Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.     

A SNP in the 5′ flanking region of the myostatin-1b gene is associated with harvest traits in Atlantic salmon (Salmo salar)

Background

Myostatin (MSTN) belongs to the transforming growth factor-β superfamily and is a potent negative regulator of skeletal muscle development and growth in mammals. Most teleost fish possess two MSTN paralogues. However, as a consequence of a recent whole genome-duplication event, salmonids have four: MSTN-1 (?1a and -1b) and MSTN-2 (?2a and -2b). Evidence suggests that teleost MSTN plays a role in the regulation of muscle growth. In the current study, the MSTN-1b gene was re-sequenced and screened for SNP markers in a commercial population of Atlantic salmon. After genotyping 4,800 progeny for the discovered SNPs, we investigated their association with eight harvest traits - four body-weight traits, two ratios of weight traits, flesh colour and fat percentage - using a mixed model association analysis.

Results

Three novel SNPs were discovered in the MSTN-1b gene of Atlantic salmon. One of the SNPs, located within the 5′ flanking region (g.1086C?>?T), had a significant association with harvest traits (p?<?0.05), specifically for: Harvest Weight (kg), Gutted Weight (kg), Deheaded Weight (kg) and Fillet Weight (kg). The haplotype-based association analysis was consistent with this result because the two haplotypes that showed a significant association with body-weight traits, hap4 and hap5 (p?<?0.05 and p?<?0.01, respectively), differ by a single substitution at the g.1086C?>?T locus. The alleles at g.1086C?>?T act in an additive manner and explain a small percentage of the genetic variation of these phenotypes.

Conclusions

The association analysis revealed that g.1086C?>?T had a significant association with all body-weight traits under study. Although the SNP explains a small percentage of the variance, our results indicate that a variation in the 5′ flanking region of the myostatin gene is associated with the genetic regulation of growth in Atlantic salmon.

     

High-throughput single nucleotide polymorphism (SNP) identification and mapping in the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) genome with genotyping by sequencing (GBS) analysis

Sesame (Sesamum indicum L. syn. Sesamum orientale L.) is considered to be the first oil seed crop known to man. Despite its versatile use as an oil seed and a leafy vegetable, sesame is a neglected crop and has not been a subject of molecular genetic research until the last decade. There is thus limited knowledge regarding genome-specific molecular markers that are indispensible for germplasm enhancement, gene identification, and marker-assisted breeding in sesame. In this study, we employed a genotyping by sequencing (GBS) approach to a sesame recombinant inbred line (RIL) population for high-throughput single nucleotide polymorphism (SNP) identification and genotyping. A total of 15,521 SNPs were identified with 14,786 SNPs (95.26 %) located along sesame genome assembly pseudomolecules. By incorporating sesame-specific simple sequence repeat (SSR) markers developed in our previous work, 230.73 megabases (99 %) of sequence from the genome assembly were saturated with markers. This large number of markers will be available for sesame geneticists as a resource for candidate polymorphisms located along the physical chromosomes of sesame. Defining SNP loci in genome assembly sequences provides the flexibility to utilize any genotyping strategy to survey any sesame population. SNPs selected through a high stringency filtering protocol (770 SNPs) for improved map accuracy were used in conjunction with SSR markers (50 SSRs) in linkage analysis, resulting in 13 linkage groups that encompass a total genetic distance of 914 cM with 432 markers (420 SNPs, 12 SSRs). The genetic linkage map constitutes the basis for future work that will involve quantitative trait locus (QTL) analyses of metabolic and agronomic traits in the segregating RIL population.     

Important photosynthetic contribution of silique wall to seed yield-related traits in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In plants, green non-foliar organs are able to perform photosynthesis just as leaves do, and the seed-enclosing pod acts as an essential photosynthetic organ in legume and Brassica species. To date, the contribution of pod photosynthesis to seed yield and related components still remains largely unexplored, and in Arabidopsis thaliana, the photosynthetic activity of the silique (pod) is unknown. In this study, an Arabidopsis glk1/glk2 mutant defective in both leaf and silique photosynthesis was used to create tissue-specific functional complementation lines. These lines were used to analyze the contribution of silique wall photosynthesis to seed yield and related traits, and to permit the comparison of this contribution with that of leaf photosynthesis. Our results showed that, together with leaves, the photosynthetic assimilation of the silique wall greatly contributed to total seed yield per plant. As for individual components of yield traits, leaf photosynthesis alone contributed to the seed number per silique and silique length, while silique wall photosynthesis alone contributed to thousand-seed weight. In addition, enhancing the photosynthetic capacity of the silique wall by overexpressing the photosynthesis-related RCA gene in this tissue resulted in significantly increased seed weight and oil content in the wild-type (WT) background. These results reveal that silique wall photosynthesis plays an important role in seed-related traits, and that enhancing silique photosynthesis in WT plants can further improve seed yield-related traits and oil production. This finding may have significant implications for improving the seed yield and oil production of oilseed crops and other species with pod-like organs.     

