Development of genic SSR markers from transcriptome sequencing of pear buds

研究目的：基于转录组数据开发具有扩增率高和跨物种转移性的基因组编码区内的SSR（genic-SSR）标记,为梨属植物的分子系统发育关系和遗传多样性相关研究提供新的方法。创新要点：首次利用梨属植物的转录组测序（RNA-seq）数据结合M-13荧光尾巴高效率地开发了104个genic-SSR标记,并成功将其应用于梨属植物的系统发育关系研究中。研究方法：应用生物信息学软件从转录组测序数据中搜索SSR位点和设计相应引物,结合高效的M-13荧光尾巴的方法筛选多态性高的SSR标记。重要结论：转录组数据能够为梨属植物分子系统发育关系和遗传多样性研究提供新的SSR标记来源。     

基于杨梅基因组鸟枪法测序开发的107个SSR标记及其在品系鉴定中应用

研究目的：开发多态性高的基因组简单重复序列（SSR）标记,为杨梅遗传多样性、遗传图谱构建及品种鉴定相关研究奠定基础。 创新要点：从杨梅全基因组鸟枪法测序数据中新开发了107个SSR标记,并应用于品种间遗传多样性分析及品系鉴定。 研究方法：应用生物信息学软件从基因组测序数据中搜索SSR位点并设计相应引物,添加通用的M13尾巴和荧光标识筛选多态性高的SSR标记。 重要结论：开发的107个基因组SSR标记可以广泛应用于杨梅种质资源和遗传育种研究,鉴定出三个杨梅新品系。     

Teaching human genetics with mustard: rapid cycling Brassica rapa (fast plants type) as a model for human genetics in the classroom laboratory

We have developed experiments and materials to model human genetics using rapid cycling Brassica rapa, also known as Fast Plants. Because of their self-incompatibility for pollination and the genetic diversity within strains, B. rapa can serve as a relevant model for human genetics in teaching laboratory experiments. The experiment presented here is a paternity exclusion project in which a child is born with a known mother but two possible alleged fathers. Students use DNA markers (microsatellites) to perform paternity exclusion on these subjects. Realistic DNA marker analysis can be challenging to implement within the limitations of an instructional lab, but we have optimized the experimental methods to work in a teaching lab environment and to maximize the “hands-on” experience for the students. The genetic individuality of each B. rapa plant, revealed by analysis of polymorphic microsatellite markers, means that each time students perform this project, they obtain unique results that foster independent thinking in the process of data interpretation.     

Determination of the genetic diversity of vegetable soybean [Glycine max (L.) Merr.] using EST-SSR markers

The development of expressed sequence tag-derived simple sequence repeats (EST-SSRs) provided a useful tool for investigating plant genetic diversity. In the present study, 22 polymorphic EST-SSRs from grain soybean were identified and used to assess the genetic diversity in 48 vegetable soybean accessions. Among the 22 EST-SSR loci, tri-nucleotides were the most abundant repeats, accounting for 50.00% of the total motifs. GAA was the most common motif among tri-nucleotide repeats, with a frequency of 18.18%. Polymorphic analysis identified a total of 71 alleles, with an average of 3.23 per locus. The polymorphism information content (PIC) values ranged from 0.144 to 0.630, with a mean of 0.386. Observed heterozygosity (H _o) values varied from 0.0196 to 1.0000, with an average of 0.6092, while the expected heterozygosity (H _e) values ranged from 0.1502 to 0.6840, with a mean value of 0.4616. Principal coordinate analysis and phylogenetic tree analysis indicated that the accessions could be assigned to different groups based to a large extent on their geographic distribution, and most accessions from China were clustered into the same groups. These results suggest that Chinese vegetable soybean accessions have a narrow genetic base. The results of this study indicate that EST-SSRs from grain soybean have high transferability to vegetable soybean, and that these new markers would be helpful in taxonomy, molecular breeding, and comparative mapping studies of vegetable soybean in the future.     

Conditional gene manipulation: Cre-ating a new biological era

To solve the problem of embryonic lethality in conventional gene knockouts,site-specific recombinase(SSR)systems(Cre-loxP,Flp-FRT,andΦC31)have been used for tissue-specific gene knockout.With the combination of an SSR system and inducible gene expression systems(tetracycline and tamoxifen),stage-specific knockout and transgenic expression can be achieved.The application of this"SSR+inducible"conditional tool to genomic manipulation can be extended in various ways.Alternatives to conditional gene targeting,such as conditional gene trapping,multipurpose conditional alleles,and conditional gene silencing,have been developed.SSR systems can also be used to construct precise disease models with point mutations and chromosomal abnormalities.With these exciting achievements,we are moving towards a new era in which the whole genome can be manipulated as we wish.     

