Marker assisted selection for seedlessness in table grape breeding

Seedlessness is one of the most appreciated traits in the table grape (Vitis vinifera L.). The development of new seedless varieties is expensive and time consuming, involving the generation and selection of thousands of hybrids each year. In seeded × seedless crosses, seedlessness commonly segregates 1:1, so molecular markers allowing for the early identification of plants that will produce seedless berries are very useful. This early selection can lead to savings in maintenance and evaluation costs, and allows additional space for larger effective progenies. The variety Sultanina has been the main source of stenospermocarpic seedlessness in table grape breeding. In a previous work, we showed a 198-bp allele at the VMC7F2 microsatellite locus as a potential marker for selection of seedless genotypes due to its close linkage to the major effect seedless QTL, SDI. In this study, we show that stenospermocarpic bred cultivars share a main haplotype around this locus not found in seeded cultivars, which facilitating the development and use of marker assisted selection (MAS) strategies for the selection of seedless plants. In this way, VMC7F2 on its own can be a very useful marker for selecting seedless individuals from segregating crosses. A MAS program based on the presence of the 198-bp allele at VMC7F2 allows the reduction of the progeny size to 54%, selecting most of the seedless individuals. In addition, our results show the existence of other possible sources of stenospermocarpic seedlessness that could provide alternative sources of genetic variation for breeding of this trait.     

A SNP-based high-density linkage map of zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.) and its use for the identification of QTL associated with winter hardiness

Zoysia japonica Steud. (2n?=?4×?=?40) is a C₄ turfgrass well-adapted for the warm-humid and transitional climatic zones of the USA. Its use is limited to warmer climates because of a relative lack of winter hardiness compared to C₃ grasses. Molecular markers associated with this trait would be useful for effective selection of winter hardy germplasm before field testing. A pseudo-F₂ mapping population of 175 individuals was developed from crosses between Z. japonica cultivars “Meyer” (freeze-tolerant) and “Victoria” (freeze-susceptible) and used to generate a high-density genetic map of 104 SSR markers and 2359 sequencing-derived SNP markers. The map covers 324 Mbp and 2520 cM as well as the 20 chromosomes for the zoysiagrass haploid genome. Phenotypic data on winter injury, establishment, and turf quality collected in North Carolina and Indiana in 2014–2016 were used in conjunction with this map to identify quantitative trait loci (QTL) associated with winter hardiness. Fifty-six QTL associated with winter injury, establishment, and turf quality were identified over six environments. Twelve of those were identified in two or more environments. Furthermore, seven regions of interest were identified on chromosomes 8, 11, and 13 where co-location of QTL for three or more traits occurred. Within these regions, analysis with NCBI basic local alignment search tool (BLAST) identified proteins related to cold and other abiotic stresses tolerance. These QTL and associated markers could be valuable in implementing marker-assisted selection for winter hardiness in zoysiagrass breeding programs.     

Mapping and pedigree analysis of the gene that controls the easy peel pellicle trait in Japanese chestnut (Castanea crenata Sieb. et Zucc.)

The Japanese chestnut (Castanea crenata Sieb. et Zucc.) has a pellicle that is difficult to peel, which increases the labor and cost for removing the pellicle from the nut during processing. Thus, a pellicle that is easier to peel has been an important objective of Japanese chestnut breeding programs. A newly released cultivar (“Porotan”) exhibits a unique, easily peeled pellicle. A previous study indicated that this trait is controlled by recessive gene p, and that several of the ancestors of Porotan (e.g., “Tanzawa” and 550-40) were P/p heterozygotes. Two F₁ populations from intra-specific crosses of Japanese chestnut, Tanzawa (P/p) × Porotan (p/p) and 550-40 (P/p) × Tanzawa (P/p), were used for genetic mapping of the gene that controls this characteristic. A total of 11 simple sequence repeat (SSR) markers were obtained that showed significant linkages to the p gene, and genetic linkage maps for the region around the p gene were established. Pedigree analysis was conducted for eight ancestors of Porotan around the pellicle-peeling locus using graphical genotypes based on the 11 SSR loci. The two recessive p alleles and surrounding haplotypes of Porotan were inherited through different intermediate cultivars: one allele was derived from “Otomune” (P/p) via Tanzawa and the other was derived from Otomune via Tanzawa, “Kunimi” (P/p), and breeding line 550-40. A recombination event was found in the flanking region close to the p gene in Kunimi. Molecular identification of the easy peel pellicle trait will lead to marker-assisted selection and will greatly improve Japanese chestnut breeding.     

