Grow-out Test Research Articles

Cucurbit Leaf Crumple Virus (CuLCrV) is Seed Transmitted in Yellow Squash (Cucurbita pepo L.).

The traditional understanding of begomovirus transmission exclusively through the whitefly Bemisia tabaci (Gennadius) has shifted with findings of seed transmission in some begomoviruses over the last decade. We investigated the seed transmissibility of cucurbit leaf crumple virus (CuLCrV), a bipartite begomovirus that has recently emerged as a severe constraint for yellow squash (Cucurbita pepo L.) production in the southeastern United States. We found high concentration of CuLCrV in male and female flower tissues of infected squash, including pollen and ovules. The virus infiltrated the fruit tissues including the endocarp and funiculus, which are anatomically positioned adjacent to the seeds. In seeds, CuLCrV was detected in the endosperm and embryo where there are no vascular connections, in addition to the seed coat. The virus was detected in the radicle, plumule, cotyledonary leaves, and true leaves of seedlings grown from seeds collected from infected fruits. In the grow-out test conducted, CuLCrV infections ranged from 17-56% of the progeny plants. To ensure that partial viral genome fragments were not being mistaken for replicative forms of the virus, we performed RCA ̶ PCR and amplified complete DNA-A and DNA-B of CuLCrV from seed tissues, seedlings, progeny plants of CuLCrV infected squash. Near complete DNA-A and DNA-B sequences of CuLCrV were recovered from a progeny plant, further validating our findings. Our results demonstrate that CuLCrV can translocate from vegetative to reproductive tissues of yellow squash, persist within the seeds, and subsequently induce infection in progeny plants, confirming its capacity for seed transmission.

Insight into selective breeding for robustness based on field survival records: New genetic evaluation of survival traits in pacific white shrimp (Penaeus vannamei) breeding line.

Survival can be considered a relatively ‘old’ trait in animal breeding, yet commonly neglected in aquaculture breeding because of the simple binary records and generally low heritability estimates. Developing routine genetic evaluation systems for survival traits however, will be important for breeding robust strains based on valuable field survival data. In the current study, linear multivariate animal model (LMA) was used for the genetic analysis of survival records from 2-year classes (BL2019 and BL2020) of pacific white shrimp (Penaeus vannamei) breeding lines with data collection of 52, 248 individuals from 481 fullsib families. During grow-out test period, 10 days intervals of survival data were considered as separate traits. Two survival definitions, binary survivability (S) and continuous survival in days (SL), were used for the genetic analysis of survival records to investigate; 1) whether adding more survival time information could improve estimation of genetic parameters; 2) the trajectory of survival heritability across time, and 3) patterns of genetic correlations of survival traits across time. Levels of heritability estimates for both S and SL were low (0.005–0.076), while heritability for survival day number was found to be similar with that of binary records at each observation time and were highly genetically correlated (r g > 0.8). Heritability estimates of body weight (BW) for BL2019 and BL2020 were 0.486 and 0.373, respectively. Trajectories of survival heritability showed a gradual increase across the grow-out test period but slowed or reached a plateau during the later grow-out test period. Genetic correlations among survival traits in the grow-out tests were moderate to high, and the closer the times were between estimates, the higher were their genetic correlations. In contrast, genetic correlations between both survival traits and body weight were low but positive. Here we provide the first report on the trajectory of heritability estimates for survival traits across grow-out stage in aquaculture. Results will be useful for developing robust improved pacific white shrimp culture strains in selective breeding programs based on field survival data.

Microsatellite marker based DNA fingerprinting of cotton (Gossypium spp.) hybrids and their parents

