分子标记技术在桉树育种研究中的应用进展

桉树是全球重要的造林树种,也是林木遗传研究的模式树种,分子标记技术在桉树遗传育种中的研究和应用具有重要的理论意义和实用价值。文中概述了几种主要的分子标记技术及其在桉树遗传育种中的应用,并提出分子标记技术在桉树遗传育种中的应用前景和研究展望。     

PRRSV2 genetic diversity defined by RFLP patterns in the United States from 2007 to 2019

The genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) increases over time. In 1998, restriction-fragment length polymorphism (RFLP) pattern analysis was introduced to differentiate PRRSV wild-type strains from VR2332, a reference strain from which a commercial vaccine (Ingelvac PRRS MLV) was derived. We have characterized here the PRRSV genetic diversity within selected RFLP families over time and U.S. geographic space, using available ISU-VDL data from 2007 to 2019. The 40,454 ORF5 sequences recovered corresponded to 228 distinct RFLPs. Four RFLPs [2-5-2 (21.2%), 1-7-4 (15.6%), 1-4-4 (11.8%), and 1-8-4 (9.9%)] represented 58.5% of all ORF5 sequences and were used for cluster analysis. Over time, there was increased detection of RFLPs 2-5-2, 1-7-4, 1-3-4, 1-3-2, and 1-12-4; decreased detection of 1-4-2, 1-18-4, 1-18-2, and 1-2-2; and different detection trends for 1-8-4, 1-4-4, 1-26-1, 1-22-2, and 1-2-4. An over-time cluster analysis revealed a single cluster for RFLP 2-5-2, supporting that sequences within RFLP 2-5-2 are still relatively conserved. For 1-7-4, 1-4-4, and 1-8-4, there were multiple clusters. State-wise cluster analysis demonstrated 4 main clusters for RFLP 1-7-4 and 1-8-4, and 6 for RFLP 1-4-4. For the other RFLPs, there was a significant genetic difference within them, particularly between states. RFLP typing is limited in its ability to discriminate among different strains of PRRSV. Understanding the magnitude of genetic divergence within RFLPs helps develop PRRSV regional control programs, placement, herd immunization strategies, and design of appropriate animal movements across borders to minimize the risk of PRRSV transmission.     

不同施肥水平下旱地土壤细菌群落多样性的RFLP分析

采用直接提取土壤微生物总DNA的方法,通过对不施肥(CK)、低量施肥(F1,N∶P2O5∶K2O为0·1gkg-1∶0·075gkg-1∶0·15gkg-1)、适量施肥(F2,N∶P2O5∶K2O为0·2gkg-1∶0·15gkg-1∶0·3gkg-1)和过量施肥(F3,N∶P2O5∶K2O为1·0gkg-1∶0·75gkg-1∶1·5gkg-1)等4种不同施肥水平土样DNA提取,扩增细菌16SrDNA基因片段,建立克隆文库。用限制性内切酶HhaI和RsaI进行PCR-RFLP分析,分别得到146、133、187、170个酶切类型。采用α多样性的测度对试验结果进行分析统计表明,不同处理间土壤细菌的多样性和物种丰富度均为F2>F3>CK>F1,表明合理施肥有利于土壤细菌的多样性;λ、R2、dMa和E指数在不同施肥处理间的变异系数达到12·86%～118·9%,尤其是Simpson指数λ变化最为敏感,处理间的差异最大。序列分析和系统发育树结果表明,不同施肥处理下供试土壤优势细菌的分布发生了改变。     

海南地方猪POU1F1基因多态性与体质量的相关性

采用 PCR–SSCP、PCR–RFLP 技术,对海南五指山猪封闭群及其近交系、海南黑猪2个品系(临高猪、屯昌猪)的 POU1F1基因外显子4、外显子5、外显子6、内含子3的多态性进行研究.结果表明:猪 POU1F1基因外显子5、外显子6不存在多态性,内含子3、外显子4存在2个等位基因和3种基因型;在外显子4基因座上,五指山猪、临高猪、屯昌猪呈 Hardy–Weinberg 平衡状态,五指山猪近交系呈不平衡状态,4个猪种群的遗传多态性均处于中度多态(0.250.5);利用 SPSS 软件分析五指山猪 POUIF1基因内含子3和外显子4基因的多态性与其3个生长阶段体质量的相关性,发现引物 P1与 P4不同基因型个体体质量的差异均不显著     

Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens

From December 2003 to July 2006, a total of 131 (28.4%) Haemophilus parasuis strains were isolated from 462 cases examined in our diagnostic laboratory. These strains were isolated from clinically diseased pigs, and 50 of them along with 15 reference strains of all known serovars were subjected to PCR–FRLP (restriction fragment length polymorphism) analysis by tbpA gene. The analysis of the 1.9-kb tbpA amplicon using TaqI, AvaI and RsaI endonucleases produced 9 RFLP patterns for the15 reference strains and 13 patterns for the 50 field isolates. And the first three prevalent genotypes in China were DBN (38%), ABN (18%) and DBP (12%). Meanwhile, co-infection of H. parasuis, PRRSV and PCV2 was examined in the 462 pig herds. It is indicated that 11.5% cases (53), 27.9% cases (129) and 4.8% cases (22) were infected only by H. parasuis, PRRSV and PCV2, respectively; and 19.2% cases (89) and 3.0% cases (14) were co-infected with two or all of the three pathogens, respectively; the rest 33.6% cases (155) were not infected by any of the three pathogens. It is confirmed that H. parasuis existed widely in southeast China with numerous genotypes.     

Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus . Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis , were studied using PCR-based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa . In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa , but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria .     

藏鸡IGF-Ⅰ基因的SNPs检测及与生长性状的关联分析

通过PCR-RFLP技术对藏鸡和隐性白羽鸡类胰岛素生长因子-Ⅰ(IGF-Ⅰ)基因的5'非翻译区(UTR)的2个位点进行了多态性检测.检测结果显示,该基因具有HinfⅠ和PstⅠ多态现象.测序结果表明,HinfⅠ识别位点发生了A→C突变,PstI识别位点发生了C→T突变,从而在2群体中分别产生了3种基因型,而出现了8种单倍型组合.单倍型组合的X2检验结果表明,2个品种间存在极显著差异(P＜0.01).方差分析结果显示,品种对所分析的17个生长发育性状都有显著影响(P＜0.05或P＜0.01),性别对6个生长发育性状有显著影响(P＜0.05或P＜0.01),而基因型或单倍型组合对5个生长发育性状(初生重、2周龄体重和胫围、7周龄体斜长以及16周龄胫围)有显著影响(P＜0.05或P＜0.01).同时,基因型或单倍型组合与品种之间的互作对鸡的部分生长发育性状也有一定的影响.另外,基因型CT在鸡初生重和7周龄体斜长上显著(P＜0.05)或极显著地(P＜0.01)高于基因型CC和TT,而单倍型组合A+T/C+T在鸡初生重、2周龄体重和胫围以及16周龄胫围上显著(P＜0.05)或极显著地(P＜0.01)高于其它6种单倍型组合.     

Characterisation of Meloidogyne species from China using Isozyme Phenotypes and Amplified Mitochondrial DNA Restriction Fragment Length Polymorphism

Forty-six Meloidogyne populations from 14 provinces of China were characterised in terms of malate dehydrogenase, esterase phenotypes and HinfI restriction profiles of amplification products from the mitochondrial DNA (mtDNA) region between the COII and lrRNA genes. Isozyme phenotyping revealed that 29 of the populations were M. incognita, six were M. javanica, six were M. arenaria, three were M. hapla and two were M. enterolobii. HinfI restriction patterns of the COII-lrRNA region correlated with nematode isozyme phenotypes and enabled reliable differentiation and identification of the five root-knot nematodes occurring in China. The size and sequence of the mtDNA amplification product were determined for the first time for M. enteroloii, a potentially economically important crop pathogen. Sequence comparison showed that the sequence of the intergenic region between the COII and lrRNA genes for M. enterolobii was identical to that reported for M. mayaguensis. Together with published observations on morphology, host range and esterase phenotype of the two nominal species, the mtDNA sequence evidence suggests that M. mayaguensis could be conspecific with M. enterolobii.     

Genetic Structure of Mycosphaerella graminicola Populations from Iran, Argentina and Australia

Restriction fragment length polymorphism (RFLP) markers were used to assess the genetic structure of populations of Mycosphaerella graminicola collected from wheat fields. A total of 585 isolates representing 10 field populations were sampled from Iran, Argentina and Australia. The genetic structure of M. graminicola populations from Iran and Argentina is described for the first time. Results were compared to previously investigated populations from Israel, Uruguay and Australia. Populations from Iran exhibited high clonality and low gene diversity, suggesting an inoculation event. Populations from uninoculated fields in Argentina had gene and genotype diversities similar to previously described European and North American populations. Genotype diversity was high for populations from Australia and tests for multilocus associations were consistent with sexual recombination in these populations. Gene diversity was low and fixed alleles were found for several loci. These findings are consistent with a relatively small founding population for Australia. These 10 new populations were integrated into a genetic distance comparison with 13 global populations that were characterized earlier.     

Discovery of two northern hemisphere Armillaria species on Proteaceae in South Africa

Armillaria root rot symptoms were observed on native Protea and Leucadendron (Proteaceae) species in Kirstenbosch Botanical Gardens in the Western Cape Province of South Africa. Intergenic spacer (IGS)-1 polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) profiling indicated the presence of at least two Armillaria species. The profiles of two isolates were identical to those of A. mellea s. str., originating in Europe. Phylogenetic analyses incorporating internal transcribed spacer (ITS) and IGS-1 sequence data identified the remaining isolates as closely related to A. calvescens , A. gallica , A. jezoensis and A. sinapina . These isolates displayed mating compatibility with A. gallica. From the RFLP profiles, sequencing results and sexual compatibility studies, it is concluded that the two species on Proteaceae in Kirstenbosch represent A. mellea and A. gallica . These are northern hemisphere fungi that have apparently been accidentally introduced into South Africa. This is the second report of Armillaria being introduced into South Africa. The introduction probably occurred early in the colonization of Cape Town, when potted plants from Europe were used to establish gardens.