Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

QTL for horticulturally important traits associated with pleiotropic andromonoecy and carpel number loci, and a paracentric inversion in cucumber.

The legendary cucumber inbred line WI2757 possesses a rare combination of resistances against nine pathogens, which is an important germplasm for cucumber breeding. However, WI2757 flowers late and does not perform well under field conditions. The genetic basis for horticulturally important traits other than disease resistances in WI2757 is largely unknown. In this study, we conducted QTL mapping using F2 and recombinant inbred line (RIL) populations from the WI2757 × True Lemon cross that were segregating for multiple traits. Phenotypic data were collected in replicated field trials across multiple years for seven traits including fruit carpel number (CN) and sex expression. A high-density SNP-based genetic map was developed with genotyping by sequencing of the RIL population, which revealed a region on chromosome 1 with strong recombination suppression. The reduced recombination in this region was due to a ~ 10-Mbp paracentric inversion in WI2757 that was confirmed with additional segregation and cytological (FISH) analyses. Thirty-six QTL were detected for flowering time, fruit length (FL), fruit diameter (FD), fruit shape (LD), fruit number (FN), CN, and powdery mildew resistance. Five moderate- or major-effect QTL for FL, FD, LD, and FN inside the inversion are likely the pleiotropic effects of the andromonoecy (m), or the cn locus. The major-effect flowering time QTL ft1.1 was also mapped inside the inversion, which seems to be different from the previously assigned delayed flowering in WI2757. Implications of these findings on the use of WI2757 in cucumber breeding are discussed.

Interaction of Lead and Cadmium Reduced Cadmium Toxicity in Ficus parvifolia Seedlings.

Potentially toxic elements (PTEs) pollution occurs widely in soils due to various anthropogenic activities. Lead (Pb) and cadmium (Cd) coexist in soil frequently, threatening plant growth. To explore the interaction effect between Pb and Cd in Ficus parvifolia and the response of plant physiological characteristics to Pb and Cd stress, we designed a soil culture experiment. The experiment demonstrated that Pb stress improved leaf photosynthesis ability, while Cd stress inhibited it. Furthermore, Pb or Cd stress increased malonaldehyde (MDA) content, but plants were able to reduce it by increasing antioxidant enzyme activities. The presence of Pb could alleviate Cd phytotoxicity in plants by inhibiting Cd uptake and accumulation as well as increasing leaf photosynthesis and antioxidant ability. Pearson correlation analysis illustrated that the variability of Cd uptake and accumulation between Pb and Cd stress was related to plant biomass and antioxidant enzyme activities. This research will offer a new perspective on alleviating Cd phytotoxicity in plants.

Genetic diversity of the wild ancient tea tree (Camellia taliensis) populations at different altitudes in Qianjiazhai.

In this study, the genetic diversity and population structure of 4 wild ancient tea tree (Camellia taliensis) populations at different altitudes (2,050, 2,200, 2,350, and 2,500 m) in Qianjiazhai Nature Reserve, Zhenyuan country, Yunnan province, were investigated using EST-SSR molecular markers to compare their genetic variation against altitude. In total, 182 alleles were detected across all loci, ranging from 6 to 25. The top one informative SSR was CsEMS4 with polymorphism information content (PIC) of 0.96. The genetic diversity of this species was high, with 100% of loci being polymorphic, an average Nei's gene diversity (H) of 0.82, and Shannon's information index (I) of 1.99. By contrast, at the population level, the genetic diversity of wild ancient tea tree was relatively low, with values of H and I being 0.79 and 1.84, respectively. Analysis of molecular variance (AMOVA) revealed a minor genetic differentiation (12.84%) among populations, and most of the genetic variation (87.16%) was detected within populations. Using population structure analysis, we found that the germplasm of wild ancient tea tree was divided into three groups, and there was a substantial gene exchange among these three groups at different altitudes. Divergent habitats caused by altitudes and high gene flow played important roles in genetic diversity of wild ancient tea tree populations, which will provide new opportunities for promoting their protection and potential utilization.

Transcriptome Analysis and Identification of a Female-Specific SSR Marker in Pistacia chinensis Based on Illumina Paired-End RNA Sequencing.

