Insights for the Captive Management of South China Tigers Based on a Large-Scale Genetic Survey.

There is an urgent need to find a way to improve the genetic diversity of captive South China tiger (SCT, Panthera tigris amoyensis), the most critically endangered taxon of living tigers, facing inbreeding depression. The genomes showed that 13 hybrid SCTs from Meihuashan were divided into two groups; one group included three individuals who had a closer relationship with pureblood SCTs than another group. The three individuals shared more that 40% of their genome with pureblood SCTs and might be potential individuals for genetic rescuing in SCTs. A large-scale genetic survey based on 319 pureblood SCTs showed that the mean microsatellite inbreeding coefficient of pureblood SCTs decreased significantly from 0.1789 to 0.0600 (p = 0.000009) and the ratio of heterozygous loci increased significantly from 38.5% to 43.2% (p = 0.02) after one individual of the Chongqing line joined the Suzhou line and began to breed in the mid-1980s, which is a reason why the current SCTs keep a moderate level of microsatellite heterozygosity and nucleotide diversity. However, it is important to establish a back-up population based on the three individuals through introducing one pureblood SCT into the back-up population every year. The back-up population should be an important reserve in case the pureblood SCTs are in danger in the future.

Chromosome-scale genomes reveal genomic consequences of inbreeding in the South China tiger: A comparative study with the Amur tiger.

The South China tiger (Panthera tigris amoyensis, SCT) is the most critically endangered subspecies of tiger due to functional extinction in the wild. Inbreeding depression is observed among the captive population descended from six wild ancestors, resulting in high juvenile mortality and low reproduction. We assembled and characterized the first SCT genome and an improved Amur tiger (P. t. altaica, AT) genome named AmyTig1.0 and PanTig2.0. The two genomes are the most continuous and comprehensive among any tiger genomes yet reported at the chromosomal level. By using the two genomes and resequencing data of 15 SCT and 13 AT individuals, we investigated the genomic signature of inbreeding depression of the SCT. The results indicated that the effective population size of SCT experienced three phases of decline, ~5.0-1.0 thousand years ago, 100 years ago, and since captive breeding in 1963. We found 43 long runs of homozygosity fragments that were shared by all individuals in the SCT population and covered a total length of 20.63% in the SCT genome. We also detected a large proportion of identical-by-descent segments across the genome in the SCT population, especially on ChrB4. Deleterious nonsynonymous single nucleotide polymorphic sites and loss-of-function mutations were found across genomes with extensive potential influences, despite a proportion of these loads having been purged by inbreeding depression. Our research provides an invaluable resource for the formulation of genetic management policies for the South China tiger such as developing genome-based breeding and genetic rescue strategy.

Molecular identification of Colpodella sp. of South China tiger Panthera tigris amoyensis (Hilzheimer) in the Meihua Mountains, Fujian, China.

A three-year-old male South China tiger died in the tiger enclosure of the China Tiger Park in the Meihua Mountains on December 2018 after being bitten by a tick. This tiger presented clinical symptoms like whole-body severe jaundice, hepatosplenomegaly, kidney, and lymph node hemorrhages. The Colpodella sp.-specific 18S rRNA gene was detected using nested PCR. Interestingly, the DNA isolated from the blood of the tiger was found to be 100% similar to that of the tick by NCBI BLAST analysis. However, the DNA fragments isolated from the tiger's blood were 90.1% similar to the Colpodella sp. strain human erythrocyte parasite (HEP, MH208621) and 90.4% similar to the Colpodella sp. strain Heilongjiang (HLJ, KT364261). To investigate the species of ticks and ticks-carried Colpodella parasites in this region, the species of ticks obtained from the grasses outside the tiger enclosure and the species of Colpodella carried by ticks were identified. The DNA from ticks as well as that from the tick-borne Colpodella sp. were amplified from each tick using PCR followed by amplicon sequencing. In total 402 adult ticks samples were collected, among which 22 were positive for Colpodella sp. (5.5%), and the species were further determined by morphology, DNA sequencing and phylogenetic analyses. Interestingly, one Colpodella sp. was found to have 94.2% sequence similarities to the Colpodella sp. strain HEP (MH208621). This strain was previously reported to infect a woman in Yunnan, China. In addition, three Colpodella sp. showed 87-91% sequence similarities to the Colpodella sp. strain HLJ (KT364261), which was previously reported to infect human in Heilongjiang, China. This study disclosed the possibility of zoonotic transmission of Colpodella sp. by ticks in China. Finally, it provides a basis for urgently determining and monitoring the repertoire of ticks-borne piroplasmid pathogens, with the ultimate aim of strategic control.

Colonization and Development of the Fecal Microflora of South China Tiger Cubs (Panthera tigris amoyensis) by Sequencing of the 16S rRNA Gene.