Genome-wide association study on chicken carcass traits using sequence data imputed from SNP array

Chicken carcass traits are economically important for the chicken industry. Detecting which genes affect chicken carcass traits is of great benefit to the genetic improvement of this important agricultural species. To investigate the genetic mechanism of carcass traits in chickens, we carried out a genome-wide association study (GWAS). A total of 435 Chinese indigenous chickens were phenotyped for carcass weight (CW), eviscerated weight with giblets (EWG), and eviscerated weight (EW) after slaughter at 91 days and were genotyped using a 600-K single nucleotide polymorphism (SNP) genotyping array. Twenty-four birds were selected for sequencing, and the 600 K SNP panel data were imputed to sequence data with the 24 birds as the reference. Univariate GWASs were performed with GEMMA software using the whole genome sequence data imputed from SNP chip data. Finally, 3, 25, and 63 suggestively significant SNPs were identified to be associated with carcass weight (CW), eviscerated weight with giblets (EWG), and eviscerated weight (EW), respectively. Six candidate genes, RNF219, SCEL, MYCBP2, ETS1, APLP2, and PRDM10 were detected. SCEL and MYCBP2 were potentially associated with these three traits, RNF219 and APLP2 were potentially associated with EWG and EW, and ETS1 and PRDM10 were only potentially associated with EWG and EW, respectively. Compared with forefathers’ research, 10 reported QTLs associated with CW were located within a 5-Mb distance near the SNPs with P value lower than 1×10^?5. This study enriched the knowledge of the genetic mechanisms of chicken carcass traits.     

A genome-wide association study uncovers novel genomic regions and candidate genes of yield-related traits in upland cotton

Key message

A total of 62 SNPs associated with yield-related traits were identified by a GWAS. Based on significant SNPs, two candidate genes pleiotropically increase lint yield.

Abstract

Improved fibre yield is considered a constant goal of upland cotton (Gossypium hirsutum) breeding worldwide, but the understanding of the genetic basis controlling yield-related traits remains limited. To better decipher the molecular mechanism underlying these traits, we conducted a genome-wide association study to determine candidate loci associated with six yield-related traits in a population of 719 upland cotton germplasm accessions; to accomplish this, we used 10,511 single-nucleotide polymorphisms (SNPs) genotyped by an Illumina CottonSNP63K array. Six traits, including the boll number, boll weight, lint percentage, fruit branch number, seed index and lint index, were assessed in multiple environments; large variation in all phenotypes was detected across accessions. We identified 62 SNP loci that were significantly associated with different traits on chromosomes A07, D03, D05, D09, D10 and D12. A total of 689 candidate genes were screened, and 27 of them contained at least one significant SNP. Furthermore, two genes (Gh_D03G1064 and Gh_D12G2354) that pleiotropically increase lint yield were identified. These identified SNPs and candidate genes provide important insights into the genetic control underlying high yields in G. hirsutum, ultimately facilitating breeding programmes of high-yielding cotton.

     

Identification of loci controlling forage yield and nutritive value in diploid alfalfa using GBS-GWAS

Key message

We attempted to identify genomic regions controlling forage yield and nutritive value in alfalfa. Several candidate genes and associated genetic markers were identified that could potentially be useful for alfalfa breeding to more efficiently develop improved cultivars.

Abstract

Alfalfa is one of the most widely cultivated forage legumes worldwide and improving alfalfa forage yield and nutritive value is a major global breeding goal. Genotyping-by-sequencing (GBS) provides cost-effective molecular marker genotyping for genome-wide association studies (GWAS). Using more than 15,000 genome-wide single nucleotide polymorphisms (SNP) identified from GBS, we conducted a GWAS to investigate forage yield and nutritive value-related traits. We have detected a number of associations for all the traits evaluated and a number of associations detected were located on the Medicago truncatula genome. The SNP in a coding region of a cell wall biosynthesis gene was associated with several cell wall-related traits, and we suggest that it may be the causative polymorphism. Two other SNPs residing in meristematic development and early growth genes were found to associate with the total biomass yield. None of the SNPs associated with regrowth after harvest or with spring regrowth were mapped to the M. truncatula genome, possibly reflecting the fact that M. truncatula is an annual species related to alfalfa that typically has limited ability to regrow. The alleles we identify with the major impact on forage yield and nutritive value can be rapidly incorporated into our breeding program.

