Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch(Sitta nagaensis)based on ddRAD-seq data

Extra-pair paternity(EPP)is commonly found in socially monogamous birds,especially in small passerine birds,and there are interspecific and intraspecific variations in the extent of EPP.The Chestnut-vented Nuthatch(Sitta nagaensis)is a socially monogamous passerine bird,and verifying whether this species has EPP relies on parentage testing-S.nagaensis is not known to have EPP.In this study,we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing(ddRAD-seq)data.A panel consisting of 50 SNP markers,with a mean heterozygosity of 0.343,was used to resolve 95% of nestlings to fathers.The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999,respectively.This panel of SNP markers is a powerful tool for parentage assignments in S.nagaensis.In addition,we found that three offspring(7.9%)from three nests(23.1%)were the result of extra-pair fertilization out of 38 offspring in 13 nests.Our study provided information on parentage analysis that has not been reported before in S.nagaensis.It also supplemented the understudied EPP behavior of birds in Asia,contributing to a general understanding of the EPP behaviors of birds.

Establishment of microsatellite-based triplex PCR for parentage analysis of Chinese shrimp Fenneropenaeus chinensis

Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp （Fenneropenaeus chinensis）, some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers （RS1101, RS0683 and H081 primers）. By adjusting the final concentration of Mg^2＋, dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1 101,RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE （probabilities of paternity exclusion） is 0.967 9,and the DP （discrimination power） is 0.999 327.Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.

Leading directions and effective distance of larch offspring dispersal at the upper treeline in the Northern and Polar Urals, Russia

Climate has changed sufficiently over the last 150 years and forced out upper treeline advance at the most studied sites around the world.The rate of advance has been extremely variable–from tens to hundreds meters in altitude.This is because the degree at which tree frontal populations respond to climate change depends on the complex interaction of biological and physical factors.The resulting stand pattern is the consequence of the interaction between dispersal and survival functions.A few publications have addressed the question of how this pattern is generated.In order to understand how the spatial structure of tree stands was formed at the upper limit of their distribution in the Ural Mountains,we assessed the distance and direction of dispersal of offspring from maternal individuals.We found that in frontal Larix sibirica Ledeb.populations,‘effective’dispersal of offspring ranges from 3 to 758 m(with a median of 20–33 m in open forest and 219 m in single-tree tundra in the Polar Urals and 107 m in open forest in the Northern Urals).We revealed that most of the offspring effectively dispersed not only in the direction of the prevailing winds,but also in the opposite direction up the slope,and the distance can reach 500–760 m.The data obtained can be used to develop an individual-based model which is capable of simulating in detail the dynamics of tree stands at the upper limit of their growth and reliably predicting the future position and pattern of treeline ecotone as growth conditions continue to improve in the face of observed climate change.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Tort. ＆ Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction （PCR） assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Torr. & Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction (PCR) assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Integrated tool for microsatellite isolation and validation from the reference genome and their application in the study of breeding turnover in an endangered avian population

Accurate individual identification is required to estimate survival rates in avian populations.For endangered species,non-invasive methods of obtaining individual identification,such as using molted feathers as a source of DNA for microsatellite markers,are preferred because of less disturbance,easy sample preparation and high efficiency.With the availability of many avian genomes,a few pipelines isolating genome-wide microsatellites have been published,but it is still a challenge to isolate microsatellites from the reference genome efficiently.Here,we have developed an integrated tool comprising a bioinformatic pipeline and experimental procedures for microsatellite isolation and validation based on the reference genome.We have identified over 95000 microsatellite loci and established a system comprising 10 highly polymorphic markers(PIC value:0.49–0.93,mean:0.79)for an endangered species,saker falcon(Falco cherrug).These markers(except 1)were successfully amplified in 126 molted feathers,exhibiting high amplification success rates(83.9–99.7%),high quality index(0.90–0.97)and low allelic dropout rates(1–9.5%).To further assess the efficiency of this marker system in a population study,we identified individual sakers using these molted feathers(adult)and 146 plucked feathers(offspring).The use of parent and offspring samples enabled us to infer the genotype of missing samples(N=28),and all adult genotypes were used to ascertain that breeding turnover is a useful proxy for survival estimation in sakers.Our study presents a cost-effective tool for microsatellite isolation based on publicly available reference genomes and demonstrates the power of this tool in estimating key parameters of avian population dynamics.