Tissue culture-induced morphological somaclonal variation in St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

Somaclonal variation has been observed in many plant species and is an alternative way to create variants and expand the germplasm pool. A large scale tissue culture experiment was conducted with St. Augustinegrass, an important turfgrass species for the southern USA, to induce somaclonal variation to enlarge the germplasm pool for breeding efforts. Using an improved protocol, approximately 7900 St. Augustinegrass plants were regenerated from cv. 'Raleigh', and 119 morphological variants were identified. Among the variants, 115 had a semi-dwarf growth habit with shorter and narrower leaves, and shortened internodes and stolons. However, 100 of them showed little vigour, which either grew very slowly or did not survive. The remaining 15 showed reasonable growth vigour and were further investigated in the field. Among them, 13 were semi-dwarf and 2 had longer leaves. In addition, 2 other variants, with variegated (yellow striping) leaves, or significantly thicker stems were also observed and characterized. The altered traits in the variant lines were stable during vegetative propagation and when grown in different environments.     

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7–14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4‐dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.     

Trifluralin-mediated polyploidization of <Emphasis Type="BoldItalic">Rosa chinensis minima</Emphasis> (Sims) Voss seedlings

Many diploid rose species and cultivars possess valuable traits that can be introgressed into modern tetraploid cultivars. Interspecific, interploidy crosses are possible, but triploid hybrids typically have limited fertility, hindering further breeding and selection. Tetraploidizing diploids before mating with tetraploids can alleviate fertility barriers. The efficiency of trifluralin was investigated for polyploidization of Rosa chinensis minima (2n = 2x = 14) seedlings. Treatments were trifluralin at 0.086% and 0.0086%, colchicine (0.5%), and distilled water and contained 2% dimethyl sulfoxide and a surfactant. Approximately 5 l of the treatment solution was applied to the apical meristem of seedlings (N = 337, 82–85 per treatment) in the process of cotyledon expansion. Guard cell length, pollen diameter, and root tip squashes of rooted cuttings were used to detect polyploidy in meristematic layer (L)I, LII, and LIII, respectively. Trifluralin (0.086%) was the most effective treatment for polyploidization (LI 20.2%, LII 12.9%, LIII 12.9%), followed by trifluralin (0.0086%) (LI 10.6%, LII 7.1%, LIII 4.7%) and colchicine (LI 2.4%, LII 0%, LIII 0%). Polyploidization consistently occurred from LI inward. Polyploids as a group had reduced pollen stainability and a lower leaflet length to width ratio than diploids. In addition, two diploid seedlings were identified which produce 2n pollen. Considerations in selecting germplasm and generating somatically-induced polyploids from seedlings versus clones for use in breeding are discussed.     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Detection of additive and additive×environment interaction effects of QTLs for yield‐component traits of rice using single‐segment substitution lines (SSSLs)

Additive effects (A) and additive‐by‐environment interactions (A×E) for five rice yield components were analysed using 20 SSSLs under mixed linear model methodology. Thirty‐one QTLs were detected. Different yield components have different QTL‐by‐environment (Q×E) interaction patterns. No A×E interaction effects were detected for the four QTLs for panicle number (PN). Four QTLs detected for spikelets per panicle (SPP) had A×E interactions. Five of seven QTLs detected for grains per panicle (GPP), two of 10 QTLs detected for 1000‐grains weight (GWT) and three of six QTLs detected for seed set ratio (SSR) showed significant A×E interaction. Most of these QTLs were distributed in clusters across the genome. The complexity of linkage and pleiotropy of these QTLs plus environmental effect may result in the diversity of the yield phenotype in the SSSLs. Only S19 exhibited a significant increase in yield with a predicted gain by 281.58 kg ha^?1. The results may be useful to design a better breeding strategy that takes advantage of QTL‐by‐environment interaction effects in each of the SSSLs.     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

Effects of substituted D genome chromosomes on photosynthetic rate of durum wheat (Triticum turgidum L. var. durum)

