Exploring almond genetic variability useful for peach improvement: mapping major genes and QTLs in two interspecific almond × peach populations

Genetic analysis of a diverse set of 42 traits for flower (5), phenology (9), fruit quality (19), leaf (8) and disease resistance (1) was carried out in two interspecific almond × peach populations, an F₂ (T × E) and a BC₁ (T1E), from the cross between ‘Texas’ almond and ‘Earlygold’ peach. Traits related to flower, phenology, fruit quality, leaf morphology and resistance to powdery mildew were phenotyped over 3 years in two locations and studied for co-segregation with a large set of SNP and SSR markers. Three maps were used, one for the T × E and two for the T1E (T1E and E) population. Nine major genes were identified and mapped: anther color (Ag/ag and Ag2/ag2), flower color (Fc2/fc2), maturity date (MD/md), almond fruit type (almond vs. peach; Alf/alf), juiciness (Jui/jui), blood flesh (DBF2/dbf2), powdery mildew resistance (Vr3) and flower type (showy/non-showy; Sh/sh). These genes were often located in genome positions different from those for major genes for similar traits mapped before. Two of them explain fundamental aspects that define the fruit of peach with respect to that of almond: Alf and Jui, for its thick and juicy mesocarp, respectively. The genetics of quantitative traits was studied, and 32 QTLs were detected, with consistent behavior over the years. New alleles identified from almond for important traits such as red skin color, blood flesh, fruit weight and powdery mildew resistance may prove useful for the introduction of new variability into the peach gene pool used in commercial breeding programs.     

Identification and pyramiding of QTLs for cold tolerance at the bud bursting and the seedling stages by use of single segment substitution lines in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Cold stress is one of the main constraints in rice production, and damage from cold can occur at different developmental stages in rice. Understanding the genetic basis of cold tolerance is the key for breeding cold-tolerant variety. In this study, we used single segment substitution lines (SSSLs) derived from a cross between cold-tolerant japonica variety “Nan-yang-zhan” and a popular indica variety “Hua-jing-xian 74” to detect and pyramid QTLs for cold tolerance at the bud bursting and the seedling stages. Evaluation of cold tolerance of these SSSLs and their recurrent parent helped identify two cold-tolerant QTLs (qCTBB-5 and qCTBB-6) at the bud bursting stage and two cold-tolerant QTLs (qCTS-6 and qCTS-12) at the seedling stage. The SSSLs carrying these QTLs showed stronger cold tolerance than their recurrent parent HJX74 did in three independent experiments. The qCTBB-6 and qCTS-6 were mapped to the same chromosomal region. QTL pyramiding was performed by intercrossing of SSSLs carrying the respective QTLs for cold tolerance at the bud bursting stage and the seedling stage and marker-assisted selection (MAS). The selected pyramiding line SC1-1 with different cold-tolerant QTLs showed cumulative effects on cold tolerance. Our results suggest that different genes (QTLs) control cold tolerance at bud bursting and seedling stages, and pyramiding of stable expression QTLs for cold tolerance at different developmental stages through MAS is a good strategy to prevent cold damage in rice.     

Branching gene expression during chrysanthemum axillary bud outgrowth regulated by strigolactone and auxin transport

