Breeding for Resistance
View abstracts in the conference PDF booklet.
A Novel Putative Oligogalacturonan-Binding Receptor-Like Kinase is Involved in Quantitative Downy Mildew Resistance in Cucumber
Jeroen Berg¹, Freddy Hermans², Wim Vriezen², Yuling Bai¹, Henk Schouten¹
¹Wageningen UR, Wageningen, 6709 PB, Netherlands. ²Bayer Crop Science Vegetable Seeds, Nunhem, 6083 AB, Netherlands
Cucurbit Downy Mildew (DM), caused by the obligate biotrophic oomycete Pseudoperonospora cubensis is a major foliar disease of cucumber. Cucumber accession PI 197088 was previously shown to be one of the most promising donors for DM resistance. The resistance in PI 197088 is controlled by multiple quantitative trait loci (QTLs), each with a relatively small effect. We recently fine-mapped one of the QTLs in order to identify potential causal genes. We combined fine mapping data with RNAseq and whole genome resequencing. In one of the mapped QTL regions, we identified a locus that contains several Receptor Like Kinase genes (RLK). Interestingly, we found evidence for the presence of a novel RLK gene at this locus in resistant genotypes. In susceptible genotypes, including the reference genotype ‘Chinese Long 9930’, this novel gene has a 551 base pair (frameshift) deletion, and was therefore not correctly predicted during the annotation of the cucumber genome. In order to functionally characterize the novel RLK gene, we cloned it from resistant and susceptible genotypes, and transiently expressed both alleles in leaves of Nicotiana benthamiana by infiltration with Agrobacterium tumefaciens. We found that whereas the functional allele of the gene triggered a strong defense reaction consisting of necrosis of infiltrated tissue, the loss-of-function allele had no effect. Comparing the RLK genes at this locus, our novel RLK gene is very similar (>90% identical) to the neighboring RLK genes concerning the predicted extracellular domain, whereas the predicted intracellular kinase domains are very different (<30%). We hypothesize, therefore, that the different RLK genes at the locus might be responsive to similar extracellular stimuli, but might trigger different intracellular signaling cascades. Interestingly, the kinase domain of the novel RLK gene is homologous to several Arabidopsis thaliana RLK genes with roles in disease resistance. The RLK genes in this locus contain predicted extracellular oligogalacturonan- binding domains. Oligogalacturonan, a breakdown product from pectin polymers in the plant cell wall, was previously shown to be a damage-associated-molecular-pattern (DAMP) which can elicit strong defence responses in plants. We are currently investigating whether genotypes with/without this novel oligogalacturonan-binding RLK gene react differently to oligogalacturonan.
Resequencing of Bottle Gourd Germplasm and Using QTL-seq to Fine-Map PRSV-W Resistance in Bottle Gourd (Lagenaria sinceraria)
Bidisha Chanda¹, Kai-Shu Ling¹, Shan Wu², Zhangjun Fei²
¹U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA. ²Boyce Thompson Institute, Ithaca, NY, USA
Bottle Gourd, a native of Africa is widely grown in the East Asian countries for food, medicine and decorative purposes. It is also used as an important rootstock to improve cold tolerance and disease resistance in other cucurbits, like watermelon and melon. Viral diseases are considered a major threat to cucurbit crop productions worldwide. The most prevalent viruses are aphid-transmitted Papaya ringspot virus watermelon strain (PRSV-W) and Zucchini yellow mosaic virus (ZYMV). Commercially available bottle gourd cultivars are susceptible to these viral diseases. Traditional breeding takes years to develop a disease-resistant cultivar. With the recent advancement in the sequencing technologies, we aim to accelerate the molecular breeding for trait improvement. We recently sequenced the bottle gourd genome and used genotyping-by-sequencing to map the PRSV-W resistance locus in an F2 population. A dominant monogenic locus Prs was mapped in a 317.8 Kb region on chromosome 1 still containing 39 annotated genes. In the current study, through QTLseq analysis on pooled libraries with the 10 most PRSV resistant or susceptible individuals from six F3 populations, we aim to fine-map the Prs locus and to identify the candidate resistance gene for PRSV-W. In addition, through screening the USDA collections of bottle gourd germplasm, significant variations in the level of resistance to PRSV-W were identified in 154 Plant Introductions (PI) evaluated. Through genome re-sequencing (GBS) of these PI accessions, we intend to use genome-wide association studies to confirm genes or SNPs linking to the PRSV-W resistance in bottle gourd. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Advances in Molecular Breeding for Disease Resistance in Cucumber
Xingfang Gu, Shengping Zhang, Han Miao, Ye Wang, Kailiang Bo, Bingyan Xie
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
Cucumber is an economically important worldwide vegetable crop. However, serious losses in yield and quality are a frequent occurrence due to a range of diseases. Many of these are not able to be controlled by conventional means, so the development of resistant cultivars offers a way to increase both production and quality. This could be achieved by traditional breeding, but a more useful approach would be to use marker-assisted selection (MAS). In 2009, CAAS- IVF completed the cucumber whole genome sequence, thus providing numerous markers for MAS. In this paper two genetic maps were constructed: a high-density map made from 248 SSR loci, and an ultra-high-density map made from 116,710 SNPs. The inheritance of resistance to several important diseases was identified and mapped. Cucumber scab resistance was found to be controlled by a single dominant gene, Ccu, which was mapped to Chr2 with an accuracy rate of 100% for the flanking marker, Indel01. For resistance to Fusarium wilt, the peak marker SSR17631 had an accuracy rate of 87.88% for detecting the major QTL, Foc2.1. Four QTLs related to powdery mildew resistance (pm5.1, pm5.2, pm5.3, and pm6.1) and five QTLs related to downy mildew resistance (dm1.1, dm5.1, dm5.2, dm5.3, and dm6.1) were detected. Fine mapping of resistance to Watermelon mosaic virus indicated that it is controlled by the single recessive gene, Csa6G421660, and the accuracy of the SNP marker WMVSNP1 has an accuracy rate of 100%. Inheritance analysis of resistance to papaya ringspot virus indicated control by a single recessive gene flanked by two SSR markers, SSR11-177 and SSR11-1 on Chr6. Marker SSR11-1 has an accuracy of 94% in resistant lines. One major QTL, cmv6.1, which was delimited by SSR9-56 and SSR11-177, explained 31.7% of the phenotypic variation. Five QTLs related to resistance to Gummy stem blight (gsb-s1.1, gsb-s2.1, gsb-s6.1, gsb-s6.2, and gsb-s6.3) were identified. The major locus, gsb-s6.2, accounted for the highest phenotypic variation of 22.7% and was flanked by markers SSR04083 and SSR02940. Using these markers, more than 30 new cucumber cultivars with multiple resistance have been developed that are grown on more than 700,000 ha throughout China.