Summary A durum wheat cultivar Langdon (LDN) and fourteen disomic D genome chromosome substitution lines of Langdon, where A or B genome chromosomes were replaced with homoeologous D genome chromosomes of Chinese Spring (CS), were used to assess the compensatory effect of the D genome chromosomes on photosynthetic rates at tetraploid level. The LDN 1D(1B) and LDN 3D(3B) lines showed significantly higher photosynthetic rates than Langdon, whereas LDN 1D(1A) and LDN 3D(3A) lines were not greatly different from Langdon. It appears that chromosomes 1B and 3B decrease photosynthesis. This suggests the differentiation of the effects on the photosynthesis within the first and third homoeologous groups. Substitution with the 2D chromosomes did not compensate the effects of either 2A or 2B chromosomes as it reduced photosynthetic rate compared to plant with either chromosomes 2A or 2B. Tetra CS had a higher photosynthetic rate than CS and Penta CS. The photosynthetic rate of CS was similar to that of Penta CS, which lacked one set of D genome. The results suggest that it may be possible to increase photosynthesis, if both sets of the D genome were entirely removed from hexaploid wheat. However, it is difficult to conclude that the lower rate of photosynthesis of the hexaploids was mainly attributable to D genome chromosome effects, because we did not find a dose dependent effect of D genome. Homoeologous differentiation of chromosomes may be involved in photosynthesis.     

Development and evaluation of chromosome segment substitution lines (CSSLs) carrying chromosome segments derived from Oryza rufipogon in the genetic background of Oryza sativa L.

The wild relatives of rice (Oryza sativa L.) are useful sources of alleles that have evolved to adapt in diverse environments around the world. Oryza rufipogon, the known progenitor of the cultivated rice, harbors genes that have been lost in cultivated varieties through domestication or evolution. This makes O. rufipogon an ideal source of value-added traits that can be utilized to improve the existing rice cultivars. To explore the potential of the rice progenitor as a genetic resource for improving O. sativa, 33 chromosome segment substitution lines (CSSLs) of O. rufipogon (W0106) in the background of the elite japonica cultivar Koshihikari were developed and evaluated for several agronomic traits. Over 90% of the entire genome was introgressed from the donor parent into the CSSLs. A total of 99 putative QTLs were detected, of which 15 were identified as major effective QTLs that have significantly large effects on the traits examined. Among the 15 major effective QTLs, a QTL on chromosome 10 showed a remarkable positive effect on the number of grains per panicle. Comparison of the putative QTLs identified in this study and previous studies indicated a wide genetic diversity between O. rufipogon accessions.     

The evaluation of adult plant resistance to powdery mildew (Erysiphe graminis f.sp. avenae) in transgressive lines of oats

Summary Previously identified segregant lines of oats with levels of adult plant resistance to powdery mildew (Erysiphe graminis f.sp. avenae) better than the resistant parent maintained this high level of resistance in field nurseries over two growing seasons. This enhancement was not expressed on inoculated detached leaves under laboratory conditions where no differences between the more resistant parent cv. Maldwyn and the most resistant segregants was detected. Reduced resistance was not detected in tests where leaf segments of the transgressive lines were inoculated with trained isolates. Problems associated with selecting for adult plant resistance under both field and laboratory conditions are discussed.     

Development of rice (Oryza sativa) lines resistant to aryloxyphenoxypropionate herbicides through induced mutation with gamma rays

The aryloxyphenoxypropionate herbicides (APPs) are graminicides with excellent control of many grass weeds species, including weedy rice (Oryza sativa L.). These herbicides block the fatty acid biosynthesis by inhibiting the enzyme acetyl‐CoA carboxylase (ACCase), resulting in the death of susceptible plants. Inducing mutation with gamma rays to rice seeds, two lines resistant to APPs herbicides were developed. Plant dose–response assays confirmed the resistance to the APPS herbicides quizalofop‐p‐ethyl and haloxyfop‐p‐methyl. The carboxyltransferase domain fragments of ACCase from the resistant biotype and susceptible control were sequenced and compared. A point mutation was detected in the amino acid position 2,027 (Rice Genome Annotation Project: Os05g22940.1). Results indicated that resistance to APPs is a consequence of an altered ACCase enzyme that confers resistance. The use of APPs herbicide‐resistant rice lines represents an innovative and promising alternative for weedy rice control in paddy rice systems.     

Parthenogenetic monohaploids (2n=x=12) from Solanum tuberosum L. and S. verrucosum Schlechtd. and the production of homozygous potato diploids

Summary Dihaploid and dihaploid derived clones of Solanum tuberosum and diploid genotypes of S. verrucosum produced 85 viable monohaploids by female parthenogenesis. All were induced using diploid S. phureja clones, homozygous for embryo spot, as pollinator. Frequency of S. tuberosum monohaploids per 100 berries was rather constant in three successive years (14, 17 and 17 respectively). No male and female fertility was found in flowering monohaploids.Colchicine-induced chromosome doubling yielded homozygous s. tuberosum diploids with low pollen quality but good seed fertility.Two diploid self-incompatible species (S. multidissectum and S. berthaultii) produced no monohaploids. The presence of genes for female parthenogenesis in some dihaploids is discussed.     