Shoot branching is essential in ornamental chrysanthemum production and determines final plant shape and quality. Auxin is associated with apical dominance to indirectly inhibit bud outgrowth. Two non-mutually exclusive models exist for indirect auxin inhibition. Basipetal auxin transport inhibits axillary bud outgrowth by limiting auxin export from buds to stem (canalization model) or by increasing strigolactone levels (second messenger model). Here we analyzed bud outgrowth in treatments with auxin (IAA), strigolactone (GR24) and auxin transport inhibitor (NPA) using a split-plate bioassay with isolated chrysanthemum stem segments. Besides measuring bud length, dividing cell percentage was measured with flow cytometry and RT-qPCR was used to monitor expression levels of genes involved in auxin transport (CmPIN1) and signaling (CmAXR2), bud dormancy (CmBRC1, CmDRM1) and strigolactone biosynthesis (CmMAX1, CmMAX3). Treatments over a 5-day period showed bud outgrowth in the control and inhibition with IAA and IAA?+?GR24. Bud outgrowth in the control coincided with high dividing cell percentage, decreased expression of CmBRC1 and CmDRM1 and increased CmPIN1 expression. Inhibition by IAA and IAA?+?GR24 coincided with low dividing cell percentage and unchanged or increased expressions of CmBRC1, CmDRM1 and CmPIN1. Treatment with GR24 showed restricted bud outgrowth that was counteracted by NPA. This restricted bud outgrowth was still concomitant with a high dividing cell percentage and coincided with decreased expression of dormancy genes. These results indicate incomplete inhibition of bud outgrowth by GR24 treatment and suggest involvement of auxin transport in the mechanism of bud inhibition by strigolactones, supporting the canalization model.     

Relationship between seasonal progression of floral meristem development and <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> expression in the deciduous tree <Emphasis Type="Italic">Fagus crenata</Emphasis>

Estimating the timing of flower bud formation in plants is essential to identify environmental factors that regulate floral transition. The presence of winter dormancy between the initiation of flowers and anthesis, characteristic of most trees in the temperate forests, hampers accurate estimation of the timing of floral transition. To overcome this difficulty, expression levels of flowering-time genes could be used as indicators of the timing of floral transition. Here, we evaluated the usefulness of molecular markers in estimating the timing of floral transition in Fagus crenata, a deciduous tree that shows intermittent and synchronized flowering at the population level. We selected FLOWERING LOCUS T (FT) as a candidate molecular marker and quantified the expression levels of its ortholog in F. crenata (FcFT). Subsequently, we analyzed the relationship between morphogenetic changes that occur between the vegetative state of the buds and the initiation of floral organs, and compared the FcFT expression levels in reproductive and vegetative buds, collected from spring to autumn. FcFT expression in leaves peaked at least two weeks before the morphological changes associated with flowering were visible in the buds in late July. FcFT expression levels were significantly higher in the reproductive buds than in the vegetative buds in July. These results suggest that the FcFT expression in July is a reliable indicator of the timing and occurrence of floral transition. This study highlights the utility of molecular tools in unraveling reproductive dynamics in plants, in combination with ecological and physiological approaches.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.     

Vetch aphid, <Emphasis Type="Italic">Megoura crassicauda</Emphasis> (Hemiptera: Aphididae), parasitism does not reduce the bean production of narrow-leaved vetch, <Emphasis Type="Italic">Vicia sativa</Emphasis> subsp. <Emphasis Type="Italic">nigra</Emphasis> (Fabaceae)

Megoura crassicauda Mordvilko (Hemiptera: Aphididae) is a dominant aphid species found on Vicia sativa subsp. nigra (L.) Ehrh. (Fabaceae) in the spring. Worker ants of Formica japonica, the dominant ant species attracted to the extrafloral nectaries of V. s. nigra, often attack ladybirds (Coccinella septempunctata), which are aphid enemies. However, the workers of F. japonica do not attack or exclude M. crassicauda, the non-myrmecophilous aphid. It appears that the “bodyguard” retained by the plant guards the plant’s herbivore by attacking the herbivores’ enemies, rather than guarding the plant itself. The relationship between V. s. nigra and M. crassicauda was observed in the field to examine and evaluate the cost of parasitism. Parasitism by M. crassicauda delayed flower bud formation markedly in V. s. nigra but did not kill the plants. V. s. nigra plants that were parasitized showed a net bean production similar to that of the non-parasitized controls. The parasitism rate of M. crassicauda increased when extrafloral nectaries were used by F. japonica. These results may indicate that M. crassicauda provides V. s. nigra with benefits by preventing other serious disadvantages.     