Relative Susceptibility of Commercial Watermelon Varieties to Powdery Mildew and Phytophthora Fruit Rot
Chandrasekar Kousik¹, Jennifer Ikerd¹, Mihir Mandal²,³
¹U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA. ²ORISE Participant, U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC. ³Department of Biology, Claflin University, Orangeburg, SC
Powdery mildew (PM, Podosphaera xanthii) and Phytophthora fruit rot (Phytophthora capsici) have been occurring frequently in recent years in commercial watermelon fields and growers routinely apply fungicides to manage these two diseases. Both these diseases are known to cause significant yield reduction. The current study was conducted in 2014, 2015 and 2016 to determine the relative susceptibility of twenty six watermelon varieties (seeded and seedless) and three pollenizers to PM and fruit rot in South Carolina. USVL677-PMS, which is highly susceptible and USVL531-MDR, which is resistant to PM and fruit rot were included as controls. A randomized complete block design with three replications was used for planting each year. Naturally occurring PM infection on plants were rated on a 0-10 scale of increasing disease severity. Mature fruit were harvested from all the variety plots and inoculated with a 7-mm plug from an actively growing colony of P. capsici. Inoculated fruit were kept on wire shelves in a large chamber (Temperature 26±2 °C) with high relative humidity (≥90%) and free moisture to enhance disease development and prevent drying of agar plugs. Five days after inoculation the lesion diameter and sporulation intensity were recorded. During all three years USVL677-PMS was the most susceptible to PM (70%, 3 year mean disease severity) with highest area under disease progress curves. In comparison, USVL531-MDR (2%) was very resistant to PM. The commercial pollenizers, SP5, SP6 and Lion were all resistant to PM (4.2%). Among the red fleshed varieties, Suprema, (seedless variety) was relatively resistant (20%) compared to other seeded and seedless varieties. Most of the seeded varieties evaluated (e.g. Malali, Black Mama, Mickey Lee) were highly susceptible to PM, however, some were relatively less susceptible (e.g. Declaration) under field conditions. Except for the resistant control USVL531-MDR (0.9 cm lesions) and the pollenizer Lion (2.5 cm), all the other watermelon varieties were highly susceptible to Phytophthora fruit rot (7-13 cm). Although resistance sources exist in watermelons, the resistance has not been bred into commercial varieties. There is a critical need to develop varieties with high levels of resistance to PM and/or Phytophthora fruit rot. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Novel Source of Resistance to Fusarium Wilt Race 1 Identified in Citron Melon
Sandra Branham, Amnon Levi, Melanie Katawczik, W. Patrick Wechter
U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA
The fungal pathogen Fusarium oxysporum Schlechtend.: Fr. f. sp. niveum (Fon) causes Fusarium wilt of watermelon, which limits production world-wide through yield loss and plant death. Thus far, the only genetic source(s) of host-plant resistance to Fon race 1 have been identified on the distal end of chromosome 1 in both Citrullus lanatus (cultivated watermelon) and Citrullus amarus (citron melon). The breakdown of host-plant resistance can be inhibited through gene pyramiding of multiple sources of resistance into a common genetic background. Cultivated watermelon and citron melon readily cross so disease resistance alleles found in citron melon can be introgressed into cultivated watermelon. Here, we identified a novel source of resistance through QTL mapping using segregating F2:3 and recombinant inbred line C. amarus populations. The Fon race 1 resistance QTL (qFon1-9) may confer non-race specific resistance to Fusarium wilt as it collocates on chromosome 9 with a previously identified QTL (qFon2-9) for Fon race 2 resistance in citron melon. Thus, qFon1-9 provides watermelon breeders with a valuable genetic resource for the improvement of Fusarium wilt resistance in watermelon.
