Showing papers in "Molecular Breeding in 2015"

Precise identification of two wheat–Thinopyrum intermedium substitutions reveals the compensation and rearrangement between wheat and Thinopyrum chromosomes

Abstract: Two wheat–Thinopyrum substitution lines X479 and X482 selected from the progenies of wheat “Mianyang26 (MY26)” × wheat–Thinopyrum intermedium ssp. trichophorum partial amphiploid were characterized by seed storage protein electrophoresis, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and PCR-based molecular markers. Seed storage protein analysis showed that X479 expressed some of Th. intermedium ssp. trichophorum-specific gliadin and glutenin bands. Chromosome counting and GISH probed by Pseudoroegneria spicata genomic DNA indicated that two pairs of Thinopyrum-derived chromosomes (St genome and St–JS translocated chromosomes) substituted for two pairs of wheat chromosomes in both X479 and X482. FISH using pAs1 and pHvG38 as probes showed that chromosomes 1B and 4B, and 4D and 6D were absent in X479 and X482, respectively. Using the newly isolated JS chromosome-specific repetitive sequence pDb12H as a probe, the FISH signals revealed that the translocation of St–JS chromosomes in X479 and X482 occurred in repetitive sequence regions of the short arm. The molecular markers based on wheat–rice colinearity confirmed that the chromosome constitutions of X479 and X482 were 1St (1B) + 4St–4JS (4B) and 4St–JS (4D) + 6St (6D), respectively. The substitution lines were both fully fertile which suggests that the Th. intermedium chromosomes in X479 and X482 substitute well for the corresponding wheat chromosomes. The rust resistance and novel agronomic traits revealed that the substitution lines will be potentially useful for genetic improvement of wheat.

Waterlogging tolerance is associated with root porosity in barley ( Hordeum vulgare L.)

Abstract: Tolerance to waterlogging is an important breeding objective for barley (Hordeum vulgare L.); however, it is a complex quantitative trait. It is difficult to screen large numbers of lines in the field due to environmental variability, and it is also challenging to screen large numbers in controlled conditions if yield data are to be collected. The direct measurement of traits that contribute to waterlogging tolerance, such as aerenchyma development in roots, may offer advantages especially if molecular markers can be developed to screen breeding populations. A doubled haploid population from a cross between Franklin and YuYaoXiangTian Erleng was screened for adventitious root porosity (gas-filled volume per unit root volume) as an indicator of aerenchyma formation. A single QTL for root porosity was identified on chromosome 4H which explained 35.7 and 39.0 % of phenotypic variation in aerated and oxygen-deficient conditions, respectively. The nearest marker was EBmac0701. This QTL is located in the same chromosomal region that contributed to tolerance when the same population was screened in an earlier independent soil waterlogging experiment. Comparative mapping revealed that this QTL is syntenic with the Qaer1.02-3 QTL in maize and the Sub1A-1 gene in rice, which are associated with aerenchyma formation (maize) and submergence tolerance (rice), respectively. This is the first report of a QTL for root porosity in barley which elucidates a major mechanism of waterlogging tolerance.

Multi-parent advanced generation inter-cross in barley: high-resolution quantitative trait locus mapping for flowering time as a proof of concept

Abstract: The choice of mapping population is one of the key factors in understanding the genetic effects of complex traits and determines the power and precision of quantitative trait locus (QTL) mapping. We present the results of the first eight-way multi-parent advanced generation inter-cross (MAGIC) doubled haploid (DH) population in barley (Hordeum vulgare ssp. vulgare) applied to mapping complex traits. The results of the genetic architecture within the barley MAGIC population allowed QTL mapping in 533 DH lines with 4,550 single nucleotide polymorphisms (SNPs) with a newly developed mixed linear model in SAS v9.2, incorporating multi-locus analysis and cross validation for flowering time. Two QTL mapping approaches, the binary approach (BA), which is widely used in QTL and association mapping, and a novel haplotype approach (HA) were compared based on their efficiency, precision for QTL detection and estimation of genetic effects. The analysis detected 17 QTLs, five of which were shared between the two approaches; five and two were specifically found with the BA and HA approaches, respectively. The combination of the two mapping approaches enabled high-precision QTL mapping for flowering time. The QTLs corresponded to the genomic regions of major flowering-time genes Vrn-H1, Vrn-H3, HvGI, Ppd-H1, HvFT2, HvFT4, Co1 and linked genes for plant height (sdw1). These results confirm the proof of concept of QTL mapping in a multi-parent population, highlight the advantages and demonstrate that the barley MAGIC DH lines in combination with an advanced QTL mapping approach are valuable resources for mapping complex traits.

Genetic analysis and gene mapping of papaya ring spot virus resistance in cucumber

Abstract: The papaya ring spot virus (PRSV) causes significant fruit yield loss in cucurbit crops. Understanding of the inheritance and molecular mapping of PRSV resistance will facilitate development of resistant varieties to control this disease. In the present study, an F2 population was developed from the cross between susceptible ‘65G’ and resistant ‘02245’ cucumber inbred lines. Genetic analysis of PRSV resistance in 144 F2:3-derived F3 families showed that resistance is controlled by a single recessive gene which was designated as prsv 02245 . Simple sequence repeat (SSR) markers were employed in polymorphism screening between PRSV-susceptible and resistant DNA pools. The PRSV resistance gene, prsv 02245 , was mapped on chromosome 6 that was flanked by two SSR markers, SSR11-177 and SSR11-1, which was 1.1 and 2.9 cM away from the prsv 02245 locus, respectively. The physical distance between the two markers was approximately 600 kb. The accuracy rate of marker-assisted selection of PRSV resistance among 35 cucumber lines using the marker, SSR11-177 was more than 80 %. Results from this study provide a valuable tool for fine mapping, gene cloning, and marker-assisted breeding for PRSV resistance in cucumber.