Pistacia chinensis Bunge (P. chinensis), a dioecious plant species, has been widely found in China. The female P. chinensis plants are more important than male plants in agricultural production, as their seeds can serve as an ideal feedstock for biodiesel. However, the sex of P. chinensis plants is hard to distinguish during the seedling stage due to the scarcity of available transcriptomic and genomic information. In this work, Illumina paired-end RNA sequencing assay was conducted to unravel the transcriptomic profiles of female and male P. chinensis flower buds. In total, 50,925,088 and 51,470,578 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 83,370 unigenes with a mean length of 1.3 kb were screened. Overall, 64,539 unigenes (77.48%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG, and GO databases, 71 of which were putatively related to the floral development of P. chinensis. Additionally, 21,662 simple sequence repeat (SSR) motifs were identified in 17,028 unigenes of P. chinensis, and the mononucleotide motif was the most dominant type of repeats (52.59%) in P. chinensis, followed by dinucleotide (22.29%), trinucleotide (20.15%). The most abundant repeats were AG/CT (13.97%), followed by AAC/GTT (6.75%) and AT/TA (6.10%). Based on these SSR, 983 EST-SSR primers were designed, 151 of which were randomly chosen for validation. Of these validated EST-SSR markers, 25 SSR markers were found to be polymorphic between male and female plants. One SSR marker, namelyPCSSR55, displayed excellent specificity in female plants, which could clearly distinguish between male and female P. chinensis. Altogether, our findings not only reveal that the EST-SSR marker is extremely effective in distinguishing between male and female P. chinensis but also provide a solid framework for sex determination of plant seedlings.

Integrated transcriptome and metabolome profiling of Camellia reticulata reveal mechanisms of flower color differentiation.

Camellia reticulata (Lindl.) is an important ornamental plant in China. Long-term natural or artificial selections have resulted in diverse phenotypes, especially for flower colors. Modulating flower colors can enhance the visual appeal and economic value in ornamental plants. In this study, we investigated the molecular mechanisms underlying flower color differentiation in C. reticulata. We performed a combined transcriptome and metabolome analysis of the petals of a popular variety C. reticulata (HHYC) (red), and its two cultivars "Xuejiao" (XJ) (pink) and "Tongzimian" (TZM) (white). Targeted metabolome profiling identified 310 flavonoid compounds of which 18 anthocyanins were differentially accumulated among the three samples with an accumulation pattern of HHYC > XJ > TZM. Likewise, transcriptome analysis showed that carotenoid and anthocyanin biosynthetic structural genes were mostly expressed in order of HHYC > XJ > TZM. Two genes (gene-LOC114287745765 and gene-LOC114289234) encoding for anthocyanidin 3-O-glucosyltransferase are predicted to be responsible for red coloration in HHYC and XJ. We also detected 42 MYB and 29 bHLH transcription factors as key regulators of anthocyanin-structural genes. Overall, this work showed that flavonoids, particularly anthocyanins contents are the major determinants of flower color differentiation among the 3 C. reticulata samples. In addition, the main regulatory and structural genes modulating anthocyanin contents in C. reticulata have been unveiled. Our results will help in the development of Camellia varieties with specific flower color and quality.

[Effects of cadmium stress on growth and cadmium enrichment of Chlorophytum comosum and Chlorophytum comosum var. variegatum.] / é•‰èƒè¿«å¯¹åŠå °åŠé“¶è¾¹åŠå °ç”Ÿé•¿åŠé•‰å¯Œé›†ç‰¹æ€§çš„å½±å“.

We examined the growth and physiological characteristics of Chlorophytum comosum and Chlorophytum comosum var. variegatum by hydroponics at different Cd2+ concentrations (0, 20, 80, 200 µmol·L-1). The results showed that 20 µmol·L-1 Cd2+ did not affect those two varieties, with no changes of single leaf area, total leaf area, chlorophyll (Chl) a content, Chl (a+b) content, carotenoid content, Chla/Chlb value, intercellular carbon dioxide concentration (Ci) and transpiration rate (Tr) compared with the control (CK). Under the 80 µmol·L-1 Cd2+ stress, the initial fluorescence (Fo) and non-photochemical quenching coefficient (NPQ) were the highest for both varieties. As for Cd2+ at 200 µmol·L-1, the biomass, chlorophyll content, maximum net photosynthetic rate (Pn), stomatal conductance (gs), maximum photochemical quantum yield (Fv/Fm), actual photochemical quantum yield Y(II), the transfer factor (TF) of both varieties and all parts biomass decreased to the lowest, while peroxidase (POD), ascorbic acid peroxidase (APX) and catalase (CAT) activities of two varieties and MDA content of C. comosum var. variegatum increased in different degrees. With the increases of Cd2+ stress, the Cd content in organs of two varieties showed an increasing trend, with higher Cd accumulation in roots. C. comosum had higher Cd content and bioconcentration factor (BCF) in all organs than C. comosum var. variegatum. It suggested that two varieties had the ability to resist Cd stress. The tolerance of C. comosum was stronger, and thus could be considered as a green plant to remediate Cd contaminated water or soil.