Linkage Map Construction and QTL Analysis for Cucurbit chlorotic yellows virus Resistance in Melon
Yoichi Kawazu¹, Mitsuhiro Sugiyama¹, Koichiro Shimomura¹, Syoichi Maeda², Yoichi Yamato², Shigenori Ueda³, Shiori Okuda4,5, Mitsuru Okuda6
¹Institute of Vegetable and Floriculture Science, NARO, Tsu, Mie 514-2392, Japan. ²Kyushu Okinawa Agricultural Research Center, NARO, Kurume, Fukuoka 839-8503, Japan. ³Hokkaido Agricultural Research Center, NARO, Kasai, Hokkaido 082-0081, Japan. 4National Institute for Environmental Studies, Tsukuba, Ibaraki 305-0053, Japan. 5Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8634, Japan. 6Central Region Agricultural Research Center, NARO, Tsukuba, Ibaraki 305-8666, Japan
Cucurbit chlorotic yellows virus (CCYV) infects many Cucurbitaceae species including important crops like melon, cucumber, watermelon and pumpkin. CCYV is transmitted by the sweet potato whitefly, Bemisia tabaci. CCYV infection induces chlorotic spots and yellowing symptoms on melon leaves and causes significant decrease in sugar contents of melon fruits, which reduces their market value. A melon accession with resistance to CCYV has been reported, but there is no genetic information on its CCYV resistance. In this study, we generated two F2 populations using the resistant accession and two susceptible cultivars. Genetic linkage maps were constructed using SSR and In-Del markers, and each map contained 12 linkage groups. CCYV resistance of each F2 plant was evaluated in a greenhouse using B. tabaci carrying CCYV. QTL analysis was performed using each F2 population, and one locus for CCYV resistance was detected on chromosome 1. Our results will accelerate developing DNA markers for marker- assisted selection for CCYV resistance in melon breeding.
New Sources of Resistance to Phytophthora Crown and Root Rot in Cucurbita moschata
Chandrasekar Kousik¹, Jennifer Ikerd¹, Mihir Mandal¹,²
¹U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA. ²Department of Biology, Claflin University, Orangeburg, SC, USA
Winter and crook neck squash (Cucurbita moschata) are important vegetable crops grown and consumed in most states in the USA. Among C. moschata, butternut type squash are the most popular and widely used across USA. Many C. moschata lines are also used to develop interspecific hybrid rootstocks for grafting watermelon in parts of Asia. However, most commercially available C. moschata varieties are highly susceptible to crown and root rot caused by the oomycete pathogen Phytophthora capsici which is prevalent in southeastern USA. As part of an USDA, NIFA SCRI grant, we evaluated all the available plant introductions (PIs) of C. moschata (319 PIs) for resistance to P. capsici. Four-week-old plants growing in 6.3- cm square pots were inoculated with 104 zoospores from a local South Carolina (SC) isolate of P. capsici. Plants were rated for disease severity two weeks after inoculation using a 0-5 rating scale. The experiments were conducted twice. Twelve potential new sources of resistance (e.g. Grif 1738, PI 438724, PI 438778, PI 442280) to crown rot caused by the local SC isolate of P. capsici were identified. Variability in resistance reaction among plants within a PI was also observed, and not all plants were resistant. Further evaluation of S1 and S2 generation from the most resistant plants indicated that highly resistant plants could be selected from the 12 PI to develop lines for use in breeding programs. In a previous study done in Florida (FL), five sources of resistance to P. capsici (e.g. PI 176531, PI 458740) were identified in C. moschata by evaluating 119 accessions (Chavez et al., 2011, HortScience 46(4):536-540). Interestingly, Grif 1738 which was resistant to the isolate from SC in the current evaluation was susceptible to a FL isolate (Chavez et al., 2011), indicating the potential for existence of host specific races of P. capsici based on C. moschata. This also suggests that new sources of resistance should be evaluated against isolates from other states. These new sources of resistance can be utilized for developing new crown and root rot resistant rootstocks for watermelon grafting and for developing resistant varieties for human consumption. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project), and by USDA, NIFA, SCRI Vegetable Grafting grant award 2016-1498-08.
Genetics of Resistance to Powdery Mildew in Watermelon Line USVL608-PMR
Chandrasekar Kousik¹, Jennifer Ikerd¹, Mihir Mandal², Phillip Wadl¹
¹U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA. ²ORISE Participant Sponsored by U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA
Powdery mildew (PM) of watermelon (Citrullus lanatus) and other cucurbits caused by Podosphaera xanthii is a major factor limiting production in greenhouse and open field. In recent years, occurrence of PM has been increasing on watermelon across the United States, and commercial watermelon cultivars with resistance are rare. Four PM resistant germplasm lines with broad resistance to isolates from South Carolina, Georgia, Florida, California and New York were developed from plant introductions and released in 2018 by USDA ARS. All four lines have red-pink flesh and hybridize readily with commercial cultivars and inbred lines. One of these, USVL608-PMR (S6), a red fleshed watermelon line with high levels of resistance to PM was used as the female parent (P1) and crossed with USVL677-PMS which is highly susceptible (P2). The parents, F1, backcrosses to both parents (BC1, BC2) and a large F2 population were inoculated with a local isolate of PM and assessed for disease severity on a 0-10 scale of increasing disease severity. All susceptible parent (USVL677-PMS) plants were rated >7 [mean disease severity (DS) = 94%], whereas most resistant parent (USVL608-PMR) plants were rated as 1 (DS=2.5%). Majority of the BC1 plants were rated ≤2 and considered as resistant. Of the 466 F2 plants, 221 were rated ≤2 (DS=3.1%). Of the 76 BC2 plants, 23 were rated ≤2 (DS=2.9%). Chi- square analyses of the observed segregation of phenotypes for the F2 plants indicated that two genes control PM resistance with a good fit for a 7:9 resistance to susceptibility ratio. The proposed model for this ratio is two genes with one recessive for high resistance and one dominant for high resistance. This is supported by a backcrossing segregation ratio of 1:3. We have observed some highly and moderately resistant plants in the F2 indicating the cumulative effect of the two genes. QTL-seq analysis on the extremes from the F2 populations and RNA-seq analysis of the parents during PM infection are being conducted to identify the chromosomal regions involved in resistance. USVL608-PMR will serve as a useful source to incorporate PM resistance into commercial cultivars. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCB
QTL-seq of Young Fruit Resistance to Phytophthora capsici in Cucumber
Ying-Chen Lin, Rebecca Grumet
Michigan State University, East Lansing, Michigan, USA
Phytophthora fruit rot caused by Phytophthora capsici, a soil-borne oomycete pathogen, can be a devastating disease for cucumber production. As young fruit are especially susceptible, the objective of this work is to identify quantitative trait loci (QTL) associated with young fruit resistance using QTL-seq analysis. A cucumber accession, PI 109483, was previously identified as a source of young fruit resistance and a resistant breeding line, MSU109483-53, was developed. Crosses were made between the susceptible pickling type cucumber breeding line, Gy14, and two resistant MSU 109483-53-derived lines: an S6 generation line, B5, and a doubled haploid line, DH A4-3. The P. capsici isolate, Barley’s 1, was used in all experiments. In the summer of 2017, F2 progeny of Gy14 x B5 (n=397) along with parental lines and F1 were grown in the field. To facilitate accurate phenotyping, plants were trellised to reduce wounding due to removal of soil during the cleaning process and lessen the possibility of contamination from other pathogens. The second population, F2 progeny of Gy14 x DH A4-3 (n=222), along with the parental lines and F1 were screened in the greenhouse in the spring of 2018. Replicate harvests were performed for each experiment providing a total of 10-50 fruits for each plant, and allowing replicated scoring for each individual. The normal distribution of disease scores of the F2 population in each experiment suggested that young fruit resistance is a quantitative trait. Individuals with extreme resistant and susceptible phenotypes were selected from each population for QTL-seq analysis. QTL-seq analysis was performed by QTLseqr using two statistical approaches: QTL-seq and G’. A major QTL was identified on chromosome 6 in the 2017 experiment, potential additional QTL were located on chromosomes 1 and 3. In the 2018 experiment, QTLs were found on chromosomes 1, 2, 3, and possibly 7. A second F2 population of Gy14 x DH A4-3 (n=362) was grown in the field and phenotyped in 2018 summer. The third population will provide additional data to identify the genomic regions associated with young fruit resistance.
Breeding for Resistance to Phytophthora Crown Rot in Squash
Geoffrey Meru, Vincent Michael, Yuqing Fu
University of Florida-Tropical Research and Education Center, Homestead, FL, USA
Breeding cultivars resistant to Phytophthora crown rot is an important goal for squash breeders worldwide. The disease is particularly severe in south Florida where heavy rainfall coupled with hurricane-driven flooding results in rapid establishment and distribution of P. capsici spores across grower fields, leading to significant crop losses. Over the last decade, the cucurbit- breeding program at the University of Florida has identified resistance to Phytophthora crown rot in C. pepo, C. moschata and C. lundelliana. This paper reports efforts to introgress resistance from breeding lines #181761-36P (C. pepo) and #394-1-27-12 (C. moschata) into various cultivar groups of summer and winter squash using conventional and molecular breeding methods. Crosses (R x S) were initiated to develop F1, F2 and backcross progeny of #181761-36P x Acorn/ Crookneck and #394-1-27-12 x Butterbush. These populations were screened for resistance to crown rot and a sub-population of 50 lines each for C. pepo and C. moschata exhibiting high resistance to crown rot were identified. These lines are currently undergoing further selection for disease resistance and horticultural performance. To facilitate efficient discrimination of resistant and susceptible genotypes, a QTL Seq. approach was employed to identify SNP markers associated with crown rot resistance in C. moschata using an F2 population (#394-1-27- 12 x Butterbush). In total, 1,334,918 SNPs were found among the parents, F1 and resistant and susceptible bulks. Of these SNPs, 10.24% were located in the coding regions. SNP-index for the resistant and susceptible bulks were determined across the genome. Delta-SNP values were determined by subtracting SNP-index values of susceptible bulk from those of the resistant bulk. Five candidate markers (delta SNP>0.6) located in coding regions were identified as potential causal SNPs for crown resistance in #394-1-27-12. Efforts to validate these SNPs in independent populations are currently underway.
Gummy Stem Blight Resistance in Melon: Screening, Inheritance Pattern and Development of Molecular Markers
Md Zahid Hassan, Md Abdur Rahim, Hoy-Taek Kim, Jong-In Park , Ill-Sup Nou
Department of Horticulture, Sunchon National University, Suncheon, 57922, Republic of Korea
Gummy stem blight (GSB) is one of the most destructive and economically important, soil borne diseases of melon caused by the ascomycete fungus, Didymella bryoniae throughout the world. In Korea, however, no GSB resistant genotype has been reported yet. We aimed to identify GSB resistant melon germplasm and to develop molecular markers linked to GSB resistance. We identified six resistant melon genotypes including inbred lines (PI 482399, PI 140471, PI 136170 and PI 420145) and cultivars (Asia Papaya and Supra) against GSB based on bioassay and molecular screening. Further, we developed 168 F2 plants from the F1 of a cross between the susceptible Cornell ZPPM 339, and the resistant PI 482399 lines. A 3:1 ratio of susceptible and resistant genotypes was observed in the F2 population, indicating control by a single recessive gene. Nucleotide-binding site leucine-rich repeat (NBS-LRR) genes confer resistance against insects and diseases in cucurbits including melon. We cloned and sequenced the TIR-NBS-LRR- type resistance gene MELO3C022157, located on melon chromosome 9, from resistant and susceptible lines. Sequence analysis revealed deletions in the first intron, a 2-bp frameshift deletion from the second exon, and a 7-bp insertion in the 4th exon of the resistant line. We developed two insertion/deletion (InDel) markers, GSB9-kh-1 and GSB9-kh-2, found in the first intron of MELO3C022157 linked to GSB resistance. We validated these markers with the F2 population and inbred lines. These InDels may be used to facilitate marker-assisted selection of GSB resistance in melon. However, functional analysis of is needed to confirm the frameshift mutation. This research was supported by the Golden Seed Project under Grant no. 213007-05- 2-CG100.
