Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

Allelic composition and associated quality traits of the <Emphasis Type="Italic">Glu-1</Emphasis> and <Emphasis Type="Italic">Glu-3</Emphasis> loci in selected modern Ethiopian durum wheat varieties

Gluten protein determines the processing quality of both durum wheat and bread wheat. The glutenin subunits compositions and associated quality traits of 20 Ethiopian durum wheat varieties were systematically analyzed using SDS-PAGE and Payne numbers. A total of 16 glutenin patterns were identified. At the Glu-A1 locus, all varieties scored the null allele. The predominant glutenin alleles at the Glu-B1 locus were Glu-B1b (7+8) and Glu-B1e (20). In Glu-3, the most abundant glutenin subunits were Glu-A3a and Glu-B3c. Based on the Payne scores, the varieties Yerer, Ginchi, Candate, and Foka were identified to have allelic composition suitable for pasta making. The cluster analysis using agglomerative hierarchical clustering (AHC) method classified the varieties into four similarity classes. Based on the findings of this experiment, suggestions were made for allelic composition improvement through introgression of superior alleles from known Glu-1 and Glu-3 sources.     

Conferring resistance to pre-harvest sprouting in durum wheat by a QTL identified in <Emphasis Type="Italic">Triticum spelta</Emphasis>

Pre-harvest sprouting (PHS) causes significant yield loss and degrade the end-use quality of wheat, especially in regions with prolonged wet weather during the harvesting season. Unfortunately, the gene pool of Triticum durum (tetraploid durum wheat) has narrow genetic base for PHS resistance. Therefore, finding out new genetic resources from other wheat species to develop PHS resistance in durum wheat is of importance. A major PHS resistance QTL, Qphs.sicau-3B.1, was mapped on chromosome 3BL in a recombinant inbred line population derived from ‘CSCR6’ (Triticum spelta), a PHS resistant hexaploid wheat and ‘Lang’, a PHS susceptible Australian hexaploid wheat cultivar. This QTL, Qphs.sicau-3B.1, is positioned between DArT marker wPt-3107 and wPt-6785. Two SCAR markers (Ph3B.1 and Ph3B.2) were developed to track this major QTL and were used to assay a BC₂F₈ tetraploid population derived from a cross between the durum wheat ‘Bellaroi’ (PHS susceptible) and ‘CSCR6’ (PHS resistant). Phenotypic assay and marker-assisted selection revealed five stable tetraploid lines were highly PHS resistant. This study has successfully established that PHS-resistance QTL from hexaploid wheat could be efficiently introgressed into tetraploid durum wheat. This tetraploid wheat germplasm could be useful in developing PHS resistant durum cultivars with higher yield and good end-use quality.     

Polymorphisms of the <Emphasis Type="Italic">FT</Emphasis> gene as a tool to identify underground rhizome types of bamboos

The Flowering Locus T (FT)-like genes of angiosperms are highly conserved. The FT-encoded proteins include a phosphatidylethanolamine-binding domain that is involved in the control of the shoot apical meristem identity and flowering time. In the present study, FT genes were investigated in 20 bamboo species that are grouped into sympodial, mixed and scattered bamboos based on their morphology. All examined orthologous FT genes consisted of four exons and three introns. Their encoded protein sequences contained the critical amino acid residues Tyr85, Glu109, Leu128, Tyr134, Trp138, Arg139, Gln140 and Asn152, of which each possesses a biological function. The DNA sequences were rich in single nucleotide polymorphism (SNP) sites. The SNP frequency was 1 SNP/16.8 bp, and the nucleotide diversity (π) equaled 0.265. Some SNPs altered restriction enzyme sites or resulted in changes in amino acid contents. The correlation analysis showed that several SNPs were informative in relation to the underground rhizome types of bamboos. Therefore, FT polymorphisms could be used as a tool to identify the underground rhizome types of bamboos. The phylogenetic tree constructed based on the FT gene sequences showed that the obtained clustering was consistent with the underground rhizome types. The SNP markers developed in the present study will provide information on the genetic diversity of bamboos and they can aid taxonomic study as well.     

