Plants Of F2 Population Research Articles

Multiplex PCR for Early Generation Identification of Tomato Segregants Carrying Ty-2, Ty-3 and Ph-3 Resistance Alleles Against Leaf Curl and Late Blight Diseases.

Deployment of different natural disease resistance alleles is the most sustainable and eco-friendly way for multiple disease management in tomato. Diagnostic molecular markers are indispensible in this effort as they offer early generation identification of resistance alleles in an environment-independent manner. Moreover, optimized multiplex polymerase chain reaction (PCR) for detecting different disease resistance alleles in a single reaction can speed-up the selection process with cost and labour-effectiveness. Here we report the optimized multiplex detection and stacking of leaf curl disease resistance alleles Ty-2 and Ty-3 along with late blight disease resistance allele Ph-3 in tomato genotypes and F2 segregants. The triplex assay could be replaced by a duplex assay (for Ty-2 and Ty-3 resistance alleles) followed by analysis at Ph-3 locus to achieve further cost-effectiveness. We identified two plants in F2 populations derived from the Arka Samrat (F1) x Kashi Chayan combination to carry the Ty-2, Ty-3 and Ph-3 resistance alleles in homozygous condition. Early generation genotyping also allowed us to identify a few morphologically better segregants, where further marker assisted selection (MAS) should identify superior multiple disease resistant lines. Thus we advocate the utility of multiplex PCR in MAS to address multiple disease resistance breeding in tomato.

Analysis of QTLs and Candidate Genes for Tassel Symptoms in Maize Infected with Sporisorium reilianum.

Heat smut is a fungal soil-borne disease caused by Sporisorium reilianum, and affects the development of male and female tassels. Our previous research found that the tassel symptoms in maize infected with Sporisorium reilianum significantly differed in inbred lines with Sipingtou blood, and exhibited stable heredity over time at multiple locations. In this study, cytological analysis demonstrated that the cellular organization structures of three typical inbred lines (Huangzao4, Jing7, and Chang7-2) showed significant discrepancies at the VT stage. QTLs that control the different symptoms of maize tassels infected with Sporisorium reilianum were located in two F2 populations, which were constructed using three typical inbred lines. The BSA (bulked segregation analysis) method was used to construct mixed gene pools based on typical tassel symptoms. The QTLs of different symptoms of maize tassels infected with Sporisorium reilianum were detected with 869 SSR markers covering the whole maize genome. The mixed gene pools were screened with polymorphic markers between the parents. Additional SSR markers were added near the above marker to detect genotypes in partially single plants in F2 populations. The QTL controlling tassel symptoms in the Huangzao4 and Jing7 lines was located on the bin 1.06 region, between the markers of umc1590 and bnlg1598, and explained 21.12% of the phenotypic variation with an additive effect of 0.6524. The QTL controlling the tassel symptoms of the Jing7 and Chang7-2 lines was located on the bin 2.07 region, between the markers of umc1042 and bnlg1335, and explained 11.26% phenotypic variation with an additive effect of 0.4355. Two candidate genes (ZmABP2 and Zm00001D006403) were identified by a conjoint analysis of label-free quantification proteome sequencings.

Genome sequencing identified novel mechanisms underlying virescent mutation in upland cotton Gossypiuma hirsutum

BackgroundVirescent mutation broadly exists in plants and is an ideal experimental material to investigate regulatory mechanisms underlying chlorophyll synthesis, photosynthesis and plant growth. Up to date, the molecular mechanisms in two virescent mutations have been clarified in cottons (Gossypiuma hirsutum). A virescent mutation has been found in the cotton strain Sumian 22, and the underlying molecular mechanisms have been studied.MethodsThe virescent mutant and wild type (WT) of Sumian 22 were cross-bred, and the F1 population were self-pollinated to calculate the segregation ratio. Green and yellow leaves from F2 populations were subjected to genome sequencing and bulked-segregant analysis was performed to screen mutations. Real-time quantitative PCR (RT-qPCR) were performed to identify genes in relations to chlorophyll synthesis. Intermediate products for chlorophyll synthesis were determined to validate the RT-qPCR results.ResultsThe segregation ratio of green and virescent plants in F2 population complied with 3:1. Compared with WT, a 0.34 Mb highly mutated interval was identified on the chromosome D10 in mutant, which contained 31 genes. Among them, only ABCI1 displayed significantly lower levels in mutant than in WT. Meanwhile, the contents of Mg-protoporphyrin IX, protochlorophyllide, chlorophyll a and b were all significantly lower in mutant than in WT, which were consistent with the inhibited levels of ABCI1. In addition, a mutation from A to T at the -317 bp position from the start codon of ABCI1 was observed in the genome sequence of mutant.ConclusionsInhibited transcription of ABCI1 might be the mechanism causing virescent mutation in Sumian 22 cotton, which reduced the transportation of protoporphyrin IX to plastid, and then inhibited Mg-protoporphyrin IX, Protochlorophyllide and finally chlorophyll synthesis. These results provided novel insights into the molecular mechanisms underlying virescent mutation in cotton.

