| Marsh tit, Parus palustris, is widely found all over the world, but relatively few studies have been conducted about this specie’s microsatellite. To develop microsatellite, next-generation sequencing technology is thought to be the most effective. This study chooses Illumina MiSeq PE300. From the obtained 2.84 M simplified sequenced reads,113,519 SLAFs were developed. MIS A was used to search the SSRs from 112558 effective fragments of the total SLAF.2471 SSRs were hunted with a density of 870.07 per M simplified sequenced reads. This result is sufficient to conduct a future study to hunt SSR locis with high polymorphism used in population genetics studies.Pairs of primers were designed for the total 80 microsatellites. After experimenting with different conditions,25 high quality (specific and stable) microsatellites locis were obtained. These high quality locis were amplified stably in a wide range of temperatures and were also tested to verify genetic diversity. Testing the 12 locis with the highest heterozygosity, polymorphism and number of alleles,11 locis were found to be the best setup for the paternity analysis. These 11 locis are R11, R16, R24, R33, R34, R37, R39, R47, R49, R69, R75. The analysis showed that the paternity exclusion 1 is 0.99669 and the paternity exclusion 2 is 0.99993. This proved that the 11 selected locis were ideal for testing paternity. Also, most of the repeat types of these 11 locis as well as most of the 25 high quality and the initial 80 ones were tetra-nucleotide. This is likely to mean that the tetra-nucleotide repeat type has higher genetical diversity than other repeat types.Testing the paternity of 14 nests with these 11 locis,42.86% were found to be extra-pair paternity nests and 24.53% of the offsprings turned out to be extra-pair offspring. Both numbers show high-level extra-pair paternity. Male species with whole paternity and males that suffered paternity loss have no significant difference in body condition parameters (Weight:t= 0.519, df= 9, P= 0.617; Tarsus length:t=-0.545, df= 9, P= 0.599). The difference between within pair offsprings and extra-pair offsprings were also not significant (Heterozygosity:t=-2.067, df= 12, P= 0.061; polymorphism:t=-1.935, df= 12, P= 0.077; Weight:t= 0.519, df= 9, P= 0.617; Tarsus length:t= 1.035, df= 104, P= 0.303). This might be explained by one of the three following reasons:1) the female might not have the tendency to always choose an extra-pair high qualtiy male; 2) the female might have other conditions, which we do not know, to assess the male’s quality; 3) the female’s extra-pair copulation does not increase the genetic diversity of offsprings. The obtained results of this study do not support the genetic benefits model, indicating that the female marsh tit does not benefit from extra-pair copulation. The study also found that one nest can have more than two paternities indicating that a female might have copulation with several different males. This leads us to conclude that the female chooses extra-pair paternity mainly to ensure full fertilization. To decide whether this conclusion is true, further studies are to be conducted. If studies showed it is, it would be aligned with the fertilization insurance hypothesis. |
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