     

Genetic variation of a global germplasm collection of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) including Italian accessions at risk of genetic erosion

Chickpea (Cicer arietinum L.) is one of the most important legumes worldwide. We addressed this study to the genetic characterization of a germplasm collection from main chickpea growing countries. Several Italian traditional landraces at risk of genetic erosion were included in the analysis. Twenty-two simple sequence repeat (SSR) markers, widely used to explore genetic variation in plants, were selected and yielded 218 different alleles. Structure analysis and hierarchical clustering indicated that a model with three distinct subpopulations best fits the data. The composition of two subpopulations, named K1 and K2, broadly reflects the commercial classification of chickpea in the two types desi and kabuli, respectively. The third subpopulation (K3) is composed by both desi and kabuli genotypes. Italian accessions group both in K2 and K3. Interestingly, this study highlights genetic distance between desi genotypes cultivated in Asia and Ethiopia, which respectively represent the chickpea primary and the secondary centres of diversity. Moreover, European desi are closer to the Ethiopian gene pool. Overall, this study will be of importance for chickpea conservation genetics and breeding, which is limited by the poor characterization of germplasm collection.     

<Emphasis Type="Italic">LIG1</Emphasis> polymorphisms: the Indian scenario

Elucidation of the genetic diversity and relatedness of the subpopulations of India may provide a unique resource for future analysis of genetic association of several critical community-specific complex diseases. We performed a comprehensive exploration of single nucleotide polymorphisms (SNPs) within the gene DNA ligase 1 (LIG1) among a multiethnic panel of Indian subpopulations representative of the ethnic, linguistic and geographical diversity of India using a two-stage design involving DNA resequencing-based SNP discovery followed by SNP validation using sequenom-based genotyping. Thirty SNPs were identified in LIG1 gene using DNA resequencing including three promoter SNPs and one coding SNP. Following SNP validation, the SNPs rs20580/C19008A and rs3730862/C8804T were found to have the most widespread prevalence with noticeable variations in minor allele frequencies both between the Indian subpopulation groups and also from those reported on other major world populations. Subsequently, SNPs found in Indian subpopulations were analysed using bioinformatics-based approaches and compared with SNP data available on major world populations. Further, we also performed genotype–phenotype association analysis of LIG1 SNPs with publicly available data on LIG1 mRNA expression in HapMap samples. Results showed polymorphisms in LIG1 affect its expression and may therefore change its function. Our results stress upon the uniqueness of the Indian population with respect to the worldwide scenario and suggest that any epidemiological study undertaken on the global population should take this distinctiveness in consideration and avoid making generalized conclusions.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Genetic Dissection of Drought and Heat Tolerance in Chickpea through Genome-Wide and Candidate Gene-Based Association Mapping Approaches

To understand the genetic basis of tolerance to drought and heat stresses in chickpea, a comprehensive association mapping approach has been undertaken. Phenotypic data were generated on the reference set (300 accessions, including 211 mini-core collection accessions) for drought tolerance related root traits, heat tolerance, yield and yield component traits from 1–7 seasons and 1–3 locations in India (Patancheru, Kanpur, Bangalore) and three locations in Africa (Nairobi, Egerton in Kenya and Debre Zeit in Ethiopia). Diversity Array Technology (DArT) markers equally distributed across chickpea genome were used to determine population structure and three sub-populations were identified using admixture model in STRUCTURE. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations (r²; when r²<0.20) was found to decay rapidly with the genetic distance of 5 cM. For establishing marker-trait associations (MTAs), both genome-wide and candidate gene-sequencing based association mapping approaches were conducted using 1,872 markers (1,072 DArTs, 651 single nucleotide polymorphisms [SNPs], 113 gene-based SNPs and 36 simple sequence repeats [SSRs]) and phenotyping data mentioned above employing mixed linear model (MLM) analysis with optimum compression with P3D method and kinship matrix. As a result, 312 significant MTAs were identified and a maximum number of MTAs (70) was identified for 100-seed weight. A total of 18 SNPs from 5 genes (ERECTA, 11 SNPs; ASR, 4 SNPs; DREB, 1 SNP; CAP2 promoter, 1 SNP and AMDH, 1SNP) were significantly associated with different traits. This study provides significant MTAs for drought and heat tolerance in chickpea that can be used, after validation, in molecular breeding for developing superior varieties with enhanced drought and heat tolerance.     