Molecular characterization and marker development for hexaploid wheat-specific HMW glutenin subunit 1By18 gene

Abstract: Wheat grain high molecular weight glutenin subunits (HMW-GS) are the major determinants of dough elasticity and viscosity, and thus of breadmaking quality. Most known HMW-GS genes in bread wheat have been already cloned except for very few such as 1By18 and 1By19. In this study, HMW-GS 1By18 gene was isolated from bread wheat for the first time. In the past, this subunit has been considered to have positive effect on gluten quality. Molecular characterization of the 1By18 subunit gene showed that its complete coding nucleotide sequence was 2163 bp long, being highly identical to 1By8, with only three single nucleotide polymorphisms (SNPs) at sites 231, 233 and 1976 of the coding sequence. The deduced amino acid sequence of 1By18 exhibited two substitutions compared to 1By8. The deduced protein was further verified by expression in E. coli and LC–MS/MS analysis. Phylogenetic analysis showed that 1By18 was more closely related to 1By8, 1By9 and 1By16 than to 1By15 and 1By20. Molecular phylogenetic analysis indicated that the Glu-B1i allele emerged after the formation of modern hexaploid wheat. Two sets of SNP-based AS-PCR markers for 1By18 gene were developed and validated on 110 bread wheat varieties with different Glu-Bly alleles, and five recombinant inbred lines. Both markers could effectively distinguish 1By18 from the other genes at the Glu-B1 locus, thus making them valuable for marker-assisted selection in wheat quality improvement programs.

Development and GBS-genotyping of introgression lines (ILs) using two wild species of rice, O. meridionalis and O. rufipogon, in a common recurrent parent, O. sativa cv. Curinga

Abstract: Two populations of interspecific introgression lines (ILs) in a common recurrent parent were developed for use in pre-breeding and QTL mapping. The ILs were derived from crosses between cv Curinga, a tropical japonica upland cultivar, and two different wild donors, Oryza meridionalis Ng. accession (W2112) and Oryza rufipogon Griff. accession (IRGC 105491). The lines were genotyped using genotyping-by-sequencing (GBS) and SSRs. The 32 Curinga/O. meridionalis ILs contain 76.73 % of the donor genome in individual introgressed segments, and each line has an average of 94.9 % recurrent parent genome. The 48 Curinga/O. rufipogon ILs collectively contain 97.6 % of the donor genome with an average of 89.9 % recurrent parent genome per line. To confirm that these populations were segregating for traits of interest, they were phenotyped for pericarp color in the greenhouse and for four agronomic traits—days to flowering, plant height, number of tillers, and number of panicles—in an upland field environment. Seeds from these IL libraries and the accompanying GBS datasets are publicly available and represent valuable genetic resources for exploring the genetics and breeding potential of rice wild relatives. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0276-7) contains supplementary material, which is available to authorized users.

Enhancing Fusarium crown rot resistance by pyramiding large-effect QTL in barley

Abstract: Fusarium crown rot (FCR) is a serious disease in wheat and barley in semiarid regions worldwide. The feasibility of enhancing FCR resistance by gene pyramiding was investigated by generating and assessing two populations segregating for three large-effect QTL located on the long arms of chromosomes 1H, 3H and 4H, respectively. Significant effects were detected for each of the three QTL in both of the populations assessed. Lines with any combination of two resistant alleles from the three QTL gave, on average, significantly better resistance than those with a single resistant allele only, and lines with resistant alleles from all three of the QTL gave the least FCR symptom. However, wide variations in FCR severity were detected for lines belonging to each of the groups with different numbers of resistant alleles. Significant effects of plant height on FCR were detected in both populations, and a significant association between heading date and FCR severity was detected in one of the two populations assessed. Results from the current study also showed that the effects of a given allele of resistance decreased with the increase in the number of resistant alleles an individual group possessed. Overall, results from this study demonstrate that gene pyramiding can be an effective approach in improving FCR resistance and those lines with resistant alleles from all of the three QTL could be valuable for breeding programs.

BARLEYMAP: physical and genetic mapping of nucleotide sequences and annotation of surrounding loci in barley

Abstract: The BARLEYMAP pipeline was designed to map both genomic sequences and transcripts against sequence-enriched genetic/physical frameworks, with plant breeders as the main target users. It reports the most probable genomic locations of queries after merging results from different resources so that diversity obtained from re-sequencing experiments can be exploited. In addition, the application lists surrounding annotated genes and markers, facilitating downstream analyses. Pre-computed marker datasets can also be created and browsed to facilitate searches and cross referencing. Performance is evaluated by mapping two sets of long transcripts and by locating the physical and genetic positions of four marker collections widely used for high-throughput genotyping of barley cultivars. In addition, genome positions retrieved by BARLEYMAP are compared to positions within a conventional genetic map for a population of recombinant inbred lines, yielding a gene-order accuracy of 96 %. These results reveal advantages and drawbacks of current in silico approaches for barley genomics. A web application to make use of barley data is available at http://floresta.eead.csic.es/barleymap . The pipeline can be set up for any species with similar sequence resources, for which a fully functional standalone version is available for download.

