seed, Japan), as well as six white- and one light-orange-colored carrots that contaminated our seed products. Within the candidate region previously reported for the Y2 locus, only one DNA marker, Y2_7, clearly distinguished white carrots from orange ones in the re-sequenced samples. The Y2_7 marker was further examined in 12 of the most popular hybrid orange cultivars in Japan, as well as 'Nantes' and 'Chantenay Red Cored 2'. The Y2_7 marker showed that all of the orange cultivars examined had the orange allele except for 'Beta-441'. False white was detected in the orange-colored 'Beta-441'. The Y2_7 marker detected white root carrot contamination in an old open-pollinated Japanese cultivar, 'Nakamura Senkou Futo'. This marker would be a useful tool in a carrot seed quality control for some cultivars.

Parentage analysis of tea cultivars in Japan based on simple sequence repeat markers.

Tea cultivars have been bred by individual selection of landraces and by crossbreeding, but the validation of the parentage is limited. In this study, we performed parentage analysis of 79 tea cultivars in Japan based on SSR markers to confirm or identify the parent-offspring relationships among them. The effectiveness of nine SSR markers for parentage analysis was validated by comparing them to the existing cleaved amplified polymorphic sequence markers. The former markers were detectable more alleles than the latter. Simulation of parentage analysis of the tea cultivars predicted biparental origins for 12 cultivars ('Houshun', 'Mie ryokuhou no. 1', 'Surugawase', 'Tenmyo', 'Yamanoibuki', 'Harumidori', 'Koushun', 'Minekaori', 'Okumusashi', 'Saemidori', 'Sofu', and 'Toyoka'), in the first five of which candidate parents of yet-to-be-defined pedigree were newly identified. Comparisons of a total of 41 SSR genotypes confirmed the newly-identified parentages of 'Asahi' for 'Tenmyo', 'Rokurou' for 'Houshun', 'Surugawase', and 'Yamanoibuki', and 'Yamatomidori' for 'Mie ryokuhou no. 1'. The maternity of seven cultivars out of the 12 was also confirmed with chloroplast DNA sequences. Uniparental origins were confirmed for 25 cultivars. This parentage analysis has improved our knowledge of tea pedigrees and will aid in the development of new cultivars.

A putative AGAMOUS ortholog is a candidate for the gene determining ease of dehulling in Tartary buckwheat (Fagopyrum tataricum).

MAIN CONCLUSION: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.

A longitudinal study on morpho-genetic diversity of pathogenic Rhizoctonia solani from sugar beet and dry beans of western Nebraska.

BACKGROUND: Root and stem rot caused by Rhizoctonia solani is a serious fungal disease of sugar beet and dry bean production in Nebraska. Rhizoctonia root rot and crown rot in sugar beet and dry bean have reduced the yield significantly and has also created problems in storage. The objective of this study was to analyze morpho-genetic diversity of 38 Rhizoctonia solani isolates from sugar beet and dry bean fields in western Nebraska collected over 10 years. Morphological features and ISSR-based DNA markers were used to study the morphogenetic diversity. RESULTS: Fungal colonies were morphologically diverse in shapes, aerial hyphae formation, colony, and sclerotia color. Marker analysis using 19 polymorphic ISSR markers showed polymorphic bands ranged from 15 to 28 with molecular weight of 100 bp to 3 kb. Polymorphic loci ranged from 43.26-92.88%. Nei genetic distance within the population ranged from 0.03-0.09 and Shannon diversity index varied from 0.24-0.28. AMOVA analysis based on ΦPT values showed 87% variation within and 13% among the population with statistical significance (p < 0.05). Majority of the isolates from sugar beet showed nearby association within the population. A significant number of isolates showed similarity with isolates of both the crops suggesting their broad pathogenicity. Isolates were grouped into three different clusters in UPGMA based cluster analysis using marker information. Interestingly, there was no geographical correlation among the isolates. Principal component analysis showed randomized distribution of isolates from the same geographical origin. Identities of the isolates were confirmed by both ITS-rDNA sequences and pathogenicity tests. CONCLUSION: Identification and categorization of the pathogen will be helpful in designing integrated disease management guidelines for sugar beet and dry beans of mid western America.

Genetic study of diversity and blast resistance in Ethiopian rice cultivars adapted to different ecosystems.