A mutation in the intron splice acceptor site of a GA3ox gene confers dwarf architecture in watermelon (Citrulluslanatus L.)

Dwarf architecture is an important trait associated with plant yield, lodging resistance and labor cost. Here, we aimed to identify a gene causing dwarfism in watermelon. The ‘w106’ (dwarf) and ‘Charleston Gray’ (vine) were used as parents to construct F1 and F2 progeny. Dwarf architecture of ‘w106’ was mainly caused by longitudinal cell length reduction and was controlled by a single recessive gene. Whole-genome sequencing of two parents and two bulk DNAs of F2 population localized this gene to a 2.63-Mb region on chromosome 9; this was further narrowed to a 541-kb region. Within this region, Cla015407, encoding a gibberellin 3β-hydroxylase (GA3ox), was the candidate gene. Cla015407 had a SNP mutation (G → A) in the splice acceptor site of the intron, leading to altered splicing event and generating two splicing isoforms in dwarf plants. One splicing isoform retained the intron sequences, while the other had a 13-bp deletion in the second exon of GA3ox transcript, both resulting in truncated proteins and loss of the functional Fe2OG dioxygenase domain in dwarf plants. RNA-Seq analysis indicated that expression of Cla015407 and other GA biosynthetic and metabolic genes were mostly up-regulated in the shoots of dwarf plants compared with vine plants in F2 population. Measurement of endogenous GA levels indicated that bioactive GA4 was significantly decreased in the shoots of dwarf plants. Moreover, the dwarf phenotype can be rescued by exogenous applications of GA3 or GA4+7, with the latter having a more distinct effect than the former. Subcellular localization analyses of GA3ox proteins from two parents revealed their subcellular targeting in nucleus and cytosol. Here, a GA3ox gene controlling dwarf architecture was identified, and loss function of GA3ox leads to GA4 reduction and dwarfism phenotype in watermelon.

Genetic analysis of morphological traits of the spike and reproductivity elements of speltoid chemomutant Triticum aestivum

Long use of the methods of direct intraspecies hybridization in the selective breeding of varieties of Triticum aestivum L. has led to narrowing of their gene fund and close similarity of the genetic potential of their selective breeding traits. Using the method of experimental mutagenesis, one can induce systemic mutants with features of other hexaploid Triticum species which can freely cross-breed with other hybridization offspring, contributing to extension of genetic potential of cultivated wheat and creation of varieties with new levels of manifestation of economically beneficial traits. We studied the pattern of inheritance of morphological traits of the ear, length of the stem and the elements of productivity of speltoid chemomutant of T. aestivum. For the genetic analysis we used hybrids F1 and F2 of soft wheat obtained by cross-breeding speltoid macromutant (Smuhlianka speltoid), induced by the impact of aqueous solution of N-nitroso-N-methylurea (NMU) in the concentration of 0.025% on the seeds of Smuhlianka variety, with plants of Smuhlianka variety (Erythrospermum variety) and Podoloanka (Lutescens variety). To determine the pattern of inheritance the spike morphology, length of the stem and the elements of productivity in F1 hybrids, we calculated the extent of phenotype domination. In populations of F2, we examined plants with different combinations of phenotype manifestation of ear morphology. In F1 hybrids, the speltoid shape of the ear, absence of awns and red colour of the glumes indicated the dominant pattern of inheritance. The high level of phenotype domination of length of the stem and ear, number of spikelets in the main ear indicates the inheritance of the features according to intermediate, partly dominant and over-dominant types. Taking into account the segregation according to the features of spike morphology, awnedness and colour of glumes, the plants of F2 population were divided into phenotype classes, that is 12 and 6 in the combinations of respectively Smuhlianka speltoid × Smuhlianka and Smuhlianka speltoid × Podolianka. We determined that the obtained results are the consequences of dihybrid linkage which corresponds to the theoretical proportion of 12 : 3 : 1. Segregation into non-aristate and aristate plants corresponds to the proportion of monogene segregation of 3 : 1. Within separately distinguished phenotype classes, no independent inheritance of the shape of the ear and awnedness was observed. Dihybrid segregation of F2 plants into speltoid, squarehead and varieties Lutescence/Erythrospermum with quantitative superiority of speltoid plants suggests the control of the trait by two non-allele genes with epistatic interaction. The red colour of the glumes indicates the dominant monogenic pattern of inheritance. Absence of independent inheritance of the shape of the ear and awnedness indicates localization of genes which determine these features in one chromosome.