QTLs detected for individual sugars and soluble solids content in apple

Abstract: Sweetness is one of the most important fruit quality traits in breeding programs, determining the overall quality and flavor perception of apples. Selecting for this trait using conventional breeding methods is challenging due to the complexity of its genetic control. In order to improve the efficiency of trait selection via DNA-based markers, extensive studies focused on the detection of quantitative trait loci (QTL) and the development of DNA-based markers associated with QTL regions for traits of interest. Newly discovered QTLs detected in multiple apple breeding populations are presented here for individual sugars (fructose, glucose, sucrose, and sorbitol) and soluble solids content (SSC) at harvest, after 10, and 20 weeks of refrigerated storage followed by 1 week at room temperature in two successive years. A total of 1416 polymorphic SNPs were filtered from the RosBreed Apple SNP Infinium® array for QTL analysis using FlexQTL™ software. QTLs for individual sugars were identified on linkage groups (LG) 1, 2, 3, 4, 5, 9, 11, 12, 13, 15, and 16, and QTLs for SSC were found on LGs 2, 3, 12, 13, and 15. One QTL region on LG 1 was consistently identified for both fructose and sucrose from harvest through storage in both years, which accounted for 34–67 and 13–41 % of total phenotypic variation, respectively. These stable QTLs with high explained phenotypic variation on LG 1 for fructose content indicate a promising genomic region for DNA-based marker development to enable marker-assisted breeding for sweetness selection in apple breeding programs.

Development of maizeSNP3072, a high-throughput compatible SNP array, for DNA fingerprinting identification of Chinese maize varieties.

Abstract: Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the maize (Zea mays L.) genome. SNPs have several advantages over simple sequence repeats, such as ease of data comparison and integration, high-throughput processing of loci, and identification of associated phenotypes. SNPs are thus ideal for DNA fingerprinting, genetic diversity analysis, and marker-assisted breeding. Here, we developed a high-throughput and compatible SNP array, maizeSNP3072, containing 3072 SNPs developed from the maizeSNP50 array. To improve genotyping efficiency, a high-quality cluster file, maizeSNP3072_GT.egt, was constructed. All 3072 SNP loci were localized within different genes, where they were distributed in exons (43 %), promoters (21 %), 3′ untranslated regions (UTRs; 22 %), 5′ UTRs (9 %), and introns (5 %). The average genotyping failure rate using these SNPs was only 6 %, or 3 % using the cluster file to call genotypes. The genotype consistency of repeat sample analysis on Illumina GoldenGate versus Infinium platforms exceeded 96.4 %. The minor allele frequency (MAF) of the SNPs averaged 0.37 based on data from 309 inbred lines. The 3072 SNPs were highly effective for distinguishing among 276 examined hybrids. Comparative analysis using Chinese varieties revealed that the 3072SNP array showed a better marker success rate and higher average MAF values, evaluation scores, and variety-distinguishing efficiency than the maizeSNP50K array. The maizeSNP3072 array thus can be successfully used in DNA fingerprinting identification of Chinese maize varieties and shows potential as a useful tool for germplasm resource evaluation and molecular marker-assisted breeding. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0335-0) contains supplementary material, which is available to authorized users.

Population structure and marker-trait association studies of iron, zinc and selenium concentrations in seed of field pea (Pisum sativum L.)

Abstract: Association analyses of iron (Fe), zinc (Zn) and selenium (Se) concentrations in seeds of field pea (Pisum sativum L.) were done using a panel of 94 diverse accessions. The panel genotypes were grown at two locations in the province of Saskatchewan, Canada, in 2011 and 2012 growing seasons. A total of 1,233 EST-based single-nucleotide polymorphism markers (SNPs) equally distributed across the pea genome as Illumina GoldenGate assay were used to genotype the panel accessions and to estimate the population structure. Eight subpopulations were identified which sufficiently explained the genetic structure of the panel genotypes. Bonferroni threshold was used to minimize the false discovery rate by combining the structured association (Q) with kinship (K) in a mixed linear model implemented in Trait Analysis by Association, Evolution and Linkage (TASSEL). A total of nine SNPs (−log10 p value ≥4.2) were significantly associated with iron, and two SNPs with zinc concentration in seeds; however, none of the markers was associated with seed Se concentration. The SNPs associated with iron and zinc concentrations in seeds were potential for use in marker-assisted selection in pea breeding programs.

QTLs for flag leaf size and their influence on yield-related traits in wheat ( Triticum aestivum L.)

Abstract: Flag leaf-related traits (FLRTs) are determinant traits affecting plant architecture and yield potential in wheat (Triticum aestivum L.). In this study, a recombinant inbred line population with 188 lines, derived from the cross between Kenong9204 and Jing411, was developed to identify quantitative trait loci (QTL) for flag leaf width (FLW), length (FLL), and area (FLA) under both low nitrogen and high nitrogen treatments. A total of 38 QTLs were detected in eight environments (year × location × treatment). Of these, two QTLs for FLW on chromosomes 4B and 6B (qFlw-4B.3 and qFlw-6B.2) and one for FLA on chromosome 5B (qFla-5B) were major stable QTLs. Both phenotypic and QTL mapping analyses indicated that FLW was the major contributor to flag leaf size. To investigate the genetic relationship between FLRTs and yield-related traits (YRTs) at the QTL level, both unconditional and multivariable conditional QTL mapping for YRTs with respect to FLRTs were conducted. Twelve QTL clusters simultaneously controlling FLRTs and YRTs were identified. In comparison with unconditional QTL mapping, conditional QTL mapping analysis revealed that most but not all the QTL for YRTs were improved by FLRTs. At the QTL level, FLA had the greatest contribution to YRTs, followed by FLW and FLL. This study provided a genetic foundation from which to obtain desirable plant architecture and improve yield potential in wheat breeding programs.