Cotton production in India by the vast majority comes from cotton hybrids whose genetic purity is of great significance in the seed production chain and trade. Therefore, there is a need to develop a rapid, reliable and reproducible technique to assess the genetic purity of cotton hybrids as the traditional, morphological traits-based ‘Grow-Out Test’ is resource intensive, time consuming, tedious and not an infallible procedure. In this regard, a study was planned to understand the genetic diversity among the hybrids and their parents and also to identify SSR markers for confirmation of genetic purity or hybridity. One intra-arboreum hybrid, CICR2 (DS 5 GMS × LD 327 Sel.), four intra-hirsutum hybrids viz., CSHH198 (CSH 19 × CSH 8), CSHH238 (SH 2379 9Y × PIL 8 Sel.), CSHH243 (CSH 2013 × CSH 43), CSHH1862 (GMS 16A × CB 33) and one hirsutum × barbadense hybrid, Phule 388 (RHC-006 × RHCb-001) along with their respective parental lines were selected for molecular characterization. Of the total 215 SSR markers surveyed, 60 markers conveyed polymorphism. The information conveyed by the polymorphic SSR markers was utilized to assess the molecular divergence among the study material. Maximum genetic dissimilarity of 0.66 was noted between Phule 388 and LD 327 (Sel.), and between RHC-006 and DS 5 (GMS). Minimum genetic dissimilarity of 0.07 was observed between CSHH1862 and CB 33, followed by 0.11 between CICR2 and DS 5 (GMS). SSR markers were highly efficient in capturing both intra-species and inter-species level diversity. The clustering and factorial analysis were  in congruence with the species of Gossypium. The diploid species genotypes were clustered separately and distinctly from the rest of the genotypes. All the hirsutum hybrids and their respective parents were found closely clustered. The inter-specific hybrid, Phule 388 along with its parents was found grouped closely. The genetic purity of the hybrids was confirmed using identified SSR markers [GH486, BNL1421, BNL3594, JESPR151 for G. hirsutum hybrids, CSHH198; GH486, BNL2449, JESPR151, TMB0436 for G. hirsutum hybrids, CSHH238; BNL2449, JESPR151, JESPR152 for G. hirsutum hybrid, CSHH243 and GH527, BNL3812, TMB1484, TMB1645, NAU1190, BNL3816 for inter-specific G. hirsutum × G. barbadense hybrid Phule 388]. The SSR markers were efficient in the analysis of hybrid seed purity. The information generated in the present study about genetic diversity and genetic purity testing will greatly facilitate quality seed production of these cotton hybrids and thus, better cotton production. Keywords: Cotton, DNA fingerprinting, Hybrids, Microsatellites, SSR markers, Diversity

SCREENING AND IDENTIFICATION OF GENETIC PURITY OF OKRA HYBRID [RANI] AND ITS PARENTAL LINES BASED ON MOLECULAR MARKERS

The present investigation was carried out to identify the genetic purity of okra hybrid and its parental line based on molecular markers. For the evaluation of genetic purity and varietal identification from collected Okra samples, molecular markers and polymerase chain reaction, as well as DNA fingerprinting approaches, were advocated. These samples were accumulated from field and nursery Adithya seeds Pvt. Ltd, Raipur (C.G) within the duration of study and research from January to July 2018. Genetic purity test over the conventional Grow-Out-Test (GOT) was achieved by using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) primers for identifying and emphasizing of hybrid. Using 3 lines of Okra cultivars, we analysed the products of PCR with RAPD and ISSR primers and discussed the design for constructing their Genetic Purity. Towards this study 64 random primers were screened between okra hybrid (Rani) and its parental lines [male line (206) and female line (189)]. Out of 64 primers only one primer (ISSR-B17898) showed polymorphic banding pattern between hybrid and its parental lines which helped in the identification and confirmation of okra hybrid. Hence, concluded that Genetic purity test is necessary for seed certification of different categories in seed multiplication chain both from agronomic as well as breeding point of view and proved to be explicit technology for researchers.

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds

BackgroundMost crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available.ResultsWe identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters.ConclusionsThis study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Genetic Purity Analysis of Maize (Zea mays L.) Hybrid Seed and Their Parents Produced in Different Seed Companies of Ethiopia