Postnatal colonization and development of the gut microbiota is linked to health and growth. A comprehensive understanding of the postnatal compositional changes and development of the microbial community is helpful to understand the gut health and improve the survival rate of South China tiger cubs (Panthera tigris amoyensis). Fecal samples from three tiger cubs were collected on the day of birth in 2018 (June 17-21 [G0], July 18 [G1], July 31 [G2], and August 7 [G3]). The 16S rRNA genes of the fecal microflora were sequenced. Results showed that 38 phyla, 58 classes, 134 orders, 272 families, and 636 genera of bacteria from 3,059 operational taxonomic units were identified from 12 fecal samples. The diversity and abundance of species of group G0 were significantly higher (p < 0.05 or 0.01) than those of groups G2 and G3. The predominant phylum was Proteobacteria in groups G0 and G1 (38.85% and 48%, respectively) and Firmicutes in groups G2 and G3 (71.42% and 75.29%, respectively). At the phylum level, the abundance of Deinococcus-Thermus was significantly decreased in groups G1, G2, and G3 as compared to group G0 (p < 0.05), while that of Firmicutes was significantly increased in groups G2 and G3 (p < 0.05). At the genus level, the abundance of Faecalibacterium, Ralstonia, and unidentified Rickettsiales was significantly decreased in groups G1, G2, and G3 as compared with group G0 (p < 0.05), while that of Pseudomonas was significantly decreased in groups G2 and G3 (p < 0.05). The composition and structure of fecal microbiota of South China tiger cubs changed after birth.

Transcriptome analysis of ovary culture-induced embryogenesis in cucumber (Cucumis sativus L.).

BACKGROUND: Ovary culture is a useful technique used to generate double haploid (DH) cucumber (Cucumis sativus L.) plants. However, cucumber ovary culture have a low rate of embryo induction and plant regeneration. Moreover, the cucumber embryogenesis mechanism remains unclear. In this study, we explored the molecular basis of cucumber embryogenesis in order to establish a foundation for a more efficient ovary culture method. Using transcriptome sequencing, we also investigated the differential expression of genes during the embryogenesis process. METHODS: Cytological and morphological observations have divided cucumber ovary culture into three stages: early embryo development (T0), embryo morphogenesis (T1, T2, T3 and T4), and shoot formation (T5). We selected six key time points for transcriptome sequencing and analysis: T0 (the ovules were cultured for 0 d), T1 (the ovules were cultured for 2 d), T2 (the embryos were cultured for 10 d), T3 (the embryos were cultured for 20 d), T4 (the embryos were cultured for 30 d), and T5 (the shoots after 60 d culture). RESULTS: We used cytology and morphology to observe the characteristics of the cucumber's developmental transformation during embryogenesis and plant regeneration. The differentially expressed genes(DEGs) at developmental transition points were analyzed using transcriptome sequencing. In the early embryogenesis stage, the cells expanded, which was the signal for gametophytes to switch to the sporophyte development pathway. RNA-seq revealed that when compared to the fresh unpollinated ovaries, there were 3,468 up-regulated genes in the embryos, including hormone signal transduction genes, hormone response genes, and stress-induced genes. The reported embryogenesis-related genes BBM, HSP90 and AGL were also actively expressed during this stage. In the embryo morphogenesis stage (from cell division to cotyledon-embryo formation), 480 genes that functioned in protein complex binding, microtubule binding, tetrapyrrole binding, tubulin binding and other microtubule activities were continuously up-regulated during the T1, T2, T3 and T4 time points. This indicated that the cytoskeleton structure was continuously being built and maintained by the action of microtubule-binding proteins and enzyme modification. In the shoot formation stage, 1,383 genes were up-regulated that were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, phenylalanine metabolism, and starch and sucrose metabolism. These up-regualted genes included six transcription factors that contained a B3 domain, nine genes in the AP2/ERF family, and two genes encoding WUS homologous domain proteins. CONCLUSIONS: Evaluation of molecular gynogenesis events may contribute to a better understanding of the molecular mechanism of cucumber ovarian culture.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

A leaf shape mutant provides insight into PINOID Serine/Threonine Kinase function in cucumber (Cucumis sativus L.).

Optimizing leaf shape is a major challenge in efforts to develop an ideal plant type. Cucumber leaf shapes are diverse; however, the molecular regulatory mechanisms underlying leaf shape formation are unknown. In this study, we obtained a round leaf mutant (rl) from an ethyl methanesulfonate-induced mutagenesis population. Genetic analysis revealed that a single recessive gene, rl, is responsible for this mutation. A modified MutMap analysis combined linkage mapping identified a single nucleotide polymorphism within a candidate gene, Csa1M537400, as the mutation underlying the trait. Csa1M537400 encodes a PINOID kinase protein involved in auxin transport. Expression of Csa1M537400 was significantly lower in the rl mutant than in wild type, and it displayed higher levels of IAA (indole-3-acetic acid) in several tissues. Treatment of wild-type plants with an auxin transport inhibitor induced the formation of round leaves, similar to those in the rl mutant. Altered expression patterns of several auxin-related genes in the rl mutant suggest that rl plays a key role in auxin biosynthesis, transport, and response in cucumber. These findings provide insight into the molecular mechanism underlying the regulation of auxin signaling pathways in cucumber, and will be valuable in the development of an ideal plant type.