Evaluation of marker-assisted selection for the stripe rust resistance gene Yr15, introgressed from wild emmer wheat.

Abstract: Stripe rust disease is caused by the fungus Puccinia striiformis f. sp. tritici and severely threatens wheat worldwide, repeatedly breaking resistance conferred by resistance genes and evolving more aggressive strains. Wild emmer wheat, Triticum dicoccoides, is an important source for novel stripe rust resistance (Yr) genes. Yr15, a major gene located on chromosome 1BS of T. dicoccoides, was previously reported to confer resistance to a broad spectrum of stripe rust isolates, at both seedling and adult plant stages. Introgressions of Yr15 into cultivated T. aestivum bread wheat and T. durum pasta wheat that began in the 1980s are widely used. In the present study, we aimed to validate SSR markers from the Yr15 region as efficient tools for marker-assisted selection (MAS) for introgression of Yr15 into wheat and to compare the outcome of gene introgression by MAS and by conventional phenotypic selection. Our findings establish the validity of MAS for introgression of Yr15 into wheat. We show that the size of the introgressed segment, defined by flanking markers, varies for both phenotypic selection and MAS. The genetic distance of the MAS marker from Yr15 and the number of backcross steps were the main factors affecting the length of the introgressed donor segments. Markers Xbarc8 and Xgwm493, which are the nearest flanking markers studied, were consistent and polymorphic in all 34 introgressions reported here and are therefore the most recommended markers for the introgression of Yr15 into wheat cultivars. Introgression directed by markers, rather than by phenotype, will facilitate simultaneous selection for multiple stripe rust resistant genes and will help to avoid escapees during the selection process.

In silico mining, characterization and cross-species transferability of EST-SSR markers for European hazelnut (Corylus avellana L.)

Abstract: The European hazelnut (Corylus avellana L.) is one of the most important nut crops. In this work, we characterize functional microsatellite or simple sequence repeat (SSR) markers for genetic analysis and molecular breeding in this species. A total of 38,454 Betulaceae EST sequences from NCBI resulted in 1,282 non-redundant EST-SSRs. Dinucleotide repeats were the most abundant (63.9 %), followed by trinucleotides (33.8 %). The putative functions of the non-redundant EST-SSRs were classified according to gene ontology (GO) categories (biological process, molecular function, and cellular component). A total of 921 sequences showed significant hits with the non-redundant protein database, and GO categories were assigned to 696 (75.5 %) of them. Flanking primer pairs were designed for 78 di- and trinucleotide EST-SSRs from Alnus glutinosa L. (29), Betula pendula Roth (26), and Betula platyphylla Suckaczev (23). Further, 41 dinucleotide repeats selected from hazelnut transcriptome sequences were added. Thirty-six out 119 primer pairs generated amplification products in six hazelnut accessions and in the samples of the species from which they were isolated. Among them, 20 were polymorphic when tested on 18 hazelnut cultivars. Fifteen loci are suitable for mapping in a F1 population of ‘Tonda Gentile delle Langhe’ × ‘Merveille de Bollwiller’ and 11 of them were functionally annotated. The cross-species transferability of 36 EST-SSR loci within nine Corylus species was also performed. The success rate of markers transferability (excluding C. avellana) ranged from 11 to 100 %, with an average of 55 %. The EST-SSRs developed increase the number of markers currently available for hazelnut.

Genetic mapping and legume synteny of aphid resistance in African cowpea (Vigna unguiculata L. Walp.) grown in California

Abstract: The cowpea aphid Aphis craccivora Koch (CPA) is a destructive insect pest of cowpea, a staple legume crop in Sub-Saharan Africa and other semiarid warm tropics and subtropics. In California, CPA causes damage on all local cultivars from early vegetative to pod development growth stages. Sources of CPA resistance are available in African cowpea germplasm. However, their utilization in breeding is limited by the lack of information on inheritance, genomic location and marker linkage associations of the resistance determinants. In the research reported here, a recombinant inbred line (RIL) population derived from a cross between a susceptible California blackeye cultivar (CB27) and a resistant African breeding line (IT97K-556-6) was genotyped with 1,536 SNP markers. The RILs and parents were phenotyped for CPA resistance using field-based screenings during two main crop seasons in a 'hotspot' location for this pest within the primary growing region of the Central Valley of California. One minor and one major quantitative trait locus (QTL) were consistently mapped on linkage groups 1 and 7, respectively, both with favorable alleles contributed from IT97K-556-6. The major QTL appeared dominant based on a validation test in a related F2 population. SNP markers flanking each QTL were positioned in physical contigs carrying genes involved in plant defense based on synteny with related legumes. These markers could be used to introgress resistance alleles from IT97K-556-6 into susceptible local blackeye varieties by backcrossing.

Mapping QTLs of resistance to head splitting in cabbage ( Brassica oleracea L.var . capitata L.)