Heterosis and combining ability of seven maize germplasm populations

Understanding the combining ability and heterosis of available germplasm is a prerequisite for successful maize improvement and breeding. The objectives of this study were to analyze the combining ability and heterosis of seven representative maize germplasm populations, and further, to evaluate their potential utility in germplasm improvement. A total of 21 crosses were made among these seven populations in a complete diallel without reciprocals. The parental populations and 21 crosses were evaluated for days to silking (DS), ear height (EH), and grain yield (GY) in the Northeast and Yellow and Huai River maize growing areas in China in 2012. Csyn5, Csyn7, Cpop.11, and Cpop.12 had desirable general combining ability (GCA) effects for DS and EH in both the Northeast China mega-environment (NCM) and the Yellow and Huai River Regions of China mega-environment (YHCM). Cpop.11 possessed a favorable GCA effect for GY in the NCM, as did Csyn5, Cpop.17, and Cpop.18 in the YHCM. Csyn6 and Csyn7 exhibited tremendous yield-enhancing potential in both mega-environments. Additionally, six combinations including Csyn7 × Csyn6, Csyn5 × Csyn6, Cpop.11 × Cpop.18, Cpop.12 × Cpop.17, Csyn7 × Cpop.17, and Csyn5 × Csyn7 exhibited better specific combining ability effects for GY, yield performance, and mid-parent heterosis in the appropriate mega-environment. These results indicated that the seven populations would be very useful for the improvement of related agronomic traits, and the six candidate combinations possessed great potential for further improvement and utilization.     

Genetics and molecular mapping of the naked grains in hexaploid oat

Avena sativa L. subsp. nudisativa has the ability to produce naked grains. Genetic studies on the naked trait of oat began over a century ago, but the genetic and molecular factors associated with the expression of this trait have not been fully clarified. The objectives of this study were to evaluate the naked trait in two oat populations of recombinant inbred lines (RILs), to determine the number of genes, to estimate the heritability, and to map genomic regions associated with the naked trait in hexaploid oat. Parental lines and RILs of each population were screened for the naked trait from plants grown in the field over a 2 year period. Based on the phenotypic data, the oat RILs were classed as naked, partially naked, partially hulled and hulled. In both populations and years, a great number of RILs showed variable expressivity for the naked trait. The genetic analysis indicated the action of a major gene (N1) with the action of modifying genes controlling the formation of naked grains. The results of the estimate of heritability show that environmental conditions do not have a great influence in determining the naked trait. The quantitative trait loci analysis detected a genomic region with a large effect on the naked trait that explained more than 50% of the phenotypic variation. Further studies are needed to validate the use of these molecular markers to assist breeding programs to select high quality and stable naked oat cultivars.     

Tall fescue forage mass in a grass-legume mixture: predicted efficiency of indirect selection

High fertilizer prices and improved environmental stewardship have increased interest in grass-legume mixed pastures. It has been hypothesized, but not validated, that the ecological combining ability between grasses and legumes can be improved by breeding specifically for mixture performance. This experiment examined the predicted efficiency of selection in a grass monoculture environment to indirectly improve tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) forage mass in a grass-legume mixture. Heritability, genetic and rank correlations, and selection efficiencies were estimated for forage mass in a tall fescue half-sib population grown as spaced-plants overseeded with either turf-type tall fescue (monoculture) or alfalfa (mixture). Heritability for tall fescue forage mass in monoculture ranged from 0.32 to 0.70 and were always similar or greater than those in mixture (range 0.27–0.55) for four successive harvests and annual total. Genetic correlations between monoculture and mixture tall fescue forage mass varied with values of 0.48, 0.92, ?0.31, 0.70, and 0.25 in June, July, August, October, and annual total, respectively. Indirect selection efficiencies exceeded or approached direct selection for mixtures only in July and October (1.29, and 0.73, respectively). Whereas, indirect selection efficiencies were low in June, August, and annual forage mass (0.58, ?0.31, and 0.28, respectively). Moreover, low Spearman’s rank correlations (?0.03 to 0.35) indicated differing half-sib family performance between the monoculture and mixture environments. Results indicate that direct selection should be used to improve tall fescue forage mass in a grass-legume mixture, and support the hypothesis of increasing ecological combining ability by breeding for mixtures per se.     

Determining resistance to <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin accumulation in African maize inbred lines resistant to <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and aflatoxins

Fusarium verticillioides and Aspergillus flavus cause Fusarium ear rot (FER) and Aspergillus ear rot (AER) of maize, respectively. Both pathogens are of concern to producers as they reduce grain yield and affect quality. F. verticillioides and A. flavus also contaminate maize grain with the mycotoxins fumonisins and aflatoxins, respectively, which has been associated with mycotoxicosis in humans and animals. The occurrence of common resistance mechanisms to FER and AER has been reported. Hence, ten Kenyan inbred lines resistant to AER and aflatoxin accumulation were evaluated for resistance to FER, F. verticillioides colonisation and fumonisin accumulation; and compared to nine South African lines resistant to FER and fumonisin accumulation. Field trials were conducted at three localities in South Africa and two localities in Kenya. FER severity was determined by visual assessment, while F. verticillioides colonisation and fumonisin content were quantified by real-time PCR and liquid chromatography tandem mass spectrometry, respectively. Significant genotype x environment interactions was determined at each location (P ≤ 0.05). Kenyan inbred CML495 was most resistant to FER and F. verticillioides colonisation, and accumulated the lowest concentration of fumonisins across localities. It was, however, not significantly more resistant than Kenyan lines CML264 and CKL05015, and the South African line RO549 W, which also exhibited low FER severity (≤5%), fungal target DNA (≤0.025 ng μL^?1) and fumonisin levels (≤2.5 mg kg^?1). Inbred lines resistant to AER and aflatoxin accumulation appear to be promising sources of resistance to F. verticillioides and fumonisin contamination.     