Genetic analysis of the blast resistance in the wild rice species Tianjinyeshengdao and preliminary mapping of the resistance gene Pi2-1

To better understand the genetic basis of blast resistance in the wild rice species Tianjinyeshengdao(TY),F2 population from the cross between TY and the rice cultivar CO39 which is highly susceptible to blast was constructed and respectively inoculated with isolates CHL1743,110–2 and 318–2 in the growth chamber.The results showed that the resistant and susceptible plants in F2 population following inoculation fitted the ratio of 3∶1,indicating the resistance to isolates CHL1743,110–2 and 318–2 in TY was controlled by a single dominant gene which was named as Pi2–1.To preliminary determine the chromosomal location of blast resistance gene,bulked segregant analysis(BSA) and recessive-class analysis(RCA) were conducted.The results indicated that Pi2–1 was located near the centromeric region of rice chromosome 6 and linked to the markers AP5659–5 and RM7213,with a distance of 0.9 cM and 1.4 cM,respectively.

PYRAMIDING IMPORTANT DISEASE-RESISTANT CHARACTERS BY HYBRIDIZATION OF TRANSGENIC AND NON-TRANSGENIC PEANUTS (ARACHIS HYPOGAEA L.)

We have produced transgenic peanut lines carrying a coat protein gene of peanut stripe virus (PStV) and showing resistance to the virus. However, their susceptibility to leafspot disease caused by Cercospora sp. and their lower productivity compared to their genetic background cultivar make them commercially less undesireable. The objective of this research was to test whether crossing the transgenic peanut plants with a non-transgenic peanut line WS, which was resistant to leafspot disease and high-yielding, could produce progenies in F2 generation that were resistant to both PStV and leafspot disease as well as of higher yield campared to their transgenic progenitor. If this test was proven, pyramiding novel transgenic and non-transgenic characters in peanut plants by hybridization would probably be a routine procedure in the future. Crosses were made between transgenic peanut plants that were resistant to PStV and non-transgenic peanut line WS. F2 population was evaluated for resistance to PStV and leafspot disease. Number of filled pods, filled pod dry weight per plant, and dry weight of each pod were measured. Result of the experiment showed that some of the plants in F2 population exhibited resistant both to PStV and leafspot disease and produced higher number of filled pods, filled pod dry weight per plant, and dry weight of each pod compared to those produced by their transgenic parent plants.

Identification of Lr24 with targeted region amplified polymorphism (TRAP) analysis in wheat

This research is aimed at developing TRAP markers, as a probe for library screening, closely linked to or co-segregated with Lr24. Ninety TRAP primer pairs were used to test the resistant and susceptible parents, as well as the resistant bulk and the susceptible bulk in our study. The polymorphic TRAP primers of TcLr24 were employed to genotype the F2 population from TcLr24×Thatcher subsequently. Ten of 90 TRAP primer pairs displayed polymorphism between TcLr24 and Thatcher, accounting for 11.11%. A further study found that primer ARBI1/RGA-2F generated a 161 bp fragment presented only in the resistance plants of F2 population. Forty-five other wheat leaf rust resistant NILs and 30 diploid materials of wheat were also tested to detect the specificity of the primer. This specific band was amplified in TcLr19, TcLr29, TcLr38, TcLr42 and TcLr44, but absented in all the 30 diploid materials. It was concluded that this marker ARBI1/RGA-2F was closely linked to Lr24, which could be used to detect Lr24 in the F2 population of TcLr24×Thatcher, and be further used as a probe for cDNA and BAC library screening of TcLr24.