Bulked segregant analysis reveals candidate genes responsible for dwarf formation in woody oilseed crop castor bean.

Plant dwarfism is a desirable agronomic trait in non-timber trees, but little is known about the physiological and molecular mechanism underlying dwarfism in woody plants. Castor bean (Ricinus communis) is a typical woody oilseed crop. We performed cytological observations within xylem, phloem and cambia tissues, revealing that divergent cell growth in all tissues might play a role in the dwarf phenotype in cultivated castor bean. Based on bulked segregant analyses for a F2 population generated from the crossing of a tall and a dwarf accession, we identified two QTLs associated with plant height, covering 325 candidate genes. One of these, Rc5NG4-1 encoding a putative IAA transport protein localized in the tonoplast was functionally characterized. A non-synonymous SNP (altering the amino acid sequence from Y to C at position 218) differentiated the tall and dwarf plants and we confirmed, through heterologous yeast transformation, that the IAA uptake capacities of Rc5NG4-1Y and Rc5NG4-1C were significantly different. This study provides insights into the physiological and molecular mechanisms of dwarfing in woody non-timber economically important plants, with potential to aid in the genetic breeding of castor bean and other related crops.

Construction of an integrated genetic linkage map for the A genome of Brassica napus using SSR markers derived from sequenced BACs in B. rapa.

BACKGROUND: The Multinational Brassica rapa Genome Sequencing Project (BrGSP) has developed valuable genomic resources, including BAC libraries, BAC-end sequences, genetic and physical maps, and seed BAC sequences for Brassica rapa. An integrated linkage map between the amphidiploid B. napus and diploid B. rapa will facilitate the rapid transfer of these valuable resources from B. rapa to B. napus (Oilseed rape, Canola). RESULTS: In this study, we identified over 23,000 simple sequence repeats (SSRs) from 536 sequenced BACs. 890 SSR markers (designated as BrGMS) were developed and used for the construction of an integrated linkage map for the A genome in B. rapa and B. napus. Two hundred and nineteen BrGMS markers were integrated to an existing B. napus linkage map (BnaNZDH). Among these mapped BrGMS markers, 168 were only distributed on the A genome linkage groups (LGs), 18 distrubuted both on the A and C genome LGs, and 33 only distributed on the C genome LGs. Most of the A genome LGs in B. napus were collinear with the homoeologous LGs in B. rapa, although minor inversions or rearrangements occurred on A2 and A9. The mapping of these BAC-specific SSR markers enabled assignment of 161 sequenced B. rapa BACs, as well as the associated BAC contigs to the A genome LGs of B. napus. CONCLUSION: The genetic mapping of SSR markers derived from sequenced BACs in B. rapa enabled direct links to be established between the B. napus linkage map and a B. rapa physical map, and thus the assignment of B. rapa BACs and the associated BAC contigs to the B. napus linkage map. This integrated genetic linkage map will facilitate exploitation of the B. rapa annotated genomic resources for gene tagging and map-based cloning in B. napus, and for comparative analysis of the A genome within Brassica species.

A missense mutation in the VHYNP motif of a DELLA protein causes a semi-dwarf mutant phenotype in Brassica napus.