Abstract: Cabbage head splitting can greatly affect both the quality and commercial value of cabbage (Brassica oleracea). To detect the genetic basis of head-splitting resistance, a genetic map was constructed using an F2 population derived by crossing “748” (head-splitting-resistant inbred line) and “747” (head-splitting-susceptible inbred line). The map spans 830.9 cM and comprises 270 markers distributed in nine linkage groups, which correspond to the nine chromosomes of B. oleracea. The average distance between adjacent markers was 3.6 cM. A total of six quantitative trait loci (QTLs) conferring resistance to head splitting were detected in chromosome 2, 4, and 6. Two QTLs, SPL-2-1 and SPL-4-1, on chromosomes 2 and 4, respectively, were detected in the experiments over 2 years, suggesting that these two potential loci were important for governing the head-splitting resistance trait. Markers BRPGM0676 and BRMS137, which were tightly linked with head-splitting resistance, were detected in the conserved QTL SPL-2-1 region using bulked segregant analysis. Synteny analysis showed that SPL-2-1 was anchored to a 3.18-Mb genomic region of the B. oleracea genome, homologous to crucifer ancestral karyotype E block in chromosome 1 of Arabidopsis thaliana. Moreover, using a field emission scanning electron microscope, significant differences were observed between the two parental lines in terms of cell structures. Line “747” had thinner cell wall, lower cell density, larger cell size, and anomalous cell wall structure compared with the resistant line “748.” The different cell structures can provide a cytological base for assessing cabbage head splitting.

Mapping of a new stripe rust resistance locus Yr57 on chromosome 3BS of wheat

Abstract: A common wheat landrace, AUS27858, from the Watkins collection showed low seedling stripe rust response against Australian Puccinia striiformis f. sp. tritici pathotypes. Genetic analysis of stripe rust resistance indicated the involvement of two independent resistance loci YrAW1 and YrAW2. YrAW1 was mapped in chromosome 4AL and formally named Yr51. Ninety seeds of a heterozygous F3 line (HSB#5474; YrAW2yrAW2) were grown individually to produce a segregating population referred to as single gene segregating population #5474 (SGSP#5474) to map YrAW2. Monogenic segregation at the YrAW2 locus was confirmed among the SGSP#5474. YrAW2 was located in chromosome 3BS through DArT-based bulked segregant analysis. SGSP#5474 was advanced to F6 generation and was phenotyped for detailed mapping. Test of allelism with Yr4, previously located on chromosome 3BS, showed 5.2 ± 1.3 % recombination between YrAW2 and Yr4. Since there is no other stripe rust resistance gene located in the distal part of chromosome 3BS, YrAW2 was formally named Yr57. Markers gwm389 and BS00062676 flanked Yr57 at genetic distances of 2.0 and 2.3 cM, proximally and distally, respectively. These markers were genotyped on a set of Australian and Indian wheat cultivars and the absence of resistance-linked alleles of gwm389 and BS00062676 markers was shown in cultivars known to lack Yr57. These markers would be useful in marker-assisted pyramiding of Yr57 with other marker-tagged major and minor genes. The genetic stock carrying Yr57 singly has been deposited with the Australian Winter Cereal Collection, Tamworth, Australia, and it is accessioned as AUS91463.

Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system

Abstract: Cisgenic apple plants of two different cultivars were developed by transferring the Rvi6 scab resistance gene of Malus floribunda 821, using a new transformation vector based on the Flp/FRT recombinase system. Transformation experiments on seven different cultivars resulted in 22 transgenic lines for the cultivars ‘Brookfield Baigent’, ‘Mitchgla’, ‘Novajo’, and ‘Pinova’, whereby 16 lines thereof were resistant to Venturia inaequalis strain 104 (race 1). Analysis of the transgenic lines revealed Rvi6 mRNA expression levels comparable to several traditional bred Rvi6 containing cultivars and identified four transgenic lines, harboring a single T-DNA insertion, as suitable for the production of cisgenic lines. The T-DNA insertion site of these lines was determined, and lines were subject to induction of the recombinase system. Two cisgenic lines originating from the cultivars ‘Brookfield Baigent’ and ‘Pinova’ were obtained for which the exact excision of the recombinase cassette was confirmed by sequencing the previously determined T-DNA integration site. Further investigations revealed both cisgenic lines as fully resistant to V. inaequalis race 1. Rvi6 mRNA expression of the cisgenic lines and traditionally bred Rvi6 harboring cultivars was still comparable. The transformation vector developed is useable for the production of cisgenic apple plants to a certain extent.

A restriction-site-associated DNA (RAD) linkage map, comparative genomics and identification of QTL for histological fibre content coincident with those for retted bast fibre yield and its major components in jute (Corchorus olitorius L., Malvaceae s. l.).

Abstract: We used RAD (restriction-site-associated DNA) sequencing to detect genome-wide SNPs and construct a dense linkage map using an intercross F2 population in jute (Corchorus olitorius). The linkage map comprising a total of 503 RAD markers in seven linkage groups spanned 358.5 cM with an average marker interval of 0.72 cM and covered 87.0 % of the genome. Genome-wide segregation distortion of the mapped loci (34.4 %) was non-random across the linkage map, with a directional bias mostly towards the female genotypes. Jute had maximum syntenic relationships with cocoa (47.5 % homology) and diploid cotton (29.2 % homology). However, synteny and collinearity were not conserved. Histological fibre content (FC; total number of fibre cell bundles in a stem cross section) was positively correlated with fibre yield (FY), plant height (PH), root weight (RW) and stem-base diameter (SBD). Broad-sense heritability estimates were high for all traits, with FC and FY showing maximum heritability (~93 %). QTL mapping based on the F2:3 phenotypes detected nine QTL across the two environments. The QTL for FC was coincident with one QTL each for FY, PH, RW and SBD on top of a single-SNP (C/T) marker at 40.2 cM on LG1, each accounting for ~7–11 % of the phenotypic variance. Two QTL linked in repulsion one each for PH and SBD, with varying degrees of overdominance, were associated with two single-SNP (C/T) markers on LG2, each accounting for ~17–18 % of the phenotypic variance. Few candidate genes were identified within the QTL regions. Our results would enable development of tools for marker-assisted selection in jute.