Genetic purity is one of the quality criteria required for successful seed production of maize. In hybrid seed production, genetic purity is contaminated due to out-crossing from other varieties or selfing events. In this study, Single nucleotide Polymorphisms (SNP) and Grow-out test (GOT) method was used for the objective to assess genetic purity of maize hybrid varieties with their parents produced by various seed growers in the country. Six three way cross hybrid (3WCH) maize varieties and seven single crosses were collected from different sources then planted with their parents in 2019. Genetic purity analysis by SNP revealed that 74% of an inbred line showed an acceptable genetic purity level (>95%). However, five inbred lines (CML395, A7033, F7215, SC22 and 124-b(113)) revealed heterogeneity >0.05 ranged from 0.13 to 0.20. All single crosses except A7033/F7215 and all the 3WCH varieties showed the genetic purity level ranging from 40 to 66% and 44 to 63% across seed sources, respectively. Based on GOT results, the level of type in single crosses and 3WCH variety across seed sources ranged from 7.1 to 46.4% and 3.6 to 35.7%, respectively. Generally, in the current result both SNP and GOT showed both heterogeneity and homogeneity of seed. This implies the presence of variation among seed producers in terms of producing high quality seed. Therefore, awareness should have to be given for each individual seed producers on quality seed production techniques and procedures that they have to follow. Similarly, genetic purity analysis could be conducted further in the seed system to provide error correction and to ensure seed quality assurance and control.

Genetic parameter estimates for resistance to Enteric Septicemia of Catfish on Pangasianodon hypophthalmus

Genetic parameters were estimated for resistant traits against Enteric Septicemia of Catfish (ESC) on the first generation of selection on striped catfish. In total, 8,207 and 5,838 individuals from 147 and 130 full-sib families were challenged by Edwardsiella ictaluri causing ESC on fingerlings and tested growth in pond accordingly. Harvest body weight (HW), length (HL) and survival (SURGROW) from grow-out test and ESC resistant traits such as binary alive-dead survival (SUR) and time to dead (TIME) at different truncated points from challenged test of fingerlings were recorded. Heritability for each trait and genetic correlations among these recorded traits were estimated. High heritabilities were found for HW (0.48) and HL (0.47), and mostly from medium to high values were estimated for SURGROW (0.23), SUR (0.13 - 0.40) and TIME (0.25 - 0.39). Genetic correlations among different truncated SUR and TIME traits were almost highly positive (0.57 - 0.99). Genetic correlations among different truncated SUR and TIME traits with HW and SURGROW were low positive (0.10 - 0.40). In summary, selection for ESC resistance would not negatively affect the growth in fingerling stage.

Assessment of Genetic Purity in Rice Using Polymorphic SSR Markers and Its Economic Analysis with Grow-Out-Test

Genetic purity is conventionally performed through grow-out-test (GOT) with morphological characters. Simple sequence repeat (SSR) markers are independent of G X E interaction. Herewith, 16 high yielding varieties of rice were analysed using 55 SSR markers for DNA fingerprinting and identification of genetic impurities: 14 were found to be polymorphic and amplified 48 alleles with an average of 3.43 alleles per each primer pair. The number of alleles amplified ranged from 2 to 6 and the size of the PCR products amplified from these 14 primer pairs ranged from 80 to 450 bp with polymorphic information content (PIC) from 0.14 (RM 346) to 0.99 (RM 5900). PICs at 0.5 or higher are highly informative SSR markers for genetic studies and the study reported (7/14) markers to be highly informative based on PIC values. Besides, the values of effective multiplex ratio, marker index and resolving power for the selected polymorphic primer pairs disclosed that the SSR markers used in the study are highly informative and could be potentially used for distinctness, uniformity and stability (DUS) testing, genetic purity analysis and DNA fingerprinting of rice varieties. Economic analysis of grow-out-test and SSR marker based genetic purity showed that the GOT process incurs Rs. 5.8 (INR) per seed (USD 0.07), while DNA-based marker analysis incurs Rs. 53.75 (INR) or $ 0.71 (USD).

Modeling of discharges from the breeding basins of rainbow trout fed with three extruded feeds (B, F1, and F2)

The main of this study is to estimate the maximum density of fish in the growing basins of rainbow trout (Oncorhynkiss Mykiss) species by mathematical equations, which will be used to introduce environmental concerns. Indeed, these equations make it possible to estimate the maximum density of fish fed by the extruded feed B and the two elaborated exruded feeds (F1 and F2) to achieve an optimal production without harming the environment. Note that the elements (PO43-, NH4 +, NO2-, NO3-, SM, COD) were analyzed during the rainbow trout grow-out test in the breeding station of 'Oum Er rbia. According to the results of density modeling as a function of physicochemical parameters, production can increase up to a density of ρF2 = 237.09 Kg / m3 without affecting the quality of the water while respecting the required standards. Our results show that the F2 formulation, containing 60% protein of animal origin and 40% of vegetable protein gives great zootechnical growth of rainbow trout with a low production of fish waste.