Locating QTLs controlling overwintering seedling rate in perennial glutinous rice 89-1 (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A new cold tolerant germplasm resource named glutinous rice 89-1 (Gr89-1, Oryza sativa L.) can overwinter using axillary buds, with these buds being ratooned the following year. The overwintering seedling rate (OSR) is an important factor for evaluating cold tolerance. Many quantitative trait loci (QTLs) controlling cold tolerance at different growth stages in rice have been identified, with some of these QTLs being successfully cloned. However, no QTLs conferring to the OSR trait have been located in the perennial O. sativa L. To identify QTLs associated with OSR and to evaluate cold tolerance. 286 F₁₂ recombinant inbred lines (RILs) derived from a cross between the cold tolerant variety Gr89-1 and cold sensitive variety Shuhui527 (SH527) were used. A total of 198 polymorphic simple sequence repeat (SSR) markers that were distributed uniformly on 12 chromosomes were used to construct the linkage map. The gene ontology (GO) annotation of the major QTL was performed through the rice genome annotation project system. Three main-effect QTLs (qOSR2, qOSR3, and qOSR8) were detected and mapped on chromosomes 2, 3, and 8, respectively. These QTLs were located in the interval of RM14208 (35,160,202 base pairs (bp))–RM208 (35,520,147 bp), RM218 (8,375,236 bp)–RM232 (9,755,778 bp), and RM5891 (24,626,930 bp)–RM23608 (25,355,519 bp), and explained 19.6%, 9.3%, and 11.8% of the phenotypic variations, respectively. The qOSR2 QTL displayed the largest effect, with a logarithm of odds score (LOD) of 5.5. A total of 47 candidate genes on the qOSR2 locus were associated with 219 GO terms. Among these candidate genes, 11 were related to cell membrane, 7 were associated with cold stress, and 3 were involved in response to stress and biotic stimulus. OsPIP1;3 was the only one candidate gene related to stress, biotic stimulus, cold stress, and encoding a cell membrane protein. After QTL mapping, a total of three main-effect QTLs—qOSR2, qOSR3, and qOSR8—were detected on chromosomes 2, 3, and 8, respectively. Among these, qOSR2 explained the highest phenotypic variance. All the QTLs elite traits come from the cold resistance parent Gr89-1. OsPIP1;3 might be a candidate gene of qOSR2.     

Life history of <Emphasis Type="Italic">Stenopsylla nigricornis</Emphasis> (Hemiptera: Psylloidea: Triozidae) and phytohormones involved in its gall induction

Many phytophagous insects have an ability to manipulate plant tissue and induce galls, but the mechanism is not yet fully understood. Some insects have multivoltine life cycles, and each generation induces galls on different plant species or different organs in the same host. Such host-use patterns are interesting study subjects to clarify the gall-inducing mechanisms of insects. We focused on a multivoltine and gall-inducing psyllid Stenopsylla nigricornis Kuwayama (Hemiptera: Psylloidea: Triozidae), which is associated with Symplocos lucida Sieb. (Symplocaceae). Based on periodic field surveys in Kyushu, Japan, S. nigricornis is revealed to have a bivoltine life history. Then, we revealed that the spring generation induces galls on leaves, while the autumn generation does so on flower buds and overwintering leaf buds. We also analyzed phytohormones in normal plant tissue, S. nigricornis nymphs, and their galls. As a result, nymphs were discovered to contain much higher concentrations of isopentenyladenosine and its possible precursor, isopentenyladenosine riboside than plant tissues, strongly suggesting that the phytohormone is involved in gall induction by S. nigricornis. Because flower bud galls contained significantly lower concentrations of abscisic acid (ABA) than normal flower bud, the autumn generation nymphs are considered to regulate the ABA level and to promote the earlier opening of host flower buds.     