Identification of New Resistance Sources in Watermelon for Anthracnose Race 2
Takshay Patel, Todd C. Wehner
North Carolina State University, Raleigh, NC, USA
Anthracnose (Colletotrichum orbiculare) is reappearing as a major problem on watermelon (Citrullus lanatus). Watermelon anthracnose has three races, 1, 2 and 3. Earlier studies screened small sets of PIs for resistance to these races, but not the whole USDA, NPGS germplasm collection. The objective of this study was to identify accessions resistant to C. orbiculare race 2. The available watermelon germplasm collection of 1408 PI accessions was screened for resistance to anthracnose race 2 using seedlings in a greenhouse. The study had 1408 accessions in 2 replications, with a spore concentration of 105 sp/ml. Seedlings were rated three times at 3, 5 and 7 days post-inoculation. Data were analyzed with a repeated measure analysis using mixed models. A retest was performed using the 30 most resistant and 20 most susceptible accessions to confirm the results. We have identified new resistance sources of Citrullus for anthracnose race 2. In the future, we will run a GWAS analysis to identify SNPs and candidate loci for resistance.
Improving Gummy Stem Blight Resistance and fruit Quality in Watermelon Germplasm
Luis Rivera-Burgos, Todd C. Wehner
North Carolina State University, Raleigh, NC, USA
Development of watermelon (Citrullus lanatus) cultivars with resistance to gummy stem blight (GSB), caused by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), has been slow, indicating complex inheritance. We have worked to combine high resistance and high fruit quality in watermelon inbreds. We developed a panel of RILs, which carries resistance genes to GSB and segregates for fruit quality traits, by crossing and intercrossing resistant plant introduction (PI) accessions and elite cultivars. The 300 RILs were evaluated for disease severity and fruit quality traits under greenhouse and field conditions in a randomized complete block design with 10 replicates in 2017 and 2018. Disease was rated on a scale of 0 (no damage) to 9 (plant dead). Severity was based on weekly ratings, as well as mean and maximum rating for each plot in the field and greenhouse. Around 200 RILs had disease severity ratings below the mean value of the disease assessment scale (4.5), indicating that they may carry some resistance genes for GSB. All disease severity ratings were correlated with each other (r=0.67 to 0.98, P < 0.001) but not correlated with fruit quality traits. The moderate heritability estimated for the RILs (69%) indicates that selection for GSB resistance in early generations would be effective. A group of resistant RILs showed good to excellent fruit quality for the market. Our results provide evidence of improved germplasm for cultivar development of GSB resistant watermelons with good fruit quality.
A Major QTL Located in Chromosome 8 of Cucurbita moschata is Responsible for Resistance to Tomato leaf curl New Delhi virus (ToLCNDV)
Cristina Sáez¹, Cecilia Martínez¹, Cristina Esteras¹, María Ferriol², Javier Montero-Pau¹, José Blanca¹, Joaquín Cañizares¹, Carmelo López¹, Belén Picó¹
¹Institute for the Conservation and Breeding of Agricultural Biodiversity (COMAV-UPV), Universitat Politècnica de València, Valencia, Spain. ²Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, Valencia, Spain
Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite whitefly-transmitted begomovirus, responsible since 2012-2013 season of severe damages in cultivated cucurbits in Southeastern Spain, mainly squash (Cucurbita pepo) and melon (Cucumis melo). The use of genetic resistances is the most efficient way to reduce the virus incidence. In previous works (Sáez et al., 2016, Ann Appl Biol 169:91–105), a core collection of Cucurbita accessions was screened against ToLCNDV using mechanical inoculation and natural infection with whiteflies. All C. pepo accessions were very susceptible to the infection, but the screening provided two C. moschata sources of resistance. In this assay, we studied the genetic control of the resistance to ToLCNDV in both resistant C. moschata accessions, crossing both with a high susceptible C. moschata control and between themselves. F1 generations were selfed and backcrossed to generate the F2 and BCs segregating populations. After mechanical inoculation with ToLCNDV, the response of the hybrids and their respective progenies was evaluated by symptoms scoring and by measuring viral titers (qPCR and molecular hybridization). The F1 and F2 offsprings of C. moschata resistant accessions cross, were symptomless and with low viral titers. Instead, F1 plants offspring of susceptible x resistant crosses were very susceptible and with high viral accumulation levels. F2 and BCs populations derived from plants of these susceptible F1 segregated, suggesting a recessive control of the resistance in both resistance sources. All plants of the different generations were genotyped with a SNPs collection covering the whole C. moschata genome. A mayor QTL was identified in chromosome 8, tightly linked to the resistance to ToLCDV. The region identify is synthetic with the region in chromosome 11 responsible of resistance to ToLCNDV in melon, described in Sáez et al., (2017, Plant Cell rep. 36:1571–1584). Since C. moschata and C. pepo are partially crossable, the SNPs linked to the resistance are facilitating the marker-assisted introgression of ToLCNDV resistance in commercial zucchini and pumpkin breeding programs. Acknowledgements to Spanish Instituto Nacional de Investigaciones Agrarias (INIA) and European Union (FEDER) for funding projects E_RTA2013-00020-C04-03 and RTA2017-00061-C03-03, and to Generalitat Valenciana for funding the project Prometeo 2017/078 and the predoctoral fellowship ACIF/2016/188.
