Myotis rufoniger genome sequence and analyses: M. rufoniger’s genomic feature and the decreasing effective population size of Myotis bats

Myotis rufoniger is a vesper bat in the genus Myotis. Here we report the whole genome sequence and analyses of the M. rufoniger. We generated 124 Gb of short-read DNA sequences with an estimated genome size of 1.88 Gb at a sequencing depth of 66× fold. The sequences were aligned to M. brandtii bat reference genome at a mapping rate of 96.50% covering 95.71% coding sequence region at 10× coverage. The divergence time of Myotis bat family is estimated to be 11.5 million years, and the divergence time between M. rufoniger and its closest species M. davidii is estimated to be 10.4 million years. We found 1,239 function-altering M. rufoniger specific amino acid sequences from 929 genes compared to other Myotis bat and mammalian genomes. The functional enrichment test of the 929 genes detected amino acid changes in melanin associated DCT, SLC45A2, TYRP1, and OCA2 genes possibly responsible for the M. rufoniger’s red fur color and a general coloration in Myotis. N6AMT1 gene, associated with arsenic resistance, showed a high degree of function alteration in M. rufoniger. We further confirmed that the M. rufoniger also has bat-specific sequences within FSHB, GHR, IGF1R, TP53, MDM2, SLC45A2, RGS7BP, RHO, OPN1SW, and CNGB3 genes that have already been published to be related to bat’s reproduction, lifespan, flight, low vision, and echolocation. Additionally, our demographic history analysis found that the effective population size of Myotis clade has been consistently decreasing since ~30k years ago. M. rufoniger’s effective population size was the lowest in Myotis bats, confirming its relatively low genetic diversity.


Introduction
Danyang, in South Korea, was sequenced using Illumina HiSeq2000 platform. A total of 124 1 Gb of paired-end short DNA sequences were produced with a read length of 100 bp, and 2 target insert sizes of 566 bp and 574 bp from two genomic libraries. After reducing low 3 sequencing quality reads and possible microbial contaminated reads, we acquired a total of 4 115 Gb of DNA sequences (Table 1; S1 Table). To confirm the species identification of the 1 containing genes were excluded, 1,239 uAACs from 929 genes were predicted to be function 1 0 altering (S9 Table). A functional enrichment analysis of the 929 genes having function- including reproductive processes in a multicellular organism (P-value: 0.00026, 39 genes, 1 6 GO: 0048609), ovulation cycle (P-value: 0.00052, 11 genes, GO: 0042698), and gamete 1 7 generation (P-value: 0.0017, 31 genes, GO: 0007276; S10 Table). In M. rufoniger, pigment 1 8 related terms were significantly enriched in the function-altered genes as in the melanin 1 9 biosynthetic process (P-value: 0.0037, 4 genes, GO: 0042438; S10 acid sequences are presented in S4 Figure. 3 As we were interested in the variants' whole gene function, all the PROVEAN variant scores 6 of each gene were summed up. We then ranked the sums of the function-altered gene 7 candidates (S12 Table). For the top 20 genes, the same analysis was carried out in the other 8 Myotis bat species for comparison (Table 3; S13, S14 Table). We ranked M. rufoniger's genes  and its demographic history showed that its population peak was 50 k years ago (Fig. 2).
1 2 period (10 ~ 50 k years ago), and it was estimated to be the lowest in present. Also, a 1 consistent decline in the effective population size of Myotis bats since ~30 k years ago was 2 found (Fig. 2). We further investigated the genomic diversity (which can be affected by  rufoniger's low effective population size (S13 Table). reproduction associated pathways. Prolonged sperm storage is a common behavior in Chalinolobus, and Plecotus) and Rhinolophidae family species [16][17][18], and thus it is 1 7 probably not M. rufoniger specific as reproduction related genes are always under strong 1 8 natural selection. Therefore, such hits can be regarded as general or even a kind of artefact.

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Therefore, further detailed functional verification is necessary to understand the roles of each 2 0 uAAC in functional categories such as reproduction.

1
The previous bat studies reported unique variants representing some general characteristics of variants. 6 We also found that the function-altering unique variants containing the genes of the M. with color diversity will be required for further analyses to understand the precise geno-2 0 phenotype associations in coloration.

1
In the M. rufoniger genome, N6AMT1 gene showed a high degree of function alteration. The   Therefore, a set of diverse species will be required to accurately model the bats' effective  Mapping sequence data 2 3 The filtered sequenced reads were mapped to the three Myotis bat genome assemblies (M. RepeatMasker (version 4.0.5) was used to identify transposable elements by aligning the M.  Table) were identified using OrthoMCL software