Gene Tagging with Random Amplified Polymorphic DNA (RAPD) Markers for Molecular Breeding in Plants

Markers are of interest to plant breeders as a source of genetic information on crops and for use in indirect selection of traits to which the markers are linked. In the classic breeding approach, the markers were invariably the visible morphological and other phenotypic characters, and the breeders expended considerable effort and time in refining the crosses as the tight linkage or association of the desired characters with the obvious phenotypic characters was never unequivocally established. Furthermore, indirect selection for a trait using such morphological markers was not practical due to (1) a paucity of suitable markers, (2) the undesirable pleiotropic effects of many morphological markers on plant phenotype, and (3) the inability to score multiple morphological mutant traits in a single segregating population. With the advancement in molecular biology, the use of molecular markers in plant breeding has become very commonplace and has given rise to “molecular breeding”. Molecular breeding involves primarily “gene tagging”, followed by “marker-assisted selection” of desired genes or genomes. Gene tagging refers to the identification of existing DNA or the introduction of new DNA that can function as a tag or label for the gene of interest. In order for the DNA sequences to be conserved as a tag, important prerequisites exist. This review also summarizes the achievements in gene tagging that have been made over the last 7 to 8 years.     

Fine mapping a major QTL for kernel number per row under different phosphorus regimes in maize (Zea mays L.)

Phosphorus (P) is one of the essential macronutrients for plant growth and development. Grain yield is the primary trait of interest in maize breeding programs. Maize grain yield and yield-related traits are seriously affected by P deficiency. Kernel number per row (KN), as one of the major components of grain yield, has attracted the attention of more and more breeders. In our previous study, one major QTL (named qKN), controlling KN under different P regimes was mapped to the interval between molecular markers bnlg1360 and umc1645 on chromosome 10 using a F _2:3 population derived from the cross between maize inbreds 178 and 5,003 (107). In order to understand its genetic basis, we developed a population of near isogenic lines (NILs) and two P regimes were used to fine map and characterize qKN. The QTL qKN was finally localized in a region of ~480 kb. A single qKN allele of inbred 178 increased KN by 6.08–10.76 % in the 5,003 (107) background; qKN acted in a partially dominant manner. Our results will be instrumental for the future identification and isolation of the candidate gene underlying qKN. The tightly linked molecular markers that we developed for qKN will be useful in maize breeding programs for improving KN applying the marker-assisted selection.     

Rapid location of Glomerella leaf spot resistance gene locus in apple by whole genome re-sequencing

Glomerella leaf spot (GLS) is a new fungal disease of apple that damages apple leaves mainly during the summer in China. For efficient GLS-resistant apple breeding by marker-assisted selection (MAS) and a better understanding of the molecular mechanisms of the resistance, it is important to find molecular markers that are tightly linked to GLS resistance genes and construct fine mapping. However, the development and selection of DNA markers are time-consuming and labor-intensive processes. Next-generation sequencing technology provides a powerful tool to overcome this limitation and is faster and more efficient in establishing the association of GLS resistance with molecular markers or searching for candidate genes. In this study, we report a method for rapid location of a GLS resistance gene locus (R _gls ) in apple by whole genome re-sequencing technology coupled with bulked segregant analysis (BSA). A total of 3,399,950 single nucleotide polymorphisms (SNPs) were identified. Through the genome-wide comparison of SNP profiles between the resistant and the susceptible bulks constructed from F₁ individuals derived from a cross between “Golden Delicious” and “Fuji,” the R _gls locus was identified on apple chromosome 15 between 2 and 5 Mb. In this region, eight SNP markers were validated using high resolution melting (HRM), and the fine genetic mapping of the eight markers was constructed. The R _gls locus was sandwiched by two flanking markers SNP4208 and SNP4257, with the recombination frequency of 0.97% (2/207). The marker SNP4236 co-segregated with R _gls . The physical size of the R _gls locus was estimated to be 49 kb. In this genetic interval, nine genes were predicted. Our study provides an effective method for rapid identification of genomic regions and development of the diagnostic markers for MAS. This strategy is potentially useful for other agronomic traits or plant species.     