Resistance screening of breeding lines and commercial tomato cultivars for <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> and <Emphasis Type="Italic">M. javanica</Emphasis> populations (Nematoda) from Ethiopia

Soil and root samples were collected from major tomato growing areas of Ethiopia during the 2012/2013 growing season to identify root-knot nematode problems. DNA-based and isozyme techniques revealed that Meloidogyne incognita and M. javanica were the predominant Meloidogyne species across the sampled areas. The aggressiveness of different populations of these species was assessed on tomato cultivars Marmande and Moneymaker. The two most aggressive populations of each species were selected and further tested on 33 tomato genotypes. The resistance screening and mechanism of resistance was performed after inoculation with 100 freshly hatched (<24 h) second-stage juveniles (J2). Eight weeks after inoculation the number of egg masses produced on each cultivar was assessed. For the resistance mechanism study, J2 penetration and their subsequent development inside the tomato roots were examined at 1, 2, 4 and 6 weeks after inoculation. On both cultivars Marmande and Moneymaker all M. incognita and M. javanica populations formed a high number of egg masses indicating highly aggressive behaviour. Populations from ‘Jittu’ and ‘Babile’ for M. incognita and ‘Jittu’ and ‘Koka’ for M. javanica were selected as most aggressive. None of the 33 tomato genotypes were immune for these M. incognita and M. javanica populations. However, several tomato genotypes were found to have a significant effect on the number of egg masses produced indicating possible resistance. For M. javanica populations there were more plants from cultivars or breeding lines on which no egg masses were found compared to M. incognita populations. The lowest number of egg masses for both populations of M. incognita was produced on cultivars Bridget40, Galilea, and Irma while for M. javanica it was on Assila, Eden, Galilea, Tisey, CLN-2366A, CLN-2366B and CLN-2366C. Tomato genotypes, time (weeks after inoculation) and their interaction were significant sources of variation for J2 penetration and their subsequent development inside the tomato roots. Differential penetration was found in breeding lines such as CLN-2366A, CLN-2366B and CLN-2366C, but many of the selected tomato genotypes resistance for the tested M. incognita and M. javanica populations were expressed by delayed nematode development. Therefore, developing a simple screening technique to be used by local farmers or extension workers is crucial to facilitate selection of a suitable cultivar.     

Identification and validation of root length QTLs for water stress resistance in hexaploid wheat (<Emphasis Type="Italic">Titicum aestivum</Emphasis> L.)

Thorough understanding of the genetic mechanisms governing drought adaptive traits can facilitate drought resistance improvement. This study was conducted to identify chromosome regions harbouring QTLs contributing for water stress resistance in wheat. A RIL mapping population derived from a cross between W7984 (Synthetic) and Opata 85 was phenotyped for root length and root dry weight under water stress and non-stress growing conditions. ANOVA showed highly significant (p ≤ 0.01) variation among the RILs for both traits. Root length and root dry weight showed positive and significant (p ≤ 0.01) phenotypic correlation. Broad sense heritability was 86% for root length under stress and 65% for root dry weight under non-stress conditions. A total of eight root length and five root dry weight QTLs were identified under both water conditions. Root length QTLs Qrln.uwa.1BL, Qrln.uwa.2DS, Qrln.uwa.5AL and Qrln.uwa.6AL combined explained 43% of phenotypic variation under non-stress condition. Opata was the source of favourable alleles for root length QTLs under non-stress condition except for Qrln.uwa.6AL. Four stress specific root length QTLs, Qrls.uwa.1AS, Qrls.uwa.3AL, Qrls.uwa.7BL.1 and Qrls.uwa.7BL.2 jointly explained 47% of phenotypic variation. Synthetic wheat contributed favourable alleles for Qrls.uwa.1AS and Qrls.uwa.3AL. Two stable root dry weight QTLs on chromosomes 4AL and 5AL were consistently found in both water conditions. Three validation populations were developed by crossing cultivars Lang, Yitpi, and Chara with Synthetic W7984 to transfer two of the QTLs identified under stress condition. The F_2.3 and F_3.4 validation lines were phenotyped under the same level of water stress as RILs to examine the effect of these QTLs. There were 13.5 and 14.5% increases in average root length due to the inheritance of Qrls.uwa.1AS and Qrls.uwa.3AL, respectively. The result indicated that closely linked SSR markers Xbarc148 (Qrls.uwa.1AS) and Xgwm391 (Qrls.uwa.3AL) can be incorporated into MAS for water stress improvement in wheat.