Seed genetic purity testing of F1 Benincasa hispida var. chieh-qua hybrids using SSR molecular marker analysis

To ensure that farmers can access high-quality seeds, it is essential to find a simple, rapid and accurate method to assess seed purity. In recent years, heterosis in chieh-qua has been widely applied to production. Using the whole genome sequence of chieh-qua, we designed simple sequence repeat (SSR) primers specific for chieh-qua. The parental lines of nine hybrids were screened using 200 SSR primers, seven of which exhibited polymorphisms. The bands were clear, stable and reproducible. We found dominant and co-dominant bands between the parents of the nine hybrids. The seven pairs of SSR primers were successfully used to assess genetic purity of the nine chieh-qua hybrids. The SSR molecular marker purity assessment results were consistent with the results obtained from a field grow-out test (GOT). However, the use of SSR markers provided a more accurate, reliable and faster method for seed purity testing than the GOT. We propose using SSR molecular marker technology to assess the genetic purity of chieh-qua hybrid seeds. With this method, the seed quality can be determined faster, which may help to accelerate the chieh-qua breeding process.

Assessment of genetic purity of bulked-seed of rice CMS lines using capillary electrophoresis.

Genetically pure cytoplasmic male sterile line (A-line) is essential to generate pure hybrid seeds in order to harness the yield heterosis in rice. Conventionally, seed purity testing is carried by grow-out test, which has many limitations. Seed purity assessments based on molecular markers reduce the time required for analysis significantly. However, it is very tedious as at least 200-400 seeds/seedlings are needed to be analyzed individually. An assay based on bulked-seed and molecular markers will be an ideal system. Keeping these points in view, in the present study, a co-dominant mitochondrial marker was used to test the purity of bulked parental line (A-line) seed utilizing CE system in a genetic analyzer. The results indicate that this method is very simple, accurate, and can be used to test purity of large number of samples rapidly in a cost-effective way compared to grow-out test and conventional molecular marker analysis.

A new seed-transmissible begomovirus in bitter gourd (Momordica charantia L.)

A begomovirus isolate collected from bitter gourd plants showing yellowing, puckering and stunting symptoms from Coimbatore district, Tamil Nadu, India was characterized. The full-length genome of the virus isolate was amplified by rolling circle amplification using phi29 DNA polymerase. The virus isolate exhibited 98% identity in the nucleotide sequence of DNA-A component with the Coccinia mosaic Virudhunagar virus (GenBank accession no. KY860899). The DNA-B component was very distinct and shared only 60% identity with the begomovirus, Coccinia mosaic Tamil Nadu virus (GenBank accession no. KM244719). The virus renamed as new species Bitter gourd yellow mosaic virus (BgYMV) was detected in seeds from infected plants and in the grow-out test seedlings by ELISA and virus-specific PCR. The seed infectivity was 79.16% and transmission rate to seedling was 32.05%. The virus titre as indicated by A405 absorption value was high (0.854–0.280) in different seed parts. Results clearly indicated seed transmission of the begomovirus, BgYMV.

Comparison of Traditional Grow-Out Test and DNA-Based PCR Assay to Estimate F1 Hybrid Purity in Cauliflower

Cauliflower ( Brassica oleracea ) is a cool-season crop belonging to the Brassicaceae family. Use of morphological differences between true-to-types and off-types in grow-out test (GOT) is the basic method for hybrid purity analysis. Traditional GOT is costly, tedious, time consuming and environment sensitive. To increase the speed and accuracy of genetic purity testing of hybrids, recent advances in DNA markers have shown promise. In the present study, the purity of cauliflower hybrid (NBH Tania-815) was assessed by traditional GOT and advanced molecular marker systems. The experiment was carried out by mixing 95% F1 hybrids with 5% female parents, individually in the sample sets of 400, 300, 200, 100, 80 and 40. For each sample size, PCR-based assay and GOT were carried out to check the hybrid purity. In the PCR-based assay, 220 pairs of SSR markers were screened, with 32 markers showing parental polymorphism including one codominant marker (BrgMS565). The purity level was determined by the co-dominant marker. A minimum sample size of 100 was standardized to confirm the hybrid purity as it showed the same result with that of higher sample sizes (200, 300 and 400). Hence, it is proposed that molecular marker-based hybrid purity assessment may serve as an effective substitute to traditional GOT.