Field assessment of a second generation backcross (BC1 × Passiflora edulis) of passion fruit for agronomic performance and resistance to CABMV

The objective of this work was to characterize and identify among the passion fruit hybrids of the second generation backcross (BC2: BC1 × P. edulis), resistance to passion fruit woodiness disease (CABMV) with desirable agronomic attributes. Ninety‐two progeny of BC2 hybrids were evaluated for severity of the virus in conditions of field, physical and chemical characterization of the fruits and precocity of the plant in relation to the vegetative and reproductive phases. The data were analysed using multivariate statistics and mixed models (REML/BLUP) to estimate genetic parameters. The was conditioned by and , for number of fruits, fruit and peel weight and soluble solids and the effect of for virus severity to CABMV and effect of on the variables fruit length, fruit diameter, peel thickness and pulp weight. For the titratable acidity it had no . The progeny BC2‐A, BC2‐C, BC2‐E, BC2‐F and BC2‐H families can be selected for use in passion fruit breeding programmes by adding high vegetative vigour, early fruit production, resistance to CABMV and also desirable agronomic attributes.     

Development of a molecular marker for self‐compatible S4′ haplotype in sweet cherry (Prunus avium L.) using high‐resolution melting

Sweet cherry (Prunus avium L.) has stylar gametophytic self‐incompatibility, which is controlled by the multi‐allelic S‐locus and encompasses the highly polymorphic genes for the S‐ribonuclease (S‐RNase) and S‐haplotype‐specific F‐box (SFB), which are female and male determinants, respectively. The self‐compatible mutant SFB4′ corresponds to an allele variant of SFB4 and presents a frameshift mutation. Even though male‐determinant molecular markers can discriminate between SFB4 and SFB4′ alleles, the methods required are laborious, time‐consuming and expensive, and not suitable for massive analysis and integration into breeding programmes. Our aim was to develop molecular markers for the evaluation of self‐compatibility alleles in sweet cherry, that could be used as a high‐throughput screening strategy to identify SFB4 and SFB4′ alleles, based on a marker for male determinacy. Our results were consistent using primers flanking the mutation responsible for the SFB4′ allele. We designed a specific molecular marker and confirmed it in sweet cherry commercial varieties. This new molecular marker is feasible for self‐compatibility alleles in the male determinant in sweet cherry‐assisted breeding programs.     

Development of drought‐tolerant breeding lines derived from Helianthus annuus × H. argophyllus interspecific crosses

Helianthus argophyllus is a wild species known as “silver sunflower”, which possesses several traits, including morphological traits that increase drought tolerance. Therefore, introgressions between chosen cultivated lines and two H. argophyllus accessions were made, and segregating generations were established. Important agronomic traits including single heading, high pollen fertility, silver canopy (indicating more cuticular wax), and content of cuticular waxes and oil were selected over six segregating generations. The resulting F₆ lines showed introgression of water saving traits, as they had lower excise leaf water loss, with comparable yield to standard checks. However, these F₆ lines were late maturing and showed poor flowering synchronization between the cultivated and introgressed lines. Introgressed line “D‐22” was particularly promising as a breeding line, with superior agronomic and drought resistance traits. This line had the potential to be used as an inbred parental line for introgression of drought resistance traits into elite sunflower germplasm. Combining ability analysis of the introgressed lines further showed their potential for heterosis breeding or to be used as parental lines in breeding programme.     

Heritability of spotted wilt resistance in a Florida‐EP™ “113”‐derived peanut (Arachis hypogaea) population

Spotted wilt, caused by tomato spotted wilt virus (TSWV), is a major disease of peanut (Arachis hypogaea ) in the south‐eastern United States. Cultivar resistance is the most important factor in disease control. However, spotted wilt resistance in current cultivars still carries risk in the absence of other practices when disease is severe. In contrast, a newly developed cultivar, Florida‐EP? “113,” has demonstrated excellent resistance even when spotted wilt is severe. Information on heritability of this resistance can help breeders better utilize it in breeding. F₂‐derived populations from the cross Florida‐EP? “113”/Georgia Valencia were developed and tested in field experiments in Florida from 2012 to 2014. Disease symptoms were evaluated visually, and the frequency of TSWV infection was measured by ImmunoStrip^®. Heritability estimated from ImmunoStrip^® was higher (0.66) compared to visual ratings (0.48). Genetic correlations among evaluation methods (r _A = 0.92–0.99) and environments (r _B = 0.86–0.99) were high. These results indicate that resistance in Florida‐EP? “113” is highly heritable and that selection in a high disease risk environment is feasible without significant erosion of genetic gain.     