Genomic organization and sequence dynamics of the AvrPiz-t locus in Magnaporthe oryzae

Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens. The R gene mediates resistance, in most cases, dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene. The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t. In this study, we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates, designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions, respectively. The sequence of the AvrPiz-t-ZB15 region is 120 966 bp whereas avrPiz-t-70-15 is 146 292 bp in length. The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus. Nevertheless, frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified, representing the major variation of the AvrPiz-t locus between different isolates. Moreover, TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions, respectively, indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus. The findings of this study suggest that M. oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.     

Developing new SSR markers from ESTs of pea (Pisum sativum L.)

The development of expressed sequence tags (ESTs) from pea has provided a useful source for mining novel simple sequence repeat (SSR) markers. In the present research, in order to find EST-derived SSR markers, 18 552 pea ESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and assembled into 10 086 unigenes. A total of 586 microsatellites in 530 unigenes were identified, indicating that merely 5.25% of sequences contained SSRs. The most abundant SSRs within pea were tri-nucleotide repeat motifs, and among all the tri-nucleotide repeats, the motif GAA was the most abundant type. In total, 49 SSRs were used for primer design. EST-SSR loci were subsequently screened on 10 widely adapted varieties in China. Of these, nine loci showed polymorphic profiles that revealed two to three alleles per locus. The polymorphism information content value ranged from 0.18 to 0.58 with an average of 0.41. Furthermore, transferable analysis revealed that some of these loci showed transferability to faba bean. Because of their polymorphism and transferability, these nine novel EST-SSRs will be valuable tools for marker-assisted breeding and comparative mapping of pea in the future.     

Development of a Meiosis Concept Inventory

We have designed, developed, and validated a 17-question Meiosis Concept Inventory (Meiosis CI) to diagnose student misconceptions on meiosis, which is a fundamental concept in genetics. We targeted large introductory biology and genetics courses and used published methodology for question development, which included the validation of questions by student interviews (n = 28), in-class testing of the questions by students (n = 193), and expert (n = 8) consensus on the correct answers. Our item analysis showed that the questions’ difficulty and discrimination indices were in agreement with published recommended standards and discriminated effectively between high- and low-scoring students. We foresee other institutions using the Meiosis CI as both a diagnostic tool and an instrument to assess teaching effectiveness and student progress, and invite instructors to visit http://q4b.biology.ubc.ca for more information.     

水稻早世代稳定株系特有分子标记研究

SSR和ISSR分子标记已广泛应用于研究遗传行为和基因定位。在对水稻早世代稳定遗传特性的研究中,选用分布于水稻12条染色体上的168对多态性较好的SSR引物,对28个稳定株系的F_1、F_2、F_3及同组合的分离株系进行了SSR标记分析；选用26个多态性较好的ISSR引物对5个组合中出现的8个稳定株系及同组合的分离株系进行了ISSR分子标记。结果显示：稳定株系的标记带型出现三种类型：(1)母本带出现,父本带消失；(2)父本带出现,母本带消失；(3)出现非父母标记带型。分离株系表现为杂合标记带。推测水稻早世代稳定是在遗传因子作用下,在杂交受精或合子早期发育中发生了双亲染色体重排,形成纯合的F_1单株。     

Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences

For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum. Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids.     

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis. The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites.     

Molecular variability and evolution of a natural population of tomato yellow leaf curl virus in Shanghai,ChinaXiu-ling YANG,Meng-ning ZHOU,Ya-juan QIAN,Yan XIE,Xue-ping ZHOU简

Tomato yellow leaf curl virus (TYLCV), belonging to the genus Begomovirus of the family Geminiviridae, is emerging as the most destructive pathogen of tomato plants. Since the first report of TYLCV in Shanghai, China in 2006, TYLCV has spread rapidly to 13 provinces or autonomous regions of China. In this study, the molecular variability and evolution of TYLCV were monitored in Shanghai from its first upsurge in 2006 until 2010. Full-length genomic sequences of 26 isolates were obtained by rolling circle amplification. Sequence analysis showed that the intergenic region was the most variable, with a mean mutation rate of 4.81×10^?3 nucleotide substitutions per site per year. Genetic differentiation was found within isolates obtained from 2006, 2009, and 2010, though a linear increase in genetic diversity over time was not evident. Whilst significant parts of TYLCV genes were under negative selection, the C4 gene embedded entirely within the C1 gene had a tendency to undergo positive selection. Our results indicate that a mechanism of independent evolution of overlapping regions could apply to the natural population of TYLCV in Shanghai, China.     