Although dwarf genes have been widely used to improve lodging resistance and enhance harvest index in cereal crops, lodging is still a serious problem in rapeseed (Brassica napus) production. A semi-dwarf B. napus mutant, ds-1, was identified through EMS mutagenesis of a microspore-cultured DH line. The mutant had a significant reduction in height due to a lower first branch position and shorter internodes when compared with wild-type cultivars. This dwarfism was inherited as a single semi-dominant gene, ds-1. DS-1 locus was mapped to chromosome A6, and co-segregated with a microsatellite marker BnEMS1125 derived from the gene BnRGA. BnRGA encodes a DELLA protein that functions as a GA signaling repressor. The expression of a mutant BnRGA allele from ds-1, Bnrga-ds, caused dwarf phenotypes in Arabidopsis. Comparative sequencing of RGA open-reading frames (ORFs) of ds-1 and wild-type cultivars revealed a single proline (P)-to-leucine (L) substitution that may lead to a gain-of-function mutation in GA signaling. The expression of the Arabidopsis homolog, Atrga-ds, bearing this site-directed mutation also rendered dwarf phenotypes in Arabidopsis, which demonstrated that the P-to-L mutation in the VHYNP motif of Bnrga-ds is responsible for the dwarfism. A yeast two-hybrid assay confirmed that this mutation inhibited the interaction between Bnrga-ds/Atrga-ds and the GA receptor, AtGID1A, in the presence of GA(3), suggesting that the conserved proline residue in the VHYNP motif of DELLA protein directly participates in DELLA-GID1 interaction. Identification and characterization of the dwarf gene ds-1 will facilitate its utilization in improving lodging resistance in Brassica breeding.

Change of rhizospheric bacterial community of the ancient wild tea along elevational gradients in Ailao mountain, China.

The rhizospheric microbial community is one of the major environmental factors affecting the distribution and fitness of plants. Ancient wild tea plants are rare genetic resource distributed in Southwest China. In this study, we investigated that rhizospheric bacterial communities of ancient wild tea plants along the elevational gradients (2050, 2200, 2350 and 2500 m) in QianJiaZhai Reserve of Ailao Mountains. According to the Illumina MiSeq sequencing of 16 S rRNA gene amplicons, Proteobacteria, Acidobacteria and Actinobacteria were the dominant phyla with the relative abundance 43.12%, 21.61% and 14.84%, respectively. The Variibacter was the most dominant genus in rhizosphere of ancient wild tea plant. Phylogenetic null modeling analysis suggested that rhizospheric bacterial communities of ancient wild tea plants were more phylogenetically clustered than expected by chance. The bacterial community at 2050 m was unique with the highest alpha diversity, tend to cluster the nearest taxon and simple co-occurrence network structure. The unique bacterial community was correlated to multiple soil factors, and the content soil ammonium nitrogen (NH4+-N) was the key factor affecting the diversity and distribution of bacterial community along the elevational gradients. This study provided the necessary basic information for the protection of ancient tea trees and cultivation of tea plants.

[Effects of exogenous abscisic acid (ABA) on growth and physiological characteristics of Machilus yunnanensis seedlings under drought stress]. / å¤–æºè„±è½é ¸å¯¹å¹²æ—±èƒè¿«ä¸‹æ»‡æ¶¦æ¥ å¹¼è‹—ç”Ÿé•¿åŠç”Ÿç†ç‰¹æ€§çš„å½±å“.

In this study, the effects of exogenous abscisic acid (ABA) on the growth and physiolo-gical characteristics of Machilus yunnanensis seedlings were examined under well water supply conditions (70%-75% field capacity, FC) and different drought stress conditions, i.e., light drought stress (50%-55% FC) and severe drought stress (30%-35% FC), respectively. The results showed that drought stress reduced leaf relative water content, plant height, and total biomass of seedlings significantly decreased, as well as net photosynthetic rate and maximal photochemistry efficiency (Fv/Fm), but enhanced root/shoot and malondialdehyde contents (MDA). Exogenous ABA improved the adaptability of seedlings under drought stress, especially under severe drought stress, with leaf relative water content being improved 21.0%. Plant height and biomass allocation were improved by exogenous ABA under drought, while root/shoot was improved by 1.1 times compared with the well watered plants. The accumulation of MDA was decreased, the activities of CAT and SOD were significantly increased, and the proline content was increased 6.7 times by exogenous ABA under drought. Exogenous ABA application alleviated the negative effect of drought on photosynthetic organs, reduced the decrease of net photosynthetic rate and stomatal conductance under drought, and enhanced Fv/Fm value. The results suggested that exogenous ABA treatment could enhance the resistance of M. yunnanensis to drought stress.

Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus.

Microsatellite or simple sequence repeat (SSR) markers are routinely used for tagging genes and assessing genetic diversity. In spite of their importance, there are limited numbers of SSR markers available for Brassica crops. A total of 627 new SSR markers (designated BnGMS) were developed based on publicly available genome survey sequences and used to survey polymorphisms among six B. napus cultivars that serve as parents for established populations. Among these SSR markers, 591 (94.3%) successfully amplified at least one fragment and 434 (73.4%) detected polymorphism among the six B. napus cultivars. No correlation was observed between SSR motifs, repeat number or repeat length with polymorphism levels. A linkage map was constructed using 163 newly developed BnGMS marker loci and anchored with 164 public SSRs in a doubled haploid population. These new markers are evenly distributed over all linkage groups (LGs). Given that the majority of these SSRs are derived from bacterial artificial chromosome (BAC) end sequences, they will be useful in the assignment of their cognate BACs to LGs and facilitate the integration of physical maps with genetic maps for genome sequencing in B. napus.

Application of a high-resolution genetic map for chromosome-scale genome assembly and fine QTLs mapping of seed size and weight traits in castor bean.

Castor bean (Ricinus communis L., Euphorbiaceae) is a critical biodiesel crop and its seed derivatives have important industrial applications. Due to lack of a high-density genetic map, the breeding and genetic improvement of castor bean has been largely restricted. In this study, based on a recombinant inbred line (RIL) population consisting of 200 individuals, we generated 8,896 high-quality genomic SNP markers and constructed a high-resolution genetic map with 10 linkage groups (LGs), spanning 1,852.33 centiMorgan (cM). Based on the genetic map, 996 scaffolds from the draft reference genome were anchored onto 10 pseudo-chromosomes, covering 84.43% of the castor bean genome. Furthermore, the quality of the pseudo-chromosome scale assembly genome was confirmed via genome collinearity analysis within the castor bean genome as well as between castor bean and cassava. Our results provide new evidence that the phylogenetic position of castor bean is relatively solitary from other taxa in the Euphorbiaceae family. Based on the genetic map, we identified 16 QTLs that control seed size and weight (covering 851 candidate genes). The findings will be helpful for further research into potential new mechanisms controlling seed size and weight in castor bean. The genetic map and improved pseudo-chromosome scale genome provide crucial foundations for marker-assisted selection (MAS) of QTL governing important agronomic traits, as well as the accelerated molecular breeding of castor bean in a cost-effective pattern.

Postconditioning with repeated mild hypoxia protects neonatal hypoxia-ischemic rats against brain damage and promotes rehabilitation of brain function.

RATIONALE: Mild hypoxia conditioning induced by repeated episodes of transient ischemia is a clinically applicable method for protecting the brain against injury after hypoxia-ischemic brain damage. OBJECTIVE: To assess the effect of repeated mild hypoxia postconditioning on brain damage and long-term neural functional recovery after hypoxia-ischemic brain damage. METHODS AND RESULTS: Rats received different protocols of repeated mild hypoxia postconditioning. Seven-day-old rats with hypoxia ischemic brain damage (HIBD) from the left carotid ligation procedure plus 2 h hypoxic stress (8% O2 at 37 °C) were further receiving repeated mild hypoxia intermittently. The gross anatomy, functional analyses, hypoxia inducible factor 1 alpha (HIF-1a) expression, and neuronal apoptosis of the rat brains were subsequently examined. Compared to the HIBD group, rats postconditioned with mild hypoxia had elevated HIF-1a expression, more Nissl-stain positive cells in their brain tissue and their brains functioned better in behavioral analyses. The recovery of the brain function may be directly linked to the inhibitory effect of HIF-1α on neuronal apoptosis. Furthermore, there were significantly less neuronal apoptosis in the hippocampal CA1 region of the rats postconditioned with mild hypoxia, which might also be related to the higher HIF-1a expression and better brain performance. Overall, these results suggested that postconditioning of neonatal rats after HIBD with mild hypoxia increased HIF-1a expression, exerted a neuroprotective effect and promoted neural functional recovery. CONCLUSIONS: Repeated mild hypoxia postconditioning protects neonatal rats with HIBD against brain damage and improves neural functional recovery. Our results may have clinical implications for treating infants with HIBD.