Effect of Two Potyviruses on Development and Yield of Tropical Pumpkin
Wilfredo Seda-Martínez, Linda Wessel-Beaver, Angela Linares-Ramírez
University of Puerto Rico at Mayagüez, Mayagüez, PR, USA
Tropical pumpkin (Cucurbita moschata Duchesne) was mechanically inoculated at the cotyledon stage with potyviruses Papaya ringspot virus, watermelon strain (PRSV-W), Zucchini yellow mosaic virus (ZYMV), and the combination of the two viruses (ZYMV+PRSV). Plants mock- inoculated with buffer were used as controls. ‘Nigerian Local’ and ‘Menina’, genotypes known to be resistant to PRSV and ZYMV, and three susceptible genotypes were used. At four weeks seedlings were transplanted from the greenhouse to the field in Puerto Rico. The 20 treatment combinations (5 genotypes x 4 inoculation treatments) were arranged in a CRD with 4 reps. Plots consisted of single plants spaced 3.7 m apart within and between rows on beds with silver plastic mulch and drip irrigation. Plants were evaluated for virus titer by ELISA (at 20 days post- inoculation in the greenhouse and at 59 and 103 days post-inoculation in the field), flowering date (male and female), number of fruits, total fruit weight, average fruit weight, fruit diameter, pulp width and color, °Brix and percentage dry matter. ‘Nigerian Local’ and ‘Menina’ tested negative for virus infection in both the greenhouse and field while the other genotypes tested positive. ELISA tests showed that some cross-infection occurred after plants were transplanted to the field potentially influencing results from control plots, but the impact was thought to be minimal. Virus-infected plants generally took more time to flower. Plants infected with PRSV produced an average of only 2.2 fruits while control plants produced an average of 3.4 fruits. Plants infected with ZYMV or PRSV+ZYMV had fewer fruits than the control but the difference was not significant. Total fruit yield was almost 50% less in infected compared to control plants of susceptible genotypes. Fruit quality traits were unaffected by virus infection. This is the first known study to document the effects of these two potyviruses in tropical pumpkin at the field level. Early infection of tropical pumpkin with PRSV or ZYMV can result in significant economic losses for growers, demonstrating the importance of developing cultivars with genetic resistance to these two potyviruses. (Supported by USDA-NIFA-SCRI no. 2015- 51181-24285, sub-award no. RC105573UPRM and Hatch accession no.1000526)
North Carolina State Cucumber Lines Developed for Downy Mildew Resistance
Emily Silverman, Todd Wehner
North Carolina State University, Raleigh, NC, USA
Downy mildew, caused by Pseudoperonospora cubensis, is a devastating foliar disease of cucumber. The use of disease resistant (low leaf damage) and tolerant cultivars (low yield loss) has proven an effective practice to develop useful lines. Since 2004, the highly resistant downy mildew accessions have become only moderately or slightly resistant, due to a shift in the pathogen population. New strains of P. cubensis that are present in midwestern and southeastern US can overcome the resistance of most cultivars. In an effort to assist the industry, we developed three populations of cucumber with moderately resistant (MR) to highly resistant (HR) families, combined with good fruit quality. The populations were NC-25 x PI 197088 (13 families), Gy 14 x PI 197088 (17 families), and Poinsett 76 x PI 197088 (15 families). The three populations were evaluated in Clinton, NC under natural disease incidence for several years using three replications in a randomized complete block design. Susceptible (S) and slightly resistant (SR), moderately resistant (MR), or highly resistant (HR) checks were included in the field trials: ‘Poinsett 76’ (MR), NC-25 (MR), Gy 14 (MR), PI 179088 (HR), ‘Coolgreen’ (S), and ‘Ashley’ (SR). Disease damage was rated weekly using a 0-9 scale based on 0-100% disease incidence. Yield was measured (number of fruit per plot) in two harvests, and fruit quality was rated on a 1-9 scale (1-3 = low, 4-6 = moderate, 7-9 = high). Data were analyzed using PROC GLM (SAS 9.4). Selections of the best families obtained from self- pollination in the greenhouse were made from the three populations based on resistance and quality. Those included three lines from NC-25 x PI 197088, four from Gy 14 x PI 197088, and two from Poinsett 76 x PI 197088. The lines were again self-pollinated in the greenhouse to produce nine lines. The nine lines will be self-pollinated again in the greenhouse, and resistant germplasm will be released after a repeat of the process in the summer of 2019.