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity

Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer (RaGAPDH) has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genomic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligand-binding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coli (EHEC), belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestifer displayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism’s virulence.     

分子标记及其在甘薯种质资源和遗传育种中的应用

植物分子生物学和植物生物技术利用分子标记技术得到了大力发展,分子标记的创新、发展与应用对其起了积极的推动作用,本文就RFLP、AFLP、RAPD、DAF、SSR、CAPS分子标记的原理与技术及其在甘薯种质资源和遗传育种中的应用作一介绍     

中国地区孢子丝菌临床分离株的分子鉴定(英文)简

In this study, we investigated the molecular phylogeny of 64 clinical isolates which were identified as Sporothrix schenckii sensu lato by morphological identification. All of the strains were isolates from patients from several provinces in China. The phylogeny was inferred by DNA sequence analyses based on datasets of the ribo- somal internal transcribed spacer （ITS） and a combined ITS and partial 13-tubulin region. Reference sequences were retrieved from GenBank. Results showed that all of the isolates were clustered in a distinct clade with a type of Spo- rothrix globosa. Our analysis showed that S. globosa is the causal agent of the tested sporotrichosis in China, rather than S. schenckii that was generally believed to be the case. The existence of S. schenckii in China remains to be confirmed. This study improved our understanding of the distribution of the species in S. schenckii complex.     

Assessment of genetic diversity by simple sequence repeat markers among forty elite varieties in the germplasm for malting barley breeding

The genetic diversity and relationship among 40 elite barley varieties were analyzed based on simple sequence repeat (SSR) genotyping data. The amplified fragments from SSR primers were highly polymorphic in the barley accessions investigated. A total of 85 alleles were detected at 35 SSR loci, and allelic variations existed at 29 SSR loci. The allele number per locus ranged from 1 to 5 with an average of 2.4 alleles per locus detected from the 40 barley accessions. A cluster analysis based on the genetic similarity coefficients was conducted and the 40 varieties were classified into two groups. Seven malting barley varieties from China fell into the same subgroup. It was found that the genetic diversity within the Chinese malting barley varieties was narrower than that in other barley germplasm sources, suggesting the importance and feasibility of introducing elite genotypes from different origins for malting barley breeding in China.     

A recessive gene controlling male sterility sensitive to short daylength/low temperature in wheat (Triticum aestivum L.)

Utilization of a two-line breeding system via photoperiod-thermo sensitive male sterility has a great potential for hybrid production in wheat (Triticum aestivum L.). 337S is a novel wheat male sterile line sensitive to both short daylength/low temperature and long daylength/high temperature. Five F₂ populations derived from the crosses between 337S and five common wheat varieties were developed for genetic analysis. All F₁’s were highly fertile while segregation occurred in the F₂ populations with a ratio of 3 fertile:1 sterile under short daylength/low temperature. It is shown that male sterility in 337S was controlled by a single recessive gene, temporarily designated as wptms3. Bulked segregant analysis (BSA) coupled with simple sequence repeat (SSR) markers was applied to map the sterile gene using one mapping population. The wptms3 gene was mapped to chromosome arm 1BS and flanked by Xgwm413 and Xgwm182 at a genetic distance of 3.2 and 23.5 cM, respectively. The accuracy and efficiency of marker-assisted selection were evaluated and proved essential for identifying homozygous recessive male sterile genotypes of the wptms3 gene in F₂ generation.     

Mothering matters: Maternal style predicts puppies’ future performance

Bray et al. (2017, Proceedings of the National Academy of Sciences, 113(32), 9128–9133) recently showed that maternal interactions between service dog mothers and their puppies were predictive of puppies’ future success as a candidate service dog. These findings prompt questions into the role of genetics and early experiences and may provide useful selection tools for working dog breeding programs.