Identification of SNPs tightly linked to the QTL for pod shattering in soybean

The pod shattering or dehiscence is essential for the propagation of pod-bearing plant species in the wild, but it causes significant yield losses during harvest of domesticated crop plants. Identifying novel molecular makers, which are linked to seed-shattering genes, is needed to employ the molecular marker-assisted selection for efficiently developing shattering-resistant soybean varieties. In this study, a genetic linkage map was constructed using 115 recombinant inbred lines (RILs) developed from crosses between the pod shattering susceptible variety, Keunol, and resistant variety, Sinpaldal. A 180 K Axiom® SoyaSNPs data and pod shattering data from two environments in 2001 and 2015 were used to identify quantitative trait loci (QTL) for pod shattering. A major QTL was identified between two flanking single nucleotide polymorphism (SNP) markers, AX-90320801 and AX-90306327 on chromosome 16 with 1.3 cM interval, 857 kb of physical range. In sequence, genotype distribution analysis was conducted using extreme phenotype RILs. This could narrow down the QTL down to 153 kb on the physical map and was designated as qPDH1-KS with 6 annotated gene models. All exons within qPDH1-KS were sequenced and the 6 polymorphic SNPs affecting the amino acid sequence were identified. To develop universally available molecular markers, 38 Korean soybean cultivars were investigated by the association study using the 6 identified SNPs. Only two SNPs were strongly associated with the pod shattering. These two identified SNPs will help to identify the pod shattering responsible gene and to develop pod shattering-resistant soybean plants using marker-assisted selection.     

Association mapping of days to flowering in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) revealed by DArT markers

In the current study, 173 common bean genotypes from several geographic regions were studied. Days to flowering (DF) was evaluated in two experimental locations in Izmir, Turkey (Bornova and Menemen) in 2 years (2015 and 2016) and was found to range from 30 to 62.7 days with a mean value of 41.5 days. A total of 22,848 SNPs based on diversity array technology were developed, and after filtering, the remaining 20,766 SNP markers were used for calculating linkage disequilibrium. Chromosomes 1–11 contained 1846, 2342, 2184, 1153, 1351, 1520, 1953, 2080, 2065, 1199, and 1511 SNPs, respectively. A total of 1562 SNPs were identified as scaffold markers. The PIC value was 0.25, ranging from 0.005 to 0.500. Common bean accessions were divided into two main subpopulations, namely POP1 (Mesoamerican) and POP2 (Andean). Mixed linear model using the Q + K model showed that three SNPs had a significant association (p?<?0.01) in Bornova in 2015 and seven SNPs had a significant association (p?<?0.01) in the same location in 2016. Five significant associations (p?<?0.01) were identified in 2015 while six (p?<?0.01) were identified in Menemen in 2016. When the data from both locations and both years was combined, six SNPs were significant (p?<?0.01). For DF, 11 putative candidate genes were predicted from the sequences representing homology to linked SNPs. We conclude that the markers, which were significantly associated with the DF of the common bean genotypes in the current study, can be used for marker-assisted selection in plant breeding program of common bean.     

Characterization of a novel y-type high molecular weight glutenin subunit at <Emphasis Type="Italic">Glu-D1</Emphasis> locus

Glu-D1y12.K as a novel y-type subunit was found in HMW-GSs encoded at the Glu-D1 locus in the JB20, which a Korean wheat line from F₉ lines crossed by Keumkang with Glu-D1d and Chinese Spring (CS) with Glu-D1a alleles. This novel subunit shows faster electrophoretic mobility and lower molecular weight than Dy12 subunit on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result of linear ion-trap and Fourier-transform mass spectrometry (LTQ-FT-MS) based on two-dimensional electrophoresis (2-DE) showed that the Dy12.K subunit has high similarity against protein ID: P08488 (GLT3_WHEAT) as ‘Glutenin, high molecular weight subunit 12’ form UniProtKB. The gene of the Glu-1Dy12.K subunit is composed of 1962 nucleotide base pairs containing open reading frame (ORF) as 652 amino acids corresponding to about 70.1 kDa. It has four indels (36 bp insertions: two repeated 18 and 24 bp deletion: two deletions with 6?+?18 bp) and 21 SNPs compared to Glu-1Dy10 (GI: 164457872 in NCBI), and one deletion (18 bp) and three SNPs compared to Glu-1Dy12 (GI: 1036031968) by DNA markers. Consequentially, in comparison with Dy10, 13 SNPs were non-synonymous SNPs and eight SNPs were synonymous SNPs of 21 SNPs. In comparison with Dy12, only one SNP was non-synonymous SNP of three SNPs. Furthermore, the deduced peptide sequences as ‘TGQGQQ’ corresponding to ‘AACAGGACAAGGGCAACA’ are deleted only in the Dy12.K subunit.     

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina''s GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.