A Loss-of-Susceptibility Mutation in the STAYGREENGene (CsSGR) Provides Durable, Broad- spectrum Disease Resistances for U.S. Cucumber Production
Yuhui Wang1, Junyi Tan¹, Zhiming Wu¹,², Kyle VandenLangenberg3, Todd Wehner3, Changlong Wen4, Xiangyang Zheng5, Ken Owens5, Alyson Thornton5, Hailey Bang5, Eric Hoeft5, Peter Kraan5, Jos Suelmann6, Junsong Pan1,7, Yiqun Weng8
¹Horticulture Department, University of Wisconsin-Madison, Madison, USA. ²Institute of Cash Crops, Hebei Academy of Agriculture & Forestry Sciences, Shijiazhuang, China. 3Horticultural Science Department, North Carolina State University, Raleigh, USA. 4Beijing Vegetable Research Center, Beijing Academy of Agricultural and Forestry Sciences, Beijing, USA. 5HM Clause Seed Company, Davis, USA. 6Bayer Vegetable Seeds, Haelen, Netherlands. 7Department of Plant Science, Shanghai Jiao Tong University, Shanghai, China. 8U.S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Research Unit, Madison, WI, USA
The Gy14 and WI2757 cucumber inbred lines are resistant to multiple diseases including the oomyceteous downy mildew (pre-2004 strains), the bacterial angular leaf spot (ALS) and the fungal anthracnose (AR) pathogens. These resistances have been widely deployed in commercial cucumber varieties and provided effective protection to cucumber production in the U.S. since the 1960s. However, the causal genes and underlying molecular mechanisms are unknown. We conducted QTL mapping for DM, AR and ALS resistances in the two lines and further map-based cloning to identify the candidate genes for the resistant loci. We show that the triple-disease resistances in Gy14 were controlled by the STAYGREEN (CsSGR) gene that encodes the magnesium dechelatase that plays important regulatory roles in senescence- inducible chlorophyll degradation. Its candidacy was validated with evidence from spatial- temporal gene expression profiling, allelic diversity and phylogenetic analysis, as well as local association studies. We found that the triple-resistance was due to a SNP in the coding region of CsSGR that resulted in a nonsynonymous amino acid substitution of Q to R amino acid in the CsSGR protein. Genes in the chlorophyll degradation pathway showed differential expression between resistant and susceptible lines in response to pathogen inoculation. The region harboring the causal SNP was significantly associated with disease resistances in natural and breeding populations, which has undergone selection in cucumber breeding. We conclude that the resistance is due to loss-of-susceptibility of the CsSGR gene. A working model to explain the molecular mechanisms of CsSGR-mediated multiple disease resistance is proposed. Supported by the Agriculture and Food Research Initiative Competitive Grants under award numbers 2013- 67013-21105 and 2015-51181-24285 from the U.S. Department of Agriculture National Institute of Food and Agriculture. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Disease Assessment in Seedlings of Tropical Pumpkin Infected with PRSV and ZYMV
Magdiel Miranda-Vélez¹, Linda Wessel-Beaver¹, Jose Carlos Rodrigues²
¹University of Puerto Rico, Mayagüez, PR, USA. ²University of Puerto Rico, Agricuiltural Experiment Station, Río Piedras, PR, USA
Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) frequently infect tropical pumpkin (Cucurbita moschata) in Puerto Rico. Breeding programs depend on efficient and reliable methods of assessing resistance. A series of experiments were conducted to determine the best method to differentiate the level of resistance among genotypes of tropical pumpkin for both PRSV and ZYMV, to determine if there are differences in seedling development of resistant vs. susceptible genotypes when infected with these viruses, and to determine if resistant genotypes inoculated with these viruses can infect susceptible genotypes. In Experiment 1, cotyledons of lines known to be either resistant or susceptible were inoculated with PRSV or ZYMV, and ELISA readings were taken from the first four leaves, sampling as each leaf expanded or sampling after all four leaves were expanded. In Experiment 2, cotyledons were inoculated with PRSV, ZYMV or PRSV+ZYMV, and then harvested and weighed at 18 days post-inoculation. In Experiment 3, susceptible genotypes were inoculated with sap from resistant genotypes ‘Nigerian Local’ and ‘Menina’ that had been previously inoculated with PRSV or ZYMV. The susceptible genotypes were tested with ELISA. ELISA readings <0.400 were considered negative for presence of virus. Small leaves, intervenal chlorosis, leaf deformation, curled leaves, and mosaic were some of the symptoms observed in susceptible genotypes inoculated with PRSV and ZYMV. Smaller leaves and chlorosis were observed in ‘Menina’ inoculated with PRSV and ZYMV and no symptoms were observed in ‘Nigerian Local’. For PRSV, sampling of the fourth leaf was required to clearly differentiate between resistant vs susceptible genotypes. For ZYMV, leaves 2, 3, and 4 can be used to differentiate resistant from susceptible genotypes. No differences were found in ELISA readings from plants with single versus double inoculation. No differences in fresh and dry weight were found between uninoculated and inoculated plants. Negative ELISA readings were obtained when susceptible genotypes were inoculated with sap of previously inoculated resistant genotypes. Both ‘Nigerian Local’ and ‘Menina’ were observed to be useful sources of resistance to ZYMV and PRSV in tropical pumpkin. (Supported by USDA-NIFA-SCRI no. 2015-51181-24285, sub-award no. RC105573UPRM and Hatch accession no.1000526)
Innovation Research on Germplasm Resources of Watermelon with Resistance to Fusarium Wilt
Wang Xiqing, Yan Wen, Jia Yunhe, Fu Yongkai, You Haibo
Horticultural Branch of Heilongjiang Academy of Agricultural Sciences, Harbin, China
