Conventional Gel Electrophoresis-Resolvable Insertion/Deletion Markers for Individual Identification and Analysis of Population Genetics in Red-Crowned Cranes in Eastern Hokkaido, Japan

Abstract

Red-crowned crane Grus japonensis is an endangered species in two separate populations: the mainland population in the Eurasian continent and the island population in eastern Hokkaido, Japan. We found 11 insertion/deletion (InDel) markers in the genome of the red-crowned crane and designed primer sets across these InDels that can be analyzed with conventional agarose gel electrophoresis. Sixty-six samples of whole blood and skeletal muscle obtained from red-crowned cranes, including 12 families in eastern Hokkaido from 1994 to 2021, showed different patterns in gel images of 11 InDel PCR reactions except for two pairs. The combined non-exclusion probability of the 11 markers indicates that individuals can be determined with a probability of 99.9%. In 39 non-relative chicks, the expected heterozygosity (He) was 0.316, suggesting low genetic diversity. This might not be caused by high levels of inbreeding since the average FIS was not significantly different from zero (0.095, p = 0.075). The results suggest that the 11 InDel primer sets can be used for fairly accurate individual identification as well as genetic population analyses in red-crowned cranes in the island population.

Possible Shifts in the Genetic Diversity of Red-crowned Cranes (Grus japonensis) in Hokkaido, Japan: Indications of Continental Gene Flow

“…The muscles of two adult cranes and blood samples of 56 chicks in southeastern Hokkaido were studied in our previous reports on the development of insertion/deletion markers of red-crowned cranes in southeastern Hokkaido [ 12 ]. Chick blood samples were collected in June and July of 2008–2021 during banding, with permission from the Japanese Ministry of the Environment (MOEJ: Tokyo, Japan) (MOEJI permission No.…”

“…The inbreeding coefficient (F IS ), which represents the degree of inbreeding, was −0.412, showing a significant deviation from zero (p = 0.0000). Compared with the 31 southeastern individuals, excluding known blood relatives [12], the pairwise fixation index (Fst) in groups of northern and southeastern Hokkaido, based on InDel type, was 0.214387, which was also significantly different (p = 0.0000) (Table 2).…”

“…While Gj13, the third Hokkaido type, was reported by Akiyama et al [ 8 ], it was detected in only two birds, including a stuffed crane, and it is already considered extinct. An examination of microsatellite [ 11 ] and InDel (insertion/deletion) types [ 12 ] also supported the very low genetic diversity of the Hokkaido population. It is thought that this may be the result of a bottleneck effect stemming from an extended period of near extinction from the late Meiji period to the Taisho period (circa 1912) [ 6 , 7 ].…”

“…In addition to these feathers, we reexamined the sex, haplotype, and insertion/deletion (InDel) type of feathers collected in the Sarobetsu Wetlands in 2021-2022 (Table S2). Possibly due to the low quantity of intact DNA that was extracted from some feathers, the frequencies of band detection for some primer sets were lower when compared to DNA extracts from blood, as reported by Kawasaki et al [12]. Feather samples were used for the population genetic analysis for the northern population, excluding possible relatives by banding record or MHC type, as described below.…”

“…An analysis of feathers collected from the nest indicated that a male with a continental haplotype (Gj5) had mated with a female with a Hokkaido haplotype (Gj2). This discovery provided the first evidence that individuals from the continental population may have reached Hokkaido and successfully interbred with the local Hokkaido population [ 12 ]. However, no information is currently available regarding the offspring of this pair.…”