Association mapping and genomic prediction for resistance to sudden death syndrome in early maturing soybean germplasm

Abstract: Sudden death syndrome (SDS), caused by Fusarium virguliforme, has spread to northern soybean growing regions in the US causing significant yield losses. The objectives of this study were to identify loci underlying variation in plant responses to SDS through association mapping (AM) and to assess prediction accuracy of genomic selection (GS) in a panel of early maturing soybean germplasm. A set of 282 soybean breeding lines was selected from the University of Minnesota soybean breeding program and then genotyped using a genome-wide panel of 1536 single-nucleotide polymorphism markers. Four resistance traits, root lesion severity (RLS), foliar symptom severity (FSS), root retention (RR), and dry matter reduction (DMR), were evaluated using soil inoculation in the greenhouse. AM identified significant peaks in genomic regions of known SDS resistance quantitative trait loci cqSDS001, cqRfs4, and SDS11-2. Additionally, two novel loci, one on chromosome 3 and another on chromosome 18, were tentatively identified. A ninefold cross-validation scheme was used to assess the prediction accuracy of GS for SDS resistance. The prediction accuracy of single-trait GS (ST-GS) was 0.64 for RLS, but less than 0.30 for RR, DMR, and FSS. Compared to ST-GS, none of multi-trait GS (MT-GS) models significantly improved the prediction accuracy due to weak correlations between the four traits. This study suggests both AM and GS hold promise for implementation in genetic improvement of SDS resistance in existing soybean breeding programs.

Overexpression of OsmiR156k leads to reduced tolerance to cold stress in rice (Oryza Sativa)

Abstract: The microRNA156 (miR156) family ‘has’ been well demonstrated to regulate plant growth and development. However, no reports focused on the roles of miR156s in environmental stress responses. In previous studies, we identified 18 cold stress responsive microRNAs in rice by using microarray analysis. Here, in the present study, we focused on the biological function of one of these cold responsive microRNAs, OsmiR156k. We generated the transgenic rice overexpressing OsmiR156k under the control of CaMV35S promoter, and verified the presence of OsmiR156k by using Southern blot analysis. We found that overexpression of OsmiR156k inhibited the seedling growth at the very early seedling stage under cold stress. Furthermore, OsmiR156k overexpression decreased plant cold tolerance at the young seedling growth stage, as evidenced by lower survival rates, chlorophyll contents and proline contents. As expected, we also suggested the down-regulated expression of the cold stress responsive genes, 01g22249 and OsP5CS, and OsmiR156k-tagated SPL genes, SPL3, SPL14 and SPL17, in the OsmiR156k transgenic lines. Taken together, our findings suggest that overexpression of OsmiR156k decreased the tolerance to cold stress in rice.

Association mapping of agronomic and quality traits in USDA pea single-plant collection

Abstract: Association mapping is an efficient approach for the identification of the molecular basis of agronomic traits in crop plants. For this purpose in pea (Pisum sativum L.), we genotyped and phenotyped individual lines of the single-plant-derived core collection of the USDA pea collection including accessions from 330 landraces and cultivars of Pisum sativum subsp. sativum var. sativum, 28 P. sativum subsp. elatius var. elatius, 16 P. sativum subsp. sativum var. arvense, four P. sativum subsp. elatius var. pumilio, three P. abyssinicum, two P. fulvum, and one P. sativum subsp. transcaucasicum. These 384 accessions were collected or donated from a total of 64 countries. The accessions were genotyped with 256 informative SNPs using a primer extension chemistry and matrix-assisted laser desorption/ionization (MALDI–TOF) mass spectrometry assay. Genetic structure analysis showed that the collection was structured into two main groups, corresponding roughly to the cultivated types/landraces and the more primitive form species and subspecies, with some intermediates. Linkage disequilibrium of pairwise loci and population structure of the collection were analyzed, and an association analysis between SNP genotypes and 25 valuable traits such as disease resistance, seed type/color, flower color, seed low molecular weight carbohydrate concentration, and seed mineral nutrient concentration was performed using a mixed linear model. A total of 71 marker–trait associations were detected as significant with 1–34 markers per trait based on the false discovery rate (FDR < 0.05). This study demonstrates the potential of using association mapping to identify markers for pea breeding.

Genetic dissection of fruit weight and size in an F2 peach (Prunus persica (L.) Batsch) progeny

Abstract: Fruit weight is a quantitative trait influenced by the combined action of several genes and environmental factors. Knowledge of the quantitative trait loci (QTLs) associated with fruit weight and size is a priority to support breeding programmes in peach (Prunus persica (L.) Batsch) because of commercial interest in larger fruits. To this end, we built a genetic map of an F2 progeny of 117 individuals from the cross PI91459 (‘NJ Weeping’) × ‘Bounty’ using a single nucleotide polymorphism (SNP) genotyping array for peach (9K SNP array v1). Data for fruit weight, height, width, and depth were recorded for the progeny and both parents over 2 years (2011, 2012). Correlations between the traits fruit weight and size were positive and significant for both years. A SNP map was constructed comprising 1,148 markers distributed over eight linkage groups. The map spans 536.6 cM with an average distance between markers of 0.52 cM, covering 93.6 % of the physical length of the peach genome, thus representing an ideal basis for QTL mapping. QTL analysis led to the identification of a total of 28 QTLs for the considered traits, eleven of which remained stable in both years. We also observed clusters of QTLs, some of which were mapped for the first time, while others correspond to loci previously identified in different progenies and following different approaches.