Fine Mapping of CsVYL, Conferring Virescent Leaf Through the Regulation of Chloroplast Development in Cucumber.

Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F2 and BC1 populations were constructed from the cross between vyl mutant and cucumber inbred line 'Hazerd' to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3.8 cM interval on chromosome 4 using these 80 F2 individuals and BSA (bulked segregation analysis) approach. Fine genetic map was conducted with 1542 F2 plants and narrowed down the vyl locus to an 86.3 kb genomic region, which contains a total of 11 genes. Sequence alignment between the wild type (WT) and vyl only identified one single nucleotide mutation (C→T) in the first exon of gene Csa4G637110, which encodes a DnaJ-like zinc finger protein. Gene Expression analysis confirmed the differences in transcription level of Csa4G637110 between wild type and mutant plants. Map-based cloning of the CsVYL gene could accelerate the study of chloroplast development and chlorophyll synthesis of cucumber.

Rapid identification of fruit length loci in cucumber (Cucumis sativus L.) using next-generation sequencing (NGS)-based QTL analysis.

The cucumber (Cucumis sativus L.) exhibits extensive variations in fruit size and shape. Fruit length is an important agronomic and domesticated trait controlled by quantitative trait loci (QTLs). Nonetheless, the underlying molecular and genetic mechanisms that determine cucumber fruit length remain unclear. QTL-seq is an efficient strategy for QTL identification that takes advantage of bulked-segregant analysis (BSA) and next-generation sequencing (NGS). In the present study, we conducted QTL mapping and QTL-seq of cucumber fruit length. QTL mapping identified 8 QTLs for immature and mature fruit length. A major-effect QTL fl3.2, which explained a maximum of 38.87% of the phenotypic variation, was detected. A genome-wide comparison of SNP profiles between two DNA bulks identified 6 QTLs for ovary length. QTLs ovl3.1 and ovl3.2 both had major effects on ovary length with a △ (SNP-index) of 0.80 (P < 0.01) and 0.74 (P < 0.01), respectively. Quantitative RT-PCR of fruit size-related homologous genes localized in the consensus QTL FL3.2 was conducted. Four candidate genes exhibited increased expression levels in long fruit genotypes. Our results demonstrated the power of the QTL-seq method in rapid QTL detection and provided reliable QTL regions for fine mapping of fruit length-related loci and for identifying candidate genes.

Association between interferon gamma 13-CA-repeats polymorphism and metastasis of nasopharyngeal carcinoma in a population of Northern China.

Interferon Gamma gamma (IFN-γ) 13-CA-repeats polymorphism is associated with a variety of diseases; here we report its association with nasopharyngeal carcinoma (NPC) metastasis in a retrospective analysis of a cohort of 220 NPC patients in the northern China. The results showed that the distributions of CA13-/CA13-genotypes were significantly higher in the patients with lymph node metastasis (P<0.05) and distant metastasis (P<0.001); there was a significant difference between NPC patients with stage I+II and those with stage III+IV regarding CA13+/CA13-(P<0.001) and CA13-/CA13- genotypes (P<0.001); further analysis showed a more pronounced difference between NPC patients with stage I+II+III and those with stage IV for CA13-/CA13-genotype (P<0.001), whereas no difference was found for CA13+/CA13- genotype (P=0.790). Thus, we identify that IFN-γ 13-CA-repeat polymorphism is significantly associated with the metastasis of NPC, which may provide insights into its prognosis and individualized treatment.

Analysis of variable sites between two complete South China tiger (Panthera tigris amoyensis) mitochondrial genomes.

In order to investigate the mitochondrial genome of Panthera tigris amoyensis, two South China tigers (P25 and P27) were analyzed following 15 cymt-specific primer sets. The entire mtDNA sequence was found to be 16,957 bp and 17,001 bp long for P25 and P27 respectively, and this difference in length between P25 and P27 occurred in the number of tandem repeats in the RS-3 segment of the control region. The structural characteristics of complete P. t. amoyensis mitochondrial genomes were also highly similar to those of P. uncia. Additionally, the rate of point mutation was only 0.3% and a total of 59 variable sites between P25 and P27 were found. Out of the 59 variable sites, 6 were located in 6 different tRNA genes, 6 in the 2 rRNA genes, 7 in non-coding regions (one located between tRNA-Asn and tRNA-Tyr and six in the D-loop), and 40 in 10 protein-coding genes. COI held the largest amount of variable sites (9 sites) and Cytb contained the highest variable rate (0.7%) in the complete sequences. Moreover, out of the 40 variable sites located in 10 protein-coding genes, 12 sites were nonsynonymous.