| In this paper, molecular markers for sex identification in ardeid birds were screened and the sex ratio in the nestling populations of Little Egret and Chinese Egret were measured. The relationships between nestling sex ratio and laying order in the broods of Little Egret and Chinese Egret were analyzed. And the sequences of CHD-W gene and CHD-Z gene in ardeid birds were analyzed and tried to study their relative and phylogenetic evolution.Molecular methods of sex identification were studied by amplifying sex gene sequences in five species of ardeidae and one species of threskiornithidae in ciconiiformes birds. Sex gene sequences of EE0.6 or CHD were amplified and compared in three pairs of Little Egret ( Egretta garzetta), Eastern Reef Heron(Egretta sacra)and Chinese Egret(Egretta eulophotes)whose sexes had been identified by dissection, and in twelve individuals of Night Heron (Nycticorax nycticorax), Cattle Egret (Bubulcus ibis),Chinese Pond Heron (Ardeola bacchus), Little Egret and Black-faced Spoonbill (Platalea minor) whose sexes were unidentified. The results showed that molecular methods of sex identification by amplifying sex gene sequence of EE0.6 or CHD are suitable for the sex identification of ardeid birds, and thus can solve the problem that the sexes of ardeid birds are difficult to be distinguished by their appearance as both male and female ardeid birds have the similar feather color. Furthermore, in both EE0.6 and CHD molecular methods of ardeid sex identification, the CHD method is not only more accuracy but also simpler than EE0.6 method.In this study, 361 ardeid birds in three populations of Little Egret at Jiyu island, Chinese Egret at Xingrentuo and Xiaocaiyu islands were sexed by amplifying CHD gene sequences using P2 and P8. When the amplified bands were not clear, the sex identifications were corrected by using 2550F and 2718R. The result showed that the population sex ratio of Little Egret (69 individuals in total at Jiyu island ) was biased to female(the female proportion was 0.6522±0.4798, P<0.05)and that of Chinese Egret (213 individuals in total in both Xingrentuo and Xiaocaiyu ) was no biased (the female proportion was 0.4507±0.4987, P>0.05)in which the population sex ratio at Xingrentuo island was no biased (the female proportion was 0.5130±0.5015, P>0.05) but the population sex ratio at Xiaocaiyu island was biased to male (the female proportion was 0.2881±0.4568, P<0.01) . The further analysis of brood sex ratio had the results similar to the population sex ratio. The brood sex ratio in the population of Little Egret (Jiyu island, 17 broods) was biased to female (brood sex ratio was 0.6333±0.2756 , P<0.05) and that of Chinese Egret (Xingrentuo population and Xiaocaiyu population, 65 broods in total) was no biased (0.4648±0.3108,P>0.05) in which the brood sex ratio in the population of Xingrentuo island (45 broods) was no biased (0.5571±0.294, P>0.05) but that of Xiaocaiyu island (20 broods) was biased to male ( 0.2708±0.2485, P<0.01).The relationship of the proportion of females among the three populations were Little Egret (Jiyu island)> Chinese Egret (Xingrentuo and Xiaocaiyu islands in total)(P<0.01), Little Egret on Jiyu island> Chinese Egret on Xingrentuo (P>0.05), Little Egret on Jiyu island> Chinese Egret on Xiaocaiyu (P<0.01), Little Egret on Jiyu island> Chinese Egret on Xiaocaiyu (P<0.05); The relationship of the brood sex ratio (proportion of females ) among the three populations were Little Egret (Jiyu island)> Chinese Egret (Xingrentuo and Xiaocaiyu islands in total)(P<0.05), Little Egret on Jiyu island> Chinese Egret on Xingrentuo (P>0.05), Little Egret on Jiyu island> Chinese Egret on Xiaocaiyu (P<0.01), Little Egret on Jiyu island> Chinese Egret on Xiaocaiyu (P<0.01).This paper also analyzed the relationship between the sex ratio (females proportion) and the laying order in the broods of Little Egret and Chinese Egret. The result found that the female proportion of Little Egret in the laying order from the first to the fifth was 0.556±0.511(n=17), 0.778±0.428(n=17), 