Development Of Polymorphic Microsatellite Loci And Study On Fine-scale Spatial Genetic Structure Of Pteroceltis Tatarinowii, AnEndangered Plant Endemic To China

Pteroceltis tatarinowii, the sole representative of the genus Pteroceltis, is an endangered deciduous tree endemic to China. It not only serves as food, medicine and timber, but also is the best source for the manufacture of Xuan paper. Due to the extensive deforestation, the distribution and size of the P. tatarinowii population have been decreasing for years, which makes it a national class Ⅲ endangered plant in China.With wide distribution in the genome, the microsatellite markers relied on expressed sequence tags (EST) can be linked to genetic traits and applied broadly in population genetics, molecular marker assisted breeding and molecular marker screening of target traits. However, the lack of P. tatarinowii genomic and transcriptional dataset limits the application of molecular markers in genetic studies of its populations. In this paper, we used Illumina high-throughput sequencing technique to sequence the P. tatarinowii leaf transcriptome, based on which polymorphic microsatellite loci is developed.According to the distribution of P. tatarinowii in Langya mountain, we chose a relatively independent and representative natural population as a sample plot. According to the diameter at breast height, P. tatarinowii population was divided into five size classes, respectively for sapling Ⅰ:DBH<5 cm, adult tree Ⅱ:5 cm≤DBH< 15 cm, Ⅲ:5 cm≤DBH<25 cm, Ⅳ:25 cm≤DBH<35 cm, and aged tree Ⅴ: 35 cm. We conducted investigation on fine-scale spatial genetic structure of this population. The main results were as follows:(1) The total number of individuals for P. tatarinowii population was 355, exhibiting a Pyramid age structure. The number of saplings was the most abundant, accounting for 65.07%. As the class increasing, the number of individuals decreased gradually, and the number of aged trees was the least, accounting for only 3.10%. Furthermore, the dynamic indices of population quantitative change were greater than zero indicating this population was in growth pattern.(2) The survivorship curve of P. tatarinowii matched Deevey Ⅲ. Below class Ⅳ, the survival quantity decreased rapidly, and the mortality was high. Above that, the survival quantity was relatively stable. The first mortality peak occurred in class I to class Ⅱ stage, where the large number and high density of saplings, restriction of habitat conditions and self thinning resulted in high mortality. The second mortality peak occurred in class V, due to the aging and physiological decline of the trees..(3) There were differences in the spatial distribution pattern of various classes. The results showed that saplings clumped around adult and aged trees, while higher classes tended to be randomly or evenly distributed.(4) The transcriptome of P. tatarinowii analysis was carried out by Illumina technology, and we obtained a total of 42477 Unigenes with an average length of 815bp. After aligning with NR, SwissProt, GO, COG and KEGG databases,23688 unigenes were assigned. These data provided the foundation for gene expression and function analysis of P. tatarinowii.(5) Among the unigenes, a total of 6543 EST-SSRs were identified.130 EST-SSRs were selected for validation as EST-SSR markers by PCR amplification. Of these,48 EST-SSRs were amplified successfully and 32 EST-SSRs were polymorphic among 47 P. tatarinowii individuals. Additionally, cross-amplifications of EST-SSR were detected in Ulmus gaussenii and Ulmus chenmoui, and the versatility and polymorphism were 25% and 87.5%, respectively. The development of P. tatarinowii microsatellite markers provided the molecular basis for the study of P. tatarinowii and other plants in family Ulmaceae.(6) Expected heterozygosity of class Ⅰ,Ⅱ,Ⅲ,Ⅳ,ⅤV and total population of P. tatarinowii were 0.442,0.439,0.426,0.435,0.471 and 0.444, respectively. Fixation index ranged from 0.015 to 0.121. The genetic diversity of P. tatarinowii were high, and no significant differences were found among the five size classes. However, there was heterozygosity deficiency in P. tatarinowii. P. tatarinowii was an outcrossing breeding, wind-dispersal seeding and long-lived plant. In addition, it is a Tertiary relic species, with plenty of time for genetic mutation and variation. For these reasons, the genetic diversity of P. tatarinowii was high. Based on sample plot investigation and analysis of gene flow in each size classes, we found that seed dispersal was limited by high density of sub canopy, and P. tatarinowii had strong sprouting ability. These led to a high degree of inbreeding and heterozygous deficiency in P. tatarinowii population.(7) Significant fine-scale SGS was found in the whole population within 28 m, and the SP was 0.0140. The Sp of class Ⅰ, Ⅱ, Ⅲ, Ⅳ and V were 0.0142,0.0158,0.0008, 0.0026 and 0.0018, respectively. Significant fine-scale SGS was found in class Ⅰ and class Ⅱ within 30 and 35 m, whose intensity was relatively high. The gene flow of Ⅰ, Ⅱ, Ⅲ and IV were 36.21,37.97,40.63 and 40.10 m. Due to the high forest canopy density, especially in the sub canopy layer, wind resistance limited the samara spread, and P. tatarinowii had strong sprouting ability. As a result, the distribution of individuals was aggregated within a small range, and class Ⅰ and class Ⅱ exhibited significant fine-scale SGS. As the plants growing, intraspecific competition enhanced the self thinning effect, and a large number of close relatives died causing significantly decrease of SGS intensity.(8) P. tatarinowii population was under growing pattern and exhibited high genetic diversity. However, saplings were clumped around adult and aged trees because of limited seed dispersal, resulting in heterozygous deficiency. Furthermore, significant fine-scale SGS was found in class Ⅰ and Ⅱ. These results indicated that P. tatarinowii population was threatened by limited gene flow, increased inbreeding and the risk of inbreeding depression. Therefore, for in situ conservation, artificial promotion of gene flow should be encouraged, while inbreeding depression should be monitored. For ex situ conservation, individuals should be sampled at apart to 28 m to reduce genetic similarity.