DIGITAL IMAGE ANALYSIS USING FLATBED SCANNING SYSTEM FOR PURITY TESTING OF RICE SEED AND CONFIRMATION BY GROW OUT TEST

<p class="abstrakinggris">The common method used for purity testing of rice seed is human visual observation. This method, however, has a high degree of subjectivity when dealing with different rice varieties which have similar morphology. Digital image analysis with flatbed scanning for purity testing of rice seed was proposed by investigating the morphology of rice seeds and confirmation by grow out test (GOT) in the field. Two extra-long seed varieties were used in this study including a red rice Aek Sibundong and an aromatic rice Sintanur. The identification on 14 parameters of morphological characteristics indicated that only six parameters were correlated, i.e. area, feret, minimum feret, aspect ratio, round, and solidity. The purity of rice seed can be effectively determined using digital image analysis of spikelet color and shape. Based on the discriminant analysis of the digital image the recognition rate of rice seed purity was higher than 99.2% for shape and 93.55% for color. The method, therefore, has a potential to be used as a complement in rice seed purity testing to increase the accuracy of human visual method and it is more sensitive than GOT.</p>

Evidence of seed transmission of dolichos yellow mosaic virus, a begomovirus infecting lablab-bean in India.

Yellow mosaic disease in field bean caused by begomoviruses belonging to the family Geminiviridae is a major threat to the cultivation of crop in South India. Appearance of bright yellow mosaic symptom in emerging seedlings in farmers field was suggestive of seed transmission of the begomovirus associated with the disease which was investigated in the present study. The begomovirus causing yellow mosaic disease was identified as dolichos yellow mosaic virus (DoYMV) and the presence of DoYMV in matured seeds was confirmed by DAS-ELISA with OD value up to 3.268. In PCR with DoYMV specific primer (DoYMV-CP) the virus was detected in different parts of the seeds viz., seed coat, endosperm and embryo. In embryo the virus was detectable up to 100 per cent followed by endosperm (69.23%). When the non symptomatic leaves of 30days old grow-out test plants were subjected to DAS-ELISA, the virus was detected up to 46.6%. This is the first evidence of seed transmission of DoYMV.

Hybrid Purity Testing of Brassica rapa Using SSR Marker Technology

Thirteen Chinese cabbage (Brassica rapa) hybrid cultivars and 26 parental inbred lines were used as experimental materials to screen for primers producing hybrid and parental complementary bands and for primers with high polymorphism information contents and low genotype frequencies. A total of 18 pairs of core primers were designed to identify the purity of Chinese cabbage. There was no significant difference in the purity percentage measured between different loci of the same strain. The fingerprint obtained by the amplification of each locus could be used to identify purity to obtain an authentic purity percentage. Curve mapping and significance analyses were conducted using the purity percentage of eight different seed samples and confirmed a sampling seed number of 96. The results of the purity test were verified by comparison with the grow-out test (GOT) using molecular markers. In conclusion, the simple sequence repeat (SSR) detection system could be used for the rapid identification of the purity of the tested Chinese cabbage hybrids.

Fertility restorer (Rf) gene linked STMS marker: An efficient tool for testing the genetic purity of hybrid rice (Oryza sativa L.) seed

A fertility restorer (Rf) gene linked STMS marker, RM 6100 as well as Grow Out Test (GOT) were used for testing the genetic purity of hybrid seed of eight popular rice hybrids. The genetic purity ranging from 96.00% to 100.00%, could be determined through marker, whereas, it varied from 97.00% to 99.50% in GOT. The comparative assessment of both methods exhibited that molecular marker was more efficient than GOT, as it could detect 0.25–2.50% more impurities very precisely as compared to GOT. It suggests that a single Rf gene linked co-dominant STMS marker appeared to be a simple, precise and quick option over GOT for testing the purity of hybrid seed lots in short span of time.