Watermelon Fusarium wilt disease caused by Fusarium oxysporum f. sp. niveum is a destructive soil-borne disease in watermelon production. Breeding for resistance is the most advocated strategy to circumvent this disease. Marker assisted backcross breeding was employed to incorporate the Fusarium oxysporum f. sp. niveum race 1 resistance gene Fon-1 from donor parents ‘Calhoun Gray’, ‘F211’ and ‘BW85’ into nine cultivated susceptible watermelon varieties (No.1 to No.9) used as recurrent parents. The molecular markers tightly linked to gene Fon-1, CAPS marker 7716_fon was published in 2013 by Beijing Vegetable Research Centre and Indel marker InDel1_fon1 was published in 2017 by Zhengzhou Fruit Research Institute. ‘Calhoun Gray’, ‘F211’ and BW85’ were used as male parent and the cultivated susceptible watermelon varieties (No.1 to No.9) were used as female parent, 25 combinations (F1) were assembled, then F1 plants were backcrossed with respective recurrent parents (No.1 to No.9). Molecular identification of Fusarium wilt resistance for the recurrent parents and F1 plants using CAPS marker (7716_fon) was carried out. The identification results of molecular detection were associated with Fusarium wilt resistance except in No.4 variety. The result indicated that 8 recurrent parents were with susceptible genotype for Fusarium wilt race, and 22 combinations expressed as heterozygous resistant genotype, No.4 variety and 3 combinations assembled using No.4 variety as female parent were Fusarium wilt resistant genotype. The marker is co- dominant marker, which can be used for the distinguishing of homozygous and heterozygous genotypes. The backcross generation BC1F1 foreground selection was carried out by the indoor seedling artificial inoculation, molecular marker detection, and screening in natural disease nursery (population levels of Fusarium in soil samples were 2.52×104 CFU·g-1 of soil), BC1F1 plants are at fruiting stage currently. The gene positive BC1F1 plants which were heterozygous genotypes chosen through molecular identification planted in natural disease nursery show different Fusarium wilt resistance, eight combinations were expressed as highly resistance with the diseased plant rate under 20 %, seven combinations were susceptible with the diseased plant rate over 60 %, it may have related to the recurrent parent genetic background. The survival BC1F1 plants will be backcrossed with respective recurrent parents, the best BC3F1 plants from each of the combinations will self to generate BC3F2 populations and plants homozygous for the target gene will be isolated. Further, selected plants will advance till BC3F4 generation and best families with resistance to Fusarium wilt from each of the combinations will be identified. At present, our team has bred 3 high quality watermelon varieties with high resistance to Fusarium wilt.
Development of a Real-Time Fluorescence-Based Microplate Assay for Pathogen Growth on Plant Tissue: Phytophthora capsici Infection of Cucumber Fruit
Chunqiu Zhang¹,², Ben Mansfeld², Rebecca Grumet²
¹Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. ²Horticulture Department, Michigan State University, East Lansing, MI, USA
Cucumber fruit rot, caused by the oomycete pathogen Phytophthora capsici, can cause significant losses for cucumber production in the Midwestern U.S. An age-related resistance (ARR) to this pathogen was observed previously in fruit of cultivar ‘Poinsett76’. Young rapidly growing fruits are highly susceptible, but become resistant as they approach full size. ARR is increasingly recognized as an important defense against pathogens, however, the basis for resistance is still unclear. Our objectives were to develop a high-throughput, reliable, and sensitive, real-time detection method for P. capsici in cucumber fruits to allow for quantitative measurement of pathogen growth in different genotypes, ages or treatments. A fluorescence- based microplate assay was developed using the P. capsici isolate NY0664-1 expressing red fluorescent protein gene tdTomato and the Spark® multimode microplate reader. Cucumber peel sections (5-6 mm thick) were sampled with a 6 mm biopsy punch placed in individual 6.5 mm microtiter plate wells, and inoculated with 5 ul of 1.0×106 P. capsici zoospores/ml. By applying this method, pathogen on the tissue could be detected immediately after inoculation. Thereafter, the increase in signal was rapid in susceptible fruit and slower in resistant fruit. The amount of pathogen quantified was highly correlated with susceptibility to P. capsici. The method developed in this study provides rapid, accurate and sensitive quantification of pathogen before symptoms were observed, allowing real-time monitoring of pathogen growth on tissue. The quantitative nature of the bioassay described in this study may be useful both in cucumber breeding and basic research.
Molecular Mechanism of CsWIN1 in regulating Cuticular Wax Biosynthesis in Grafted Cucumber
Jian Zhang, Jingjing Yang, Changlong Wen
Beijing Vegetable Research Center, Beijing Academy of Agricultural and Forestry Sciences, Beijing, China
Fruit appearance quality is one of the important commercial quality characters for cucumber. Previous studies showed that cucumber bloom on the fruit surface was not only affected by environmental factors but also regulated genes including those related to wax synthesis related genes and silicon transport. Grafting to suit pumpkin was considered as an effective method to decrease bloom on cucumber fruit. To understand the molecular regulation mechanism of de- blooming by grafting, a cucumber ‘Jingyan 116’ was grafted on pumpkin ‘Jingxinzhen 6’ in the present study. RNA-seq and DNA methylation sequencing analysis were conducted on grafted and self-rooted cucumber. A total of 69 different expression genes were identified between grafted and self-rooted cucumber, including five up-regulated wax synthesis related genes (Csa3G127750, Csa6G079750, Csa6G151790, Csa6G151810, Csa6G302180) and four down-regulated aquaporin transporters (Csa3G743400, Csa5G162580, Csa5G199270, Csa5G623360). We also found 20 genes with DNA methylation in grafted cucumber. Consequently, we isolated an important candidate gene CsWIN1(Csa3G017320), which encodes an AP2/ERF-type transcription factor. CsWIN1is a homologous gene in Arabidopsis thaliana and positively regulates the biosynthesis of wax. Our results demonstrated that grafted cucumber fruit surface contained more wax and lower silicon than self-rooted cucumber. This study also provided valuable information for breeding glossy cucumber varieties by MAS and to facilitate decrease bloom research in other cucurbit crops.