Heterotic loci for various morphological traits of maize detected using a single segment substitution lines test-cross population

Abstract: Heterosis has been exploited to increase grain yield, quality, and resistance in many crops, and it plays an important role in plant breeding. However, the genetic mechanism of heterosis remains unclear. To dissect the genetic basis of heterosis, a set of 203 single segment substitution lines (SSSLs) was developed, and its test-cross population was used to identify heterotic loci (HL) for plant morphological traits in maize, including plant height (PH), ear height (EH), leaf number (LN), tassel main axis length (TMAL), and tassel branch number (TBN). A total of 41 QTLs and 37 HL were identified for five morphological traits in the test-cross population derived from the 203 SSSLs and the parent, Xu178. Nine HL for PH, nine HL for EH, seven HL for LN, seven HL for TMAL, and five HL for TBN were detected in three different environments, respectively. Eight HL, ph1a, ph1b, ph2, ph5, eh3a, eh3b, eh10, and tmal1b, were simultaneously detected in the three environments. Among the 37 HL, only 10 (27.03 %; for PH, EH, LN, and TBN) had a corresponding QTL (24.39 %) sharing the same chromosomal region. Of all the HL, 21.4 % showed dominance effects, 76.8 % showed over-dominance effects, and only one (1.8 %) showed a partial-dominance effect. This result illustrated that heterosis and performance was controlled by different genetic mechanisms, and over-dominance effects were the main contributors to heterosis for plant-related traits at the single-locus level in maize.

Fine mapping of DTH3b, a minor heading date QTL potentially functioning upstream of Hd3a and RFT1 under long-day conditions in rice

Abstract: Flowering time or heading date in rice is an important agronomic trait that determines cultivation area and cropping season of a given variety. The genes/loci that have minor effect on the heading date are believed to play a critical role in adaptation of rice to different geographical regions and are preferred by breeders. Previously, we detected a stable minor-effect quantitative trait locus, qDTH-3b (for Days to heading 3b; hereafter referred to as DTH3b), using a small population consisting of recombinant inbred lines derived from a cross between the japonica cv. Asominori (Aso) and the indica cv. IR24. However, its precise location remains to be defined. In this study, we fine-mapped DTH3b by using advanced backcrossing lines and explored its role in regulating the heading date. First, we constructed a BC4F2 population by backcrossing a chromosome segment substitution line (CSSL23) with Aso as a recurrent parent. Then, we developed a near-isogenic line (NIL) from this population by marker-assisted selection. This NIL has the genetic background of Aso but carries a 12-cM DTH3b-containing chromosome segment from IR24. Compared with Aso, the NIL showed 6.9-day delay in flowering time and 63.8 % lower seed maturation rate under long-day (LD) conditions, whereas there was no significant difference between the NIL and Aso under short-day conditions. Using a total of 1500 Asominori/NIL F2:3 or F3:4 late-heading families grown under LD conditions, we finally dissected DTH3b to a single Mendelian factor and delimited it to a 46-kb genomic region which contains seven open reading frames. Further, our quantitative real-time PCR analysis indicated that transcription level of Hd3a (Heading date 3a) and RFT1 (RICE FLOWERING LOCUS T 1), the two florigen genes, was significantly lower in the NIL than in Aso, suggesting that DTH3b functions upstream of Hd3a and RFT1 under LD conditions. We propose that DTH3b Aso positively regulates flowering time and contributes to adaptation of rice to the north. Cloning and then manipulation of DTH3b can be a useful approach to optimize flowering time in rice breeding.

Construction of a genetic map for pearl millet, Pennisetum glaucum (L.) R. Br., using a genotyping-by-sequencing (GBS) approach

Abstract: Pearl millet is the main component of traditional farming systems and a staple grain in the diet of sub-Saharan Africa and India. To facilitate breeding work in this crop, a genetic map consisting of single nucleotide polymorphism (SNP) markers was constructed using an F2 population of 93 progenies, from a wild × cultivated pearl millet cross. We used a modified genotyping-by-sequencing (GBS) protocol involving two restriction enzymes (PstI–MspI) and PCR amplification with primers including three selective bases to generate 3,321 SNPs. Of these, 2,809 high-quality SNPs exhibited a minor allele frequency ≥0.3. In total, 314 non-redundant haplotypes and 85 F2 individuals were used to construct a genetic map spanning a total distance of 640 cM. These SNPs were evenly distributed over seven linkage groups ranging considerably in size (62–123 cM). The average density for this map was 0.51 SNP/cM, and the average interval between SNP markers was 2.1 (±0.6) cM. Finally, to establish bridges between the linkage groups of this and previous maps, 19 SSR markers were examined for polymorphism between the parents of this population. We could only tentatively suggest a correspondence between four of our linkage groups and those of previous maps. Overall, GBS enabled us to quickly produce a genetic map with a density and uniformity of markers greater than previously published maps. The availability of such a map will be useful for the identification of genomic regions associated with Striga resistance and other important agronomic traits.