Interval Mapping of Quantitative Trait Loci Underlying Endosperm Traits with NC III and TTC Designs

The endosperm of cereal crops is a triploid tissue produced by a male gamete from the pollen grain fusing with two female nuclei. Endosperm traits refer to those characteristics that determined by the physical and chemical properties of grain endosperm. Because many quality traits are closely related to endosperms, the genetic study of endosperm traits plays a funda-mental and vital role in improving grain quality. There have been several publications detailing the unique properties of endosperm traits. A series of QTL mapping models and approaches specific for triploid traits have been proposed, using single grain observation or more practical one that obtained from bulked samples. However, current methods based on bulked samples often fail to estimate two dominance effects of endosperm QTL separately, or involve a heavy work of crossing. On the basis of quanti-tative genetic models for triploid endosperm traits, a new mapping approach based on North Carolina Design III (NC III) and Triple Test Cross (TTC) was proposed in this study. Feasibility and efficacy of the method were investigated through simulated data. Experimental factors considered in the simulations include QTL heritability, number of plants in F2 population, and number of endosperms collected per family. The results suggested that the two methods had satisfactorily high detection powers, which reached 100% even when the QTL heritability was 5%. But the TTC-based approach tended to have slightly higher power than the NC III-based one. Both approaches can distinguish two dominance effects successfully. But overall, the TTC-based approach can offer better estimation of either QTL position or effect, though this superiority waned as the heritability and sample size increase.

The inheritance of resistance to beet necrotic yellow vein virus (BNYVV) in B. vulgaris subsp. maritima, accession WB42: Statistical comparisons with Holly-1-4

In this study, the inheritance of resistance to Beet necrotic yellow vein virus (BNYVV) in accessions Holly-1-4and WB42 was investigated. Crosses between both resistant sources and susceptible parents were carried out and F1F2 and BC1 populations were obtained. Virus concentrations in WB42and its F1 populations were lower than in Holly-1-4. Observed ratios of susceptible and resistant plants in segregating populations of Holly-1-4 as well as WB42 were in agreement with hypothesis of one dominant major gene. Segregation of plants in F2 populations obtained from crosses betweenHolly-1-4 and WB42 revealed that the resistance genes in Holly-1-4 and WB42 were nonallelic and linked loci.

Detection of linkage between restriction fragment length polymorphism markers and quantitative traits

Methodologies commonly used to detect linkage of marker loci to loci affecting quantitative traits are discussed. It is shown that variances for the quantitative trait differ among marker genotypes when using F2 or pooled backcross data if linkage exists. Hence, to analyze this type of data by single factor ANOVA or other statistical techniques that assume a common variance is inadequate. Restriction fragment length polymorphism (RFLP) markers are a powerful tool in plant breeding but cost is an important drawback; hence, a methodology is suggested to obtain the minimum number of plants in F2 populations to detect such linkage.

Evaluation of parent selection methods for yield improvement of cowpea (Vigna unguiculata (L.) Walp.)

The mean aim of this study was to identify an effective method of predicting cross-potential in respect of yield improvement of the cowpea (Vigna unguiculata (L.) Walp.) through hybridization and subsequent selection. Three prediction tests based on data collected from experiments with ten cowpea lines and the F1 and F2 generations of all possible crosses between these lines were evaluated. The three prediction tests were based on: i Yields of prospective parents. ii. General combining abilities estimated from F1 and parental data iii. Frequencies of superior plants in F2 populations Each of the three procedures was used to identify the crosses with the lowest or highest yield potential, i.e. those crosses most likely to produce the greatest number of lines with yields higher or lower than those of either parent. Crosses selected on the basis of these criteria were evaluated in the following growing season on the basis of biometrical analysis of F2 and backcross populations and the performance of the respective F3 and F4 generations. Six different crosses were selected from the 45 tested on the basis of the three prediction tests. Analyses of F2 and backeross populations from each cross provided estimates of [d] the difference in parental means and 1/2D the additive genetic component which, in turn, were used to predict the frequency of transgressive inbred lines obtained by single-seed descent from the F2. Both crosses selected on the basis of F2 data were expected to produce a much higher frequency of transgressive lines than those selected on the basis of parental yields. The high potential cross selected on the basis of general combining ability was expected to produce a higher frequency of transgressive lines than the corresponding low yield potential cross. Replicated trails of F3 bulks of the selected crosses showed that high yield potential crosses were significantly higher yielding than low yield potential crosses for all prediction criteria. In a trial with 22F4 lines of each of the two crosses selected on the basis of parental yields and frequency of high yielding F2 plants, the high yield potential cross in each case had a greater mean and variance for seed yield than the corresponding low yield potential cross. It was concluded that parental yields provide a sound basis for an initial screening of prospective parents. This screening should identify a high yielding line which can be crossed with a number of contrasting lines to produce F2 populations for the final screening phase.