Potential of advanced breeding lines of bread‐making wheat to accumulate grain minerals (Ca,Fe, Mg and Zn) and low phytates under Mediterranean conditions

About 3 billion people may suffer from micronutrient deficiency such as Ca, Fe, Mg or Zn, caused not only by a mineral deficiency in staple food but also by a high content of phytates which bind those minerals and inhibit their absorption. With the aim of evaluating the potential of new cultivars of bread‐making wheat to accumulate those minerals and low phytates, nine advanced breeding lines from an ongoing Portuguese breeding program were studied during 2 years in a field experiment. A wide genetic variability was found between the studied genotypes in all the parameters studied, especially grain yield (ranging on average between 2,027 and 3,209 kg/ha) and grain Mg and Zn concentrations (ranging on average between 1,070 and 1,336 mg/kg, and 23.4 and 30.7 mg/kg, respectively). In global terms, the cultivars with best performance, and therefore, the most potentially suitable to be used in a breeding program, were the Cultivars 3 and 4. However, such a potential varied depending on the analysed trait, and it was clearly influenced by the climatic conditions. The consumption of 100 g of Cultivar‐4 produced under the most favourable conditions might provide a 5.2% of Ca, 26.4% of Fe, 38.9% of Mg and 31.9% of Zn of the recommended daily intakes, with a very good bioavailability for Fe and Ca, but low for Mg and Zn.     

西藏三联小穗小麦中三联小穗性状控制基因的SSR分子标记定位

西藏三联小穗小麦是中国西藏地区一种独特的小麦地方品种,拥有特殊的三联小穗性状,超多的小穗数和小花数。分子定位控制三联小穗基因的基因座,发掘与之紧密连锁的分子标记,可为小麦高产育种提供分子标记辅助选择工具。本研究利用西藏三联小穗小麦的衍生系TTSW-5与普通穗型小麦,间3和川麦55,分别构建F2群体,成熟后进行穗部性状的表型分析和SSR基因型鉴定。性状表型遗传分析表明,西藏三联小穗小麦的三联小穗性状由两个独立遗传的隐性基因控制;通过SSR标记鉴定来自TTSW-5/间3组合的F2群体,在2A染色体上检测到1个与三联小穗性状相关的QTL,定位于SSR标记Xgwm275和Xgwm122之间,两标记间的遗传距离为6.6cM,该QTL的LOD值为6.19,可解释的表型变异值为33.1%,初步命名为qTS2A-1。我们推测qTS2A-1可能是控制三联小穗性状相关的主效QTL,SSR标记Xgwm275和Xgwm122可能可用于三联小穗性状的辅助选择。     

Validation of a set of informative simple sequence repeats markers for variety identification in Pak‐choi (Brassica rapa L. ssp. chinensis var. communis)

A core set of 21 simple sequence repeats (SSR) markers was developed for Pak‐choi (Brassica rapa ssp. chinensis var. communis) variety identification. We initially selected 74 SSR markers which exhibited high polymorphism and reproducibility in SSR detection from 2129 SSRs. Using the 74 SSR‐based dendrogram for 45 inbred lines as calibration, 21 core SSRs were selected out. The utility of this core set SSRs was firstly tested in 45 inbred lines and finally verified in 102 commercial varieties. We also constructed a molecular ladder for each core SSR as a reference standard. Diversity analysis of this core SSR panel in 102 varieties demonstrated that each marker generates 2–3 alleles (averaged 2.33), with polymorphism information content values ranging from 0.01 to 0.56 (averaged 0.31). The averaged values of Shannon information index, observed heterozygosity, expected heterozygosity and Wright's fixation index were 0.59, 0.43, 0.38 and −0.09, respectively. Furthermore, the 21 SSR‐based classifications for 102 varieties were consistent with traditional classification based on morphology. This core SSR panel represents an effective tool for genetic variation analysis in Pak‐choi.