Genetic mapping of microsatellite markers around the arcelin bruchid resistance locus in common bean

The deployment in common beans (Phaseolus vulgaris L.) of arcelin-based bruchid resistance could help reduce post-harvest storage losses to the Mexican bean weevil [(Zabrotes subfasciatus (Boheman)]. Arcelin is a member of the arcelin-phytohemagglutinin-α-amylase inhibitor (APA) family of seed proteins, which has been extensively studied but not widely used in bean breeding programs. The purpose of this study was to evaluate microsatellite markers for genetic analysis of arcelin-based bruchid resistance and to determine the orientation of markers and the rate of recombination around the APA locus. A total of 10 previously developed microsatellites and 22 newly developed markers based on a sequenced BAC from the APA locus were screened for polymorphism and of these 15 were mapped with an F₂ population of 157 individuals resulting from a susceptible × resistant cross of SEQ1006 × RAZ106 that segregated for both the arcelin 1 allele and resistance to the bruchid, Z. subfasciatus. Microsatellites derived from APA gene sequences were linked within 0.8 cM of each other and were placed relative to the rest of the b04 linkage group. In a comparison of genetic to physical distance on the BAC sequence, recombination was found to be moderate with a ratio of 125 kb/cM, but repressed within the APA locus itself. Several markers were predicted to be very effective for genetic studies or marker-assisted selection, based on their significant associations with bruchid resistance and on low adult insect emergence and positions flanking the arcelin and phytohemagglutinin genes.     

A naturally occurring insertion in the <Emphasis Type="Italic">RsFLC2</Emphasis> gene associated with late-bolting trait in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

Premature flowering reduces the yield and quality of the harvested fleshy taproot in radish. However, there has been little molecular marker research on the radish late-bolting trait. In this study, F₂ and F_2:3 populations derived from a cross of “Ninengo” (late-bolting) and “Maer” (early-bolting) were analyzed to map late-bolting genes. Five hundred insertion and deletion (InDel) markers were designed according to the whole-genome resequencing data of the two parents. A genetic map was constructed based on the F₂ population, and a late-bolting gene was detected in a 1.1-cM region between the markers InDel520 and InDel535 on chromosome R02 that explained the highest (76.4%) phenotypic variance. RsFLC2 was identified as a candidate gene in this region. Notably, “Ninengo” contains a 1627-bp insertion near the 5′ end of the first intron of RsFLC2. Allelic variation analyses in the F₂ population further validated that RsFLC2was associated with the late-bolting trait in radish. The expression pattern of RsFLC2 was significantly different between “Ninengo” and “Maer” during vernalization. Vernalization suppressed RsFLC2 expression, and the 1627-bp insertion in the first intron weakened gene repression in “Ninengo” plants, resulting in late-bolting. This study lays a foundation for uncovering the molecular mechanism of late-bolting and marker-assisted selection for breeding late-bolting varieties of radish.     

Genetic control of soybean seed oil: I. QTL and genes associated with seed oil concentration in RIL populations derived from crossing moderately high-oil parents

Soybean seed is a major source of oil for human consumption worldwide and the main renewable feedstock for biodiesel production in North America. Increasing seed oil concentration in soybean [Glycine max (L.) Merrill] with no or minimal impact on protein concentration could be accelerated by exploiting quantitative trait loci (QTL) or gene-specific markers. Oil concentration in soybean is a polygenic trait regulated by many genes with mostly small effects and which is negatively associated with protein concentration. The objectives of this study were to discover and validate oil QTL in two recombinant inbred line (RIL) populations derived from crosses between three moderately high-oil soybean cultivars, OAC Wallace, OAC Glencoe, and RCAT Angora. The RIL populations were grown across several environments over 2 years in Ontario, Canada. In a population of 203 F_3:6 RILs from a cross of OAC Wallace and OAC Glencoe, a total of 11 genomic regions on nine different chromosomes were identified as associated with oil concentration using multiple QTL mapping and single-factor ANOVA. The percentage of the phenotypic variation accounted for by each QTL ranged from 4 to 11 %. Of the five QTL that were tested in a population of 211 F_3:5 RILs from the cross RCAT Angora × OAC Wallace, a “trait-based” bidirectional selective genotyping analysis validated four QTL (80 %). In addition, a total of seven two-way epistatic interactions were identified for oil concentration in this study. The QTL and epistatic interactions identified in this study could be used in marker-assisted introgression aimed at pyramiding high-oil alleles in soybean cultivars to increase oil concentration for biodiesel as well as edible oil applications.     