Rice (Oryza sativa L.) has been considered one of the most important crops in Ethiopia. Landraces and improved accessions in Ethiopia were characterized on the basis of polymorphism data for SSR markers, and classified into two groups: I and II. Cluster I was further divided into two sub-clusters, Ia and Ib. Cluster Ia corresponded to Japonica-like type, Cluster Ib to Japonica type, and Cluster II to Indica type with some Indica-like type. Many landraces and improved varieties belonged to Cluster Ia. Superior landraces were included in Cluster Ib. Further categorization based on blast resistance demonstrated three groups: Clusters A, B1, and B2. Cluster A comprised accessions with relatively high resistance, whereas Clusters B1 and B2 included susceptible accessions. Most of the improved varieties were found in Cluster A. Superior landraces, X-Jigna classified into Ib or DNA type tended to be susceptible in Cluster B2 for blast resistance. These results demonstrated that traditional landraces preferred by farmers should be improved for disease resistance using blast-resistant varieties. In order to avoid hybrid sterility occurring in cross-hybridizing breeding between Indica and Japonica types, desirable parental accessions can be chosen within the same DNA cluster. The clustering information among accessions may be useful in breeding schemes for selection of counterparts in cross-breeding programs.

DNA marker-assisted evaluation of cooked bean hardness of three soybean progeny lines.

Cooked bean hardness is an important trait for the processing of soybean products such as nimame, natto, miso, and soy sauce. Previously, we showed that cooked bean hardness is primarily affected by the pectin methylesterase gene Glyma03g03360, and that calcium content has a secondary effect on this trait. To establish a simple and timely method for the evaluation of cooked bean hardness, primers of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were designed to detect a single-nucleotide polymorphism of Glyma03g03360 and subsequently used to evaluate three soybean progeny lines. The determined genotypes were compared to those identified using the cleaved amplified polymorphic sequence (CAPS) method. Seven out of 284 lines presented different genotypes, which were determined using the two methods: A genotypes were incorrectly assigned as heterozygous by CAPS, suggesting that ARMS-PCR is more reliable. Glyma03g03360 genotypes could be used to evaluate cooked bean hardness, except for intermediate values. Cooked bean hardness within the same genotype groups was significantly correlated with calcium contents. These findings indicate that ARMS-PCR is useful for a marker-assisted selection of soybean with soft-cooked beans and that calcium content may be used for additional selection.

DNA marker development by the allele-specific detection of powdery mildew resistance loci derived from Japanese domestic tobacco cultivar 'Kokubu'.

Japanese domestic tobacco (Nicotiana tabacum L.) cultivar 'Kokubu' shows high powdery mildew resistance controlled by recessive alleles at two loci, and these alleles have been widely used as a resource for powdery mildew resistance in tobacco breeding. However, the introduction of this trait by conventional breeding takes much work because of the requirement for test crosses with the parental strains and inoculation tests using active fungi to confirm the introduction of two recessive alleles during back-crossing. Recently, we found that powdery mildew resistance in 'Kokubu' is caused by splice site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. Here, we report DNA markers that detect mutations of the NtMLO1/2 genes based on the cleaved amplified polymorphic sequence (CAPS) or allele-specific polymerase chain reaction (AS-PCR) methods. These markers can be used as co-dominant markers that detect heterozygotes of the NtMLO genes at the seedling stage in back-crossed progenies, and will contribute to the simplification of breeding.

Marker-trait association for grain weight of spring barley in well-watered and drought environments.

Climate change will increase the effect of drought stress which is one of major constrains for barley production and productivity in Egypt. Identification and development new cultivars having a high drought tolerance combined with a high yield are urgently needed. In this study, a set of 60 highly homozygous and diverse barley genotypes was evaluated in well-watered (N) and dry (D) environments for two successive seasons. Five yield traits were scored; plant height, spike length, days to flowering, grain yield per spike (GYPS), and thousand kernel weight (TKW). High genetic variation was found among genotypes in all studied traits under N and D. High heritability for all traits was observed in both seasons. The drought susceptibility index (DSI) for GYPS and TKW was estimated to determine the tolerant and susceptible genotypes in both seasons. As a result, four spring barley genotypes were considered drought tolerant for TKW and GYPS in both seasons. A set of ten single sequence repeats primers, developed from wheat genome, were tested in the 60 genotypes. All SSR primers had a high polymorphism among the genotypes producing 82 marker alleles. Single marker analysis was performed for DSI, TKW, and GYPS in both seasons. Twenty QTLs were found to be associated with low DSI and high GYPS and TKW in N and D. The marker alleles associated with the 20 QTL were screened in the four tolerant genotypes. PNBYT15 included only one marker allele associated with one QTL, while, SCYT-28 included six marker alleles controlling nine QTL. The high genetic variation and heritability for the studied traits indicated that these traits could be used for selection for high yielding and drought tolerance. The four drought tolerant genotypes can be used for a further breeding program to improve drought tolerance in barley.