Exploitation And Genetic Diversity Analysis Of Sour Orange Germplasm

Sour orange(Citrus aurantium L.)originates in the southeast of Eurasia.China was supposed to be the main area of sour orange origin.Sour orange is widely distribute in the areas along Yangtze River and Pearl River,and has been introduced to citrus producing areas all over the world.Sour orange is rich in variation and plays an important role in the systematic classification of citrus.It is an important material to study the evolution,genetics and improvement of citrus.In this study,the germplasm of sour orange was surveyed and collected from all over China;later they were evaluated and preserved.About 500 G genome data via the second generation whole genome sequencing technology were analyzed for the genetic diversity of the collected sour orange germplasm.A total of 80 accessions of local sour orange and related resources have been collected,and eight of them are from abroad.More than 280 plants were preserved by grafting and seedling.In this study,we evaluate the traits including acid content,saccharinity,peel thickness,seed quantity and embryony characteristic.Leaf phenotypic traits such as leaf length,leaf width,and leaf perimeter and leaf area were measured and analyzed by variance analysis,cluster analysis and principal component analysis.The results showed that the variety of sour orange are rich in genetic diversity.According to the color of pericarp,they can be divided into three types including red,orange and yellow fruits.The transverse diameter of fruit varies from 44 mm to 104 mm,the fruit shape index is 0.74-1.08,the peel thickness is 1.76mm-9.03 mm,saccharinity7.60-16.87%,the acid content is 1.27%-8.62%,and the average number of seed is 26,monoembryony type accounted for18.5%.The variation of leaf type traits is extremely rich.The highest variation coefficient is 86.06% for winged-petiole width and lowest coefficient 13.57% for the leaf index of length /width.Eleven leaf type traits show significant genetic variance.According to the clustering and principal component analysis of leaf type traits,the collection accessions can be divided into four categories.From the analysis of fruit and leaf traits,the genetic diversity acrossthe sour orange population is very high,and mutations are abundant.Through the nSSR analysis,we got 33 representative materials from 80 accessions and the whole genome resequence depth is 30 X.Using “Citrus ID”,21 species were identified as sour orange,10 suspected sour orange hybrids and 2(HP,GXSJ)identified as neither sour orange nor sour orange hybrids.In 10 sour orange hybrids,8 may be backcross species of pomelo and hybrids from mandarin and pomelo.The other two may be backcross species of mandarin and hybrids from mandarin and pomelo.After preliminary analysis,we got 56,525,679 SNP,the average SNP is 1,823,409.The highest is Red ZhuLuan 1,975,839 and the lowest is QLJ 1,564,037.Based on nucleotide diversity,genetic structure and principal component analysis(PCA)and combining with NJ(Neighbor-Joining)cluster tree,the genetic diversity and population structure of representative sour oranges were preliminarily analyzed.The study shows that sour orange can be divided into 3groups,Hunan sour orange as main group A,Jiangxi,Zhejiang sour orange as group B and “DaiDai" sour orange as main group C.Group A and group B have a great overlap in the genetic structure,principal component analysis and NJ clustering tree,showing very frequent genetic flow and close genetic relationship,but group C has greater differentiation and genetic variance than the other two types and showing single genetic background.Group B has the highest genetic diversity?= 0.0052,and group C the lowest?= 0.0037.The Tajima's D of three groups are greater than zero,indicating that there are a large number of medium frequency alleles,which is closely related to the development of the cultivated sour orange with pressure of artificial selection and the reduction of the population groups of sour orange;“DaiDai” sour orange has the greatest pressure.The Fst(Fixation index)of intrapopulation all less than 0.05(Fst=0.0040,0.0193,0.0031),indicating that there is a small genetic differentiation and the relationship of intrapopulation is close.The genetic divergence between group B and group C is very high(Fst = 0.2417),but the genetic divergence is low between group A and group B,and between group A and group C with Fst =0.0561 and 0.0998 respectively.Thus parents of “DaiDai” are more likely wild or ancient cultivated species similar to Hunan sour orange than Jiangxi and Zhejiangsour orange.Based on previous studies and geographical position,it is speculated that sour orange population may originate from the Nanling mountains and spread to other area by river flow and human activity.Both Hunan and Jiangxi are the main genetic diversity centers of sour orange,while other regions such as Chongqing,Zhejiang,Hubei and its surrounding areas are the secondary transmission areas.The results also indicated that eight foreign introduced sour orange are also derived from China as clustered with landraces of China's sour orange,suggesting that China is the core origin of sour orange.“DaiDai” as a special variety of sour orange,has been involved in the formation of European cultivated lemon in the process of propagation.