Identification of quantitative trait loci (QTL) for fruit-quality traits and number of weeks of flowering in the cultivated strawberry

Fruit quality and repeat flowering are two major foci of several strawberry breeding programs. The identification of quantitative trait loci (QTL) and molecular markers linked to these traits could improve breeding efficiency. In this work, an F₁ population derived from the cross ‘Delmarvel’ × ‘Selva’ was used to develop a genetic linkage map for QTL analyses of fruit-quality traits and number of weeks of flowering. Some QTL for fruit-quality traits were identified on the same homoeologous groups found in previous studies, supporting trait association in multiple genetic backgrounds and utility in multiple breeding programs. None of the QTL for soluble solids colocated with a QTL for titratable acids, and, although the total soluble solid contents were significantly and positively correlated with titratable acids, the correlation coefficient value of 0.2452 and independence of QTL indicate that selection for high soluble solids can be practiced independently of selection for low acidity. One genomic region associated with the total number of weeks of flowering was identified quantitatively on LG IV-S-1. The most significant marker, FxaACAO2I8C-145S, explained 43.3 % of the phenotypic variation. The repeat-flowering trait, scored qualitatively, mapped to the same region as the QTL. Dominance of the repeat-flowering allele was demonstrated by the determination that the repeat-flowering parent was heterozygous. This genomic region appears to be the same region identified in multiple mapping populations and testing environments. Markers linked in multiple populations and testing environments to fruit-quality traits and repeat flowering should be tested widely for use in marker-assisted breeding.     

Inheritance and QTL mapping of Fusarium wilt race 4 resistance in cotton

Diseases such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] represent expanding threats to cotton production. Integrating disease resistance into high-yielding, high-fiber quality cotton (Gossypium spp.) cultivars is one of the most important objectives in cotton breeding programs worldwide. In this study, we conducted a comprehensive analysis of gene action in cotton governing FOV race 4 resistance by combining conventional inheritance and quantitative trait loci (QTL) mapping with molecular markers. A set of diverse cotton populations was generated from crosses encompassing multiple genetic backgrounds. FOV race 4 resistance was investigated using seven parents and their derived populations: three intraspecific (G. hirsutum × G. hirsutum L. and G. barbadense × G. barbadense L.) F₁ and F₂; five interspecific (G. hirsutum × G. barbadense) F₁ and F₂; and one RIL. Parents and populations were evaluated for disease severity index (DSI) of leaves, and vascular stem and root staining (VRS) in four greenhouse and two field experiments. Initially, a single resistance gene (Fov4) model was observed in F₂ populations based on inheritance of phenotypes. This single Fov4 gene had a major dominant gene action and conferred resistance to FOV race 4 in Pima-S6. The Fov4 gene appears to be located near a genome region on chromosome 14 marked with a QTL Fov4-C14 ₁ , which made the biggest contribution to the FOV race 4 resistance of the generated F₂ progeny. Additional genetic and QTL analyses also identified a set of 11 SSR markers that indicated the involvement of more than one gene and gene interactions across six linkage groups/chromosomes (3, 6, 8, 14, 17, and 25) in the inheritance of FOV race 4 resistance. QTLs detected with minor effects in these populations explained 5–19 % of the DSI or VRS variation. Identified SSR markers for the resistance QTLs with major and minor effects will facilitate for the first time marker-assisted selection for the introgression of FOV race 4 resistance into elite cultivars during the breeding process.     

Validation of a functional molecular marker suitable for marker-assisted breeding for fruit texture in apple (Malus × domestica Borkh.)