Assessment of genetic purity in hybrid lines by field grow-out test and molecular markers

Tomato is a significant vegetable crop of special economic importance, ranking second to potato in the world. As genetic composition of a variety is its ultimate identity, achieving and maintaining genetic purity in crops is an important both from agronomic as well as breeding point of view. The present investigation was carried out to identify polymorphic primers where it can distinguish the hybrid parental line and utilization of this marker for further genotyping in the hybrid population. Towards this, 18 random decamer primers (RAPD), were screened between hybrid 2 parental lines, out of which only one primer (OPAO9) showed polymorphism and also study the tomato hybrid 2 parental lines consisted of 42 plants were analysed by using SSR Marker and only one primer (TGS0100) was found to be polymorphic which showed difference in banding pattern with hybrid 2 parental lines (male and female) and to check the genetic purity through protein analysis in the given tomato hybrid 2 parental lines (male and female). It was successfully reproduced in the hybrids. Thus, the present investigation shows the efficiency of RAPD and SSR marker as a molecular tool for genetic purity analysis. Hence, this marker was employed in the hybrid population. Furthermore DUS characters were also taken in the hybrids and parental lines. The results were correlating with the molecular data. Therefore, molecular GOT can be effectively employed in hybrid lots for purity checking. This could be better and fast technology than field GOT and it will be more useful for the quality assessment in the seed industries.

The Rate of Virus Spread in New Potato Cultivars in the North of Poland

The aim of this research was to assess the rate of increase in the level of tuber infection by PVY, PVM, and PLRV during three consecutive years of multiplication in the field for 17 new cultivars registered in Poland in 2009–2011 and for two cultivars not listed in the registry but popularly cultivated. The research was conducted in Bonin near Koszalin (north-western Poland) in 2010–2013. Tuber infection was assessed using DAS ELISA in a grow-out test in the winter-spring period. Among the 19 cultivars examined, eight had high resistance to PVY (above grade 8 on a scale of 1–9); during the 3-year research period, they were not infected. Also, cv. Gawin seemed more resistant than previously assumed. In contrast, cvs. Hermes and Sylvana, which were rated in the Netherlands as quite resistant, were clearly very susceptible (grade 3–4) in Polish conditions, as within 2 years 100% of tubers were infected with this virus. The greatest susceptibility to PVM was shown by cvs. Danuta and Stasia, 50% of which were infected, despite moderate exposure to the virus. Cultivars Zenia, Etiuda, Jubilat, and Viviana appeared very resistant to PVM as the number of infected tubers did not exceed 5%. PVM was not detected in the tubers of cvs. Bursztyn, Gustaw, and Legenda, which confirmed that these cultivars possess the Rm gene. Not all cultivars regarded susceptible (grade 3–4) to PLRV were infected. The virus was not found in tubers of cv. Bursztyn, while in cv. Hermes, assessed in the Netherlands as being quite resistant, almost 40% of tubers were infected.

박과 작물에 과일썩음병을 일으키는 Acidovorax citrulli 검출을 위한 nested-PCR 검사법 개발

The specific and sensitive nested-PCR method to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch on cucurbitaceae, was developed. PCR primers were designed from the draft genome sequence which was obtained with the Next Generation Sequencing of A. citrulli KACC10651, and the nested-PCR primer set (Ac-ORF 21F/Ac-ORF 21R) were selected by checking of specificity to A. citrulli with PCR assays. The selected nested-PCR primer amplified the 140 bp DNA only from A. citrulli strains, and detection sensitivity of the nested PCR increased 10,000 times of 1 PCR detection limit (10 ng genomic DNA/PCR). The nested PCR detected A. citrulli from the all samples of seed surface wash (external seed detection) of the artificially inoculated watermelon seeds with 10 cfu/ml and above population of A. citrulli while the nested PCR could not detected A. citrulli from the mashed seed suspension (internal seed detection) of the all artificially inoculated watermelon seeds. When the naturally infested watermelon seeds (10% seed infested rate with grow-out test) used, the nested PCR detected A. citrulli from 2 seed samples out of 10 replication samples externally and 5 seed samples out of 10 replication samples internally. We believe that the nested-PCR developed in this study will be useful method to detect A. citrulli from the Cucurbitaceae seeds.