Marker-assisted introgression of bacterial blight and blast resistance into DRR17B, an elite, fine-grain type maintainer line of rice

Abstract: DRR17A is a stable wild-abortive cytoplasmic male sterile line with medium-slender grain type. DRR17A and its maintainer line DRR17B are highly susceptible to two of the major rice diseases, bacterial blight (BB) and blast. To improve DRR17B for resistance against BB and blast, we have introgressed a major dominant gene each conferring resistance against BB (Xa21) and blast (Pi54) into the maintainer line through marker-assisted backcross breeding using RP-Bio-Patho-2 (a near-isogenic line of Samba Mahsuri possessing Xa21 and Pi54) as the donor parent. PCR-based molecular markers tightly linked to Xa21 and Pi54 were used for foreground selection of the resistance plants at each backcross generation, while molecular markers tightly linked to the major fertility restorer genes, Rf3 and Rf4, were used for negative selection (i.e. selection of plants possessing non-fertility-restoring alleles at the two loci) at BC1 generation. After foreground selection for the target genes at each backcross generation, the ‘positive’ plants were screened with parental polymorphic markers for identifying backcross plants possessing maximum recovery of DRR17B genome. Marker-assisted backcrossing was continued till BC3 generation, and a single BC3F1 plant possessing the target genes with ~94 % recovery of recurrent parent genome was identified and selfed to generate BC3F2s. A total of six homozygous BC3F2 plants were identified and advanced. At BC3F5, six promising, stable, backcross-derived lines possessing high level of resistance against BB and blast, high yield, short plant stature, fine-grain type, have been identified; their maintenance ability and heterotic potential validated through test crosses and these lines are being converted to CMS lines through marker-assisted breeding.

Gene targeting and editing in crop plants: a new era of precision opportunities

Abstract: With increasing global food demands in the face of challenging biotic and abiotic pressures on crop production, there is a vital need for good crop improvement strategies. Gene editing and gene targeting using designer nucleases are relatively new, sophisticated approaches that can be used for crop improvement. Designer nucleases are molecules that can be engineered to cleave virtually any endogenous DNA target sequence, making this technology inherently more powerful over current, essentially random mutation strategies. These molecules can also be used to promote targeted DNA insertions and homologous recombination. Further modifications of these molecules can convert them into designer transcription factors that can activate or suppress a gene of choice. Four designer nuclease platforms are currently available: meganucleases, zinc finger nucleases, TALENs and the more recently developed CRISPR/Cas9 system. All four of these systems have been shown to function in crop plants and have been used for site-specific gene targeting and gene editing. Herein, we describe the basis of each designer nuclease platform, highlighting the advantages and disadvantages of each, and give examples of their application in crop improvement.

OsEXPB2, a β-expansin gene, is involved in rice root system architecture

Abstract: Expansins are cell wall loosening proteins which generally play important roles as endogenous regulators in plants. Here we report a rice (Oryza sativa L.) β-expansin gene, OsEXPB2, which encodes a 28.6-kDa protein of 261 amino acids. Sequence alignment revealed that the N-terminal and C-terminal of OsEXPB2 share six discontinuous cysteine residues and four intermittent tryptophan residues, respectively. The OsEXPB2 promoter contains conserved root hair-specific elements. Subcellular localization assay revealed that OsEXPB2 was localized in the cell wall. Analysis of spatial and temporal expression patterns demonstrated that OsEXPB2 was predominantly expressed in root of rice. OsEXPB2 expression levels were up-regulated by abiotic stresses, such as phosphate or iron deficiency, and also suppressed by abscisic acid. A clear difference was observed between RNA interference (RNAi) lines and wild-type in root system architecture and plant height, and the suppression of OsEXPB2 resulted in a visible alteration of the width of the leaf blade. Anatomical analysis found that the cell size of root cortical cells in OsEXPB2-suppressed lines was significantly smaller than that of their counterparts in wild-type plants. Furthermore, cryo-scanning electron microscopy analysis showed that the development of root hair was suppressed in RNAi lines. All these results suggest that OsEXPB2 is a root-predominant gene with a key role in root-hair formation and has the potential to be utilized in transgenic root breeding to improve abiotic stress tolerance.

QTL mapping of pomological traits in peach and related species breeding germplasm

Abstract: Peach is an economically important fruit tree crop that exhibits high phenotypic variability yet suffers from diversity-limited gene pool. Genetic introgression of novel alleles from related species is being pursued to expand genetic diversity. This process is, however, challenging and requires the incorporation of innovative genomic and statistical tools to facilitate efficient transfer of these exotic alleles across the multiple generations required for introgression. In this study, pedigree-based analysis (PBA) in a Bayesian QTL mapping framework was applied to a diverse peach pedigree introgressed with almond and other related Prunus species. The aim was to investigate the genetic control of eight commercially important fruit productivity and fruit quality traits over two subsequent years. Fifty-two QTLs with at least positive evidence explaining up to 98 % of the phenotypic variance across all trait/year combinations were mapped separately per trait and year. Several QTLs exhibited variable association with traits between years. By using the peach genome sequence as a reference, the intrachromosomal positions for several QTLs were shown to differ from those previously reported in peach. The inclusion of introgressed germplasm and the explicit declaration of the genetic structure of the pedigree as covariate in PBA enhanced the mapping and interpretation of QTLs. This study serves as a model study for PBA in a diverse peach breeding program, and the results highlight the ability of this strategy to identify genomic resources for direct utilization in marker-assisted breeding.