0.611±0.502(n=17), 0.463±0.497(n=13), and 1.000(n=1) respectively, and the female proportion of Chinese Egret in the laying order from the first to the fifth was 0.403±0.494(n=68), 0.508±0.504(n=62), 0.482±0.504(n=48), 0.539±0.508(n=22), 1.000(n=3) respectively. In two populations of Chinese Egret, the female proportion in the laying order from the first to the fifth was 0.438±0.501(n=45), 0.652±0.482(n=42), 0.595±0.498(n=34), 0.565±0.507(n=20), 1.000(n=3) for Xingrentuo population and 0.333±0.476(n=23), 0.158±0.375(n=20), 0.235±0.437(n=14), 0.5±0.577(n=2) for Xiaocaiyu population. The results showed that the sex of last egg in the brood was female when the clutch size of the brood was 5. The female proportion of Little Egret in the laying order from the first to the third was higher than that of Chinese Egret (Xingrentuo and Xiaocaiyu islands in total, (P>0.05) or that of Chinese Egret at Xingrentuo island (P >0.05) or that of Chinese Egret at Xiaocaiyu island (P <0.05) except that the female proportion of Little Egret in the fourth laying order was lower than that of Chinese Egret (Xingrentuo and Xiaocaiyu islands in total, (P>0.05) or that of Chinese Egret at Xingrentuo island (P>0.05) or that of Chinese Egret at Xiaocaiyu island (P<0.05) . The female proportion of Chinese Egret at Xingrentuo island in the laying order from the first to the fourth was higher than that of Chinese Egret at Xiaocaiyu island (P <0.05).The female proportion of the second laid egg was the highest in Little Egret at Jiyu island or in Chinese Egret at Xingrentuo island but was the lowest in Chinese Egret at Xiaocaiyu island.At last in this paper, the sequences of CHD-W gene in Chinese Egret, Cattle Egret and Black-faced Spoonbill,and the sequences of CHD-Z gene in Chinese Egret, Large Egret (Egretta alba ), Intermediate Egret (Egretta intermediate), Cattle Egret, Night Heron and Black-faced Spoodbill were analyzed and used to study their relative and phylogenetic evolution . It was found that the sequences amplified from the CHD-W gene using primers P2,P8 were more conservative, the sequences amplified from the CHD-Z gene had many variation, and that the length of Z chromosome had polymorphyism during the sex identification of Chinese Egret. The results indicated that these sequences were not the suitable molecule markers for their relative and phylogenetic study.In summary, the quick, simple and accurate molecular methods for sex identification of ardeid birds were established in this paper to solve the problem that the ardeid birds are difficult to be sexed by their appearance as both their males and females has the similar feather color. The population sex ratio and brood sex ratio (female proportion) of Little Egret was biased to female(P<0.05)and higher than those of Chinese Egret ( P<0.05 ) might explain why the population reproductivity and increase rate in Little Egret was higher than that in Chinese Egret. That the population sex ratio and brood sex ratio (female proportion) of Little Egret was higher than those of Chinese Egret might result from that the female proportion of Little Egret in the laying order from the first to the third was higher than that of Chinese Egret. It is suggested that the population size had the positive effect on the female proportion as the population sex ratio and brood sex ratio (female proportion) of Little Egret was higher than those of Chinese Egret, the population sex ratio and brood sex ratio in the population of Xingrentuo island was higher than that of Xiaocaiyu island(P<0.05), the parent population size of Little Egret at Jiyu island was higher than that of Chinese Egret at Xingrentuo island, and the parent population size of Chinese Egret at Xingrentuo island was higher than that of Chinese Egret at Xiaocaiyu island. The results above supplied a gap in the study of sex identification and sex ratio in ardeid birds and supported the academical and technical groundwork for the further researches on the reproductivity and protection of the ardeid birds.
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