Molecular markers are nowadays considered fundamental tools in breeding programs, supporting the selection of the most favourable offspring. This role is invaluable in the case of complex agronomic traits in tree fruit crop species, such as fruit texture in apple (Malus × domestica Borkh.). This work presents the validation of a previously identified functional simple sequence repeat marker, named Md-PG1_SSR10kd, suitable for the advanced selection of high texture performance seedlings. Two independent populations were chosen by marker-assisted parent selection, and a specific set of seedlings was selected by marker-assisted seedling selection, to validate the predictive power of this marker. The two groups of seedlings, further phenotyped for fruit texture, showed a clear difference in texture behaviour. The selection of this marker also showed a higher efficiency than Md-ACS1 and Md-ACO1, two functional markers currently implemented in different breeding programs. Finally, the allelic effect was estimated by computing the breeding values in a collection of 83 different apple cultivars. The results reported here confirmed the association of Md-PG1_SSR10kd with texture sub-traits, proposing this as a novel promising selection strategy suitable for apple fruit texture.     

Identification of a major QTL for Xanthomonas arboricola pv. pruni resistance in apricot

Xanthomonas arboricola pv. pruni causes bacterial spot of stone fruit resulting in severe yield losses in apricot production systems. Present on all continents, the pathogen is regulated in Europe as a quarantine organism. Host resistance is an important component of integrated pest management; however, little work has been done describing resistance against X. arboricola pv. pruni. In this study, an apricot population derived from the cross “Harostar” × “Rouge de Mauves” was used to construct two parental genetic maps and to perform a quantitative trait locus analysis of resistance to X. arboricola pv. pruni. A population of 101 F1 individuals was inoculated twice for two consecutive years in a quarantine greenhouse with a mixture of bacterial strains, and disease incidence and resistance index data were collected. A major QTL for disease incidence and resistance index accounting respectively for 53 % (LOD score of 15.43) and 46 % (LOD score of 12.26) of the phenotypic variation was identified at the same position on linkage group 5 of “Rouge de Mauves.” Microsatellite marker UDAp-452 co-segregated with the resistance, and two flanking microsatellites, namely BPPCT037 and BPPCT038A, were identified. When dividing the population according to the alleles of UDAp-452, the subgroup with unfavorable allele had a disease incidence of 32.6 % whereas the group with favorable allele had a disease incidence of 21 %, leading to a reduction of 35.6 % in disease incidence. This study is a first step towards the marker-assisted breeding of new apricot varieties with an increased tolerance to X. arboricola pv. pruni.     

High-resolution genetic mapping of rice bacterial blight resistance gene Xa23

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world’s population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7 cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F₂ mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4 cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8 kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.     

SSR-based genetic linkage map construction in pistachio using an interspecific F<Subscript>1</Subscript> population and QTL analysis for leaf and shoot traits

Pistachio is one of the most commercially important nut trees in the world. To characterize the genetic controls of horticultural traits and facilitate marker-assisted breeding in pistachio, we constructed an SSR-based linkage map using an interspecific F₁ population derived from a cross between the cultivar “Siirt” (Pistacia vera L.) and the monoecious Pa-18 genotype of Pistacia atlantica Desf. This population was also used for the first QTL analysis in pistachio on leaf and shoot characters. In total, 1312 SSR primers were screened, and 388 loci were successfully integrated into parental linkage maps. The Siirt maternal map contained 306 markers, while the “Pa-18” paternal map included 285 markers along the 15 linkage groups. The Siirt map spanned 1410.4 cM, with an average marker distance of 4.6 cM; the Pa-18 map covered 1362.5 cM with an average marker distance of 4.8 cM. Phenotypic data were collected during the growing seasons of 2015 and 2016 for four traits: leaf length (LL), leaf width (LW), leaf length/leaf width ratio (LWR), number of leaflet pairs (NLL), and young shoot color (YSC). A total of 17 QTLs were identified in the parental maps. Four QTLs for LL and LW were located on LG2 and LG4, while four QTLs for LWR ratio on LG13 and LG14, two QTLs for NLL and two QTLs for YSC were on LG7 and LG9, respectively, with similar positions in both parental maps. The SSR markers, linkage maps, and QTLs reported here will provide a valuable resource for future molecular and genetic studies in pistachio.     

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.     