Effect of exogenous abscisic acid on cold acclimation in two Magnolia species

In northern China, freezing injury is observed frequently in the rare species Magnolia wufengensis but not in the more common species Magnolia denudata. To investigate the role of the phytohormone abscisic acid (ABA) on frost tolerance in these two species, exogenous ABA was applied to the seedlings and then physiological and biochemical responses were measured during cold acclimation. Shoot growth cessation was stimulated by ABA in M. wufengensis but not in M. denudata. Abscisic acid inhibited shoot growth in M. wufengensis but not in M. denudata. Treatment with ABA stimulated leaf senescence in both species, and this effect was greater in M. denudata. For both species, ABA-treated plants exhibited bud dormancy sooner and had an increased tolerance to freezing, decreased water content and increased accumulation of proline, glucose, and fructose in shoots. These effects were generally greater for M. denudata. Freezing tolerance was significantly correlated with content of water, proline, glucose, and fructose for both species, but freezing tolerance was significantly correlated with raffinose content only in M. wufengensis. We conclude that exogenous ABA could increase cold acclimation and improve cold hardiness of both Magnolia species, although M. denudata was more responsive to ABA than M. wufengensis, which might result from a greater dehydration and accumulation of proline and certain soluble sugars.     

Adenosine monophosphate-activated protein kinase from the mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>: cDNA cloning and profiles under cold stress

Adenosine monophosphate-activated protein kinase (AMPK), an important energy sensor, is crucial for organism survival under adverse conditions. In this study, the roles of this gene under cold stress in a warm-water mud crab, Scylla paramamosain was investigated. The full-length cDNA (SpAMPK) was 1884 bp and its open reading frame of 1566 bp was isolated and characterized. The expressions of SpAMPK detected by quantitative real-time PCR (qRT-PCR) in various tissues revealed that the highest expression was in the hepatopancreas. The profiles of SpAMPK gene in the hepatopancreas, chela muscle and gill were detected when the subadult crabs were exposed to the four temperature conditions of 10, 15, 20 and 25^°C. The results showed that the expression patterns of SpAMPK mRNA in the three tissues were significantly higher when crabs were exposed to 15^°C than the other three temperature treatments, while at 10^°C treatment, the SpAMPK mRNA was lowest among the four temperature treatments. These findings suggested that the high expression of SpAMPK mRNA might initiate ATP-producing pathways to generate energy to cope with cold stress at 15^°C treatment, which was slightly below the range of optimum temperatures; while treatment at 10^°C, far lower than optima, the low expression of SpAMPK mRNA could reduce the energy expenditure and thus induce the crabs into cold anesthesia. The results of SpAMPK in this study might contribute to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain.     

Genome-wide identification and expression analysis of the GRAS family proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The GRAS gene family performs a variety of functions in plant growth and development processes, and they also play essential roles in plant response to environmental stresses. Medicago truncatula is a diploid plant with a small genome used as a model organism. Despite the vital role of GRAS genes in plant growth regulation, few studies on these genes in M. truncatula have been conducted to date. Using the M. truncatula reference genome data, we identified 68 MtGRAS genes, which were classified into 16 groups by phylogenetic analysis, located on eight chromosomes. The structure analysis indicated that MtGRAS genes retained a relatively constant exon–intron composition during the evolution of the M. truncatula genome. Most of the closely related members in the phylogenetic tree had similar motif compositions. Different motifs distributed in different groups of the MtGRAS genes were the sources of their functional divergence. Twenty-eight MtGRAS genes were expressed in six tissues, namely root, bud, blade, seedpod, nodule, and flower tissues, suggesting their putative function in many aspects of plant growth and development. Nine MtGRAS genes were upregulated under cold, freezing, drought, ABA, and salt stress treatments, indicating that they play vital roles in the response to abiotic stress in M. truncatula. Our study provides valuable information that can be utilized to improve the quality and agronomic benefits of M. truncatula and other plants.