Three EST-SSR Markers Associated with QTL for the Growth of the Clam Meretrix meretrix Revealed by Selective Genotyping

The clam Meretrix meretrix is a member of widely cultured, commercially important clams. A marker–trait association analysis was performed using expressed sequence tag (EST) simple sequence repeat (SSR) markers for marker-assisted selection in M. meretrix. Three markers, MM1272, MM2034, and MM7721, were found to be significantly associated with quantitative trait loci (QTLs) controlling shell length (P?<?0.0001) in clams of a fast-growing population (JSF) and a control population (JSC). The 144-bp allele of MM1272, the 154-bp allele of MM2034, and the 152- and 165-bp alleles of MM7721 showed a significantly higher frequency in the JSF population (17.65, 36.41, 28.67, and 29.33 %) than in the JSC population (4.65, 8.33, 3.47, and 5.56 %). The three markers showed lower values for the number of alleles and observed heterozygosity as well as a higher proportion of homozygotes in JSF than in JSC population. The three markers have been further confirmed in the high and low tails of another population (09G₃SPSB); similarly, lower values for the number of alleles and observed heterozygosity as well as a higher proportion of homozygotes were found in 09G₃SPSB_H. The putative functions of the three gene fragments containing MM1272, MM2034, and MM7721 also suggested that the three SSR-containing genes might be involved in growth of M. meretrix. All the results suggest that the three EST-SSR markers associated with growth QTLs would be useful for marker-assisted selection in M. meretrix breeding.     

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F₈ recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F₈ population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of “false positives” (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.     

Chromosome location and allele-specific PCR markers for marker-assisted selection of the oat crown rust resistance gene Pc91

Race-specific seedling resistance genes are the primary means of controlling crown rust of oat caused by Puccinia coronata Corda f. sp. avenae Eriks in Canada. Pc91 is a seedling crown rust resistance gene that is highly effective against the current crown rust population in North America. A number of race-specific resistance genes have been mapped and markers that are closely linked to them have been identified. However, the use of these markers in oat breeding has been limited by the economics of marker-assisted selection (MAS). A crucial step in the successful application of MAS in breeding programs is the development of inexpensive and easy-to-use molecular markers. The primary objective of this study was to develop co-dominant KBioscience competitive allele-specific PCR (KASP) markers linked to Pc91 for deployment in high-throughput MAS in oat breeding programs. The allele-specific marker showed consistent diagnostic polymorphism between the selected 16 North American oat breeding lines. The developed co-dominant marker was also validated on three F₂ populations (AC Morgan × Stainless; SW Betania × Stainless; AC Morgan × CDC Morrison) and one recombinant inbred line population (CDC Sol-Fi × HiFi) segregating for Pc91 using KASP genotyping technology. We recommend the simple, low-cost marker as a powerful tool for pyramiding Pc91 with other effective crown rust resistance loci into a single line. The mapping results indicate that crown rust resistance gene Pc91 resides on the translocated oat chromosome 7C-17A.     

Inheritance and gene mapping of spotted to non-spotted trait gene <Emphasis Type="Italic">CmSp-1</Emphasis> in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L<Emphasis Type="Italic">.</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis> Pangalo)

Melon (Cucumis melo L.) is one of the most popular and highly nutritious vegetable species within Cucurbitaceae. Because appearance is used as an important indicator of quality, the spotted to non-spotted trait associated with this product somewhat influences the buying habits of consumers. We tested a six-generation family to determine the inheritance and genetic basis of this trait. Genetic groups F₁, F₂, BC₁P₁, and BC₁P₂ were from a cross between “IM16559” (non-spotted) and “IM16553” (spotted). Our genetic analysis showed that the spotted to non-spotted trait was controlled by a single dominant gene that we named CmSp-1. Whole-genome resequencing-bulked segregant analysis (WG-BSA) demonstrated that this gene was located on the end of chromosome 2, in the intersections of 22,160,000 to 22,180,000 bp and 22,260,000 to 26,180,000 bp, an interval distance of 3.94 Mb. Insertion-deletion (InDel) markers designed based on WG-BSA data were used to map this gene. Using 13 InDel markers, we produced a genetic map indicating that CmSp-1 was tightly linked to markers I734-2 and I757, with genetic distances of 1.8 and 0.4 cM and an interval distance of 280.872 kb. The closest marker was I757. Testing of 107 different melon genotypes presented an accuracy of 84.11% in predicting the phenotype. By being able to locate CmSp-1 in melon, we can now use the findings to identify potential targets for further marker-assisted breeding and cloning projects.