Parentage analysis of indigenous citrus species ‘Zhi Que' based on nuclear and chloroplast SSR markers

Abstract：【Objective】‘Zhi Que'is a natural citrus species cultivated in southern Shaanxi Province, with more than 500 years of cultivation for its usage of dried fruits as. Herb medicine and fresh fruits as well. It is also famous for strong cold endurance with a potential of being a cold-resistant rootstock.However, the origin of‘Zhi Que'still remains unclear. Therefore, cpSSR and n SSR markers were employed to analyze the origin of‘Zhi Que's for further improvement and utilization.【Methods】21 citrus materials including Citrus, Poncirus and Fortunella were used in this study. The collected fresh leaves were taken to the laboratory in iced cassettes and frozen in liquid nitrogen and stored at-80 ℃until testing. The improved CTAB method was used to extract the total DNA genome, 10 pairs of cpSSR primers and 14 pairs of nSSR primers previously reported were used. The total PCR reaction vol-ume was 15 μ L: 10 × Buffer (Mg2+free) 2 μL, 25 μmol· L^-1 MgCl21.2 μL, 10 μ mol·L^-1 forward and reverse primers each 0.5 μL, 5 U· µL^-1 Taq DNA polymerase 0.2 μL, 100-150 ng· µL^-1 DNA template 1 μL, 2 μmol·L^-1 dNTPs 2 μL, ddH2 O 8 μL. PCR amplification was performed on the Biometra PCR instrument and the cycles were programmed as follows: one initial denaturing cycle at 94 ℃ for 7 min, 32 cycles of 30 s denaturing at 94 ℃, 30 s annealing at 55 ℃, 45 s elongation at 72 ℃ and one final cycle of 10 min at 72 ℃, stored at 4 ℃. The products were electrophoresised on 6% denaturing polyacrylamide gel and visualized by silver staining. The polymorphism was determined according to the presence (scored as“1”) or absence (scored as“0”) of the SSR band. The data format was converted using GenAlex 6.5. The average allele number (Na) , Shannon information index were calculated by Popgene1.31, and the observed heterozygosity (Ho) , expected heterozygosity (He) , and polymorphic information content (PIC) were calculated by Cervus version 3.0. UPGMA (unweighted pair group method arithmetic averages) based on the DICE similarity coefficient was constructed by NTSYSpc.【Results】According to our resource investigation, the leaf and fruit phenotypes were similar to pummelo. The embryoes were mainly polyembryonic. 10 pairs of cpSSR primer amplified a total of 37 bands with 3.7 bands per locus.The average allele number (Na) , average effective allele number (Ne) , average expected heterozygosity (He) , and average Shannon information index (I) were 3.7, 2.61, 0.54 and 0.98, respectively. UPGMA analysis of cpSSR showed that 21 materials could be divided into 7 groups at the similarity coefficient of 0.589. Red tangerine, ‘Nianju'‘Ruju'and‘Bendiguangju'were classified into group Ⅰ, Citron (YN 11) and trifoliate orange group Ⅱ, Ichange papeda, Fragrant citrus (CQ7) and‘Ziyangxiangcheng'group Ⅲ, Red limonia, Hongkong kumquat and Citron (YN 5) group Ⅳ, Ⅴ andⅦ separately. Group Ⅵ was consisted of two subgroup, one was composed of all pummelo varieties (Sour pummelo, ‘Shuijingmiyou'‘Wusuanyou'‘Guanximiyou') , the another subgroup was composed of‘Goutoucheng', Newhall navel orange, Citrange, Citrumelo and‘Zhi Que'. Further more, Citrange, Citrumelo, ‘Goutoucheng'and Newhall navel orange all had maternal genetic background of pummelo. 14 pairs of nSSR primers amplified a total of 62 bands with 4.43 bands per locus. The average allele number (Na) , average observed heterozygosity (Ho) , average expected heterozygosity (He) , and polymorphic information content (PIC) were 4.43, 0.31, 0.63 and 0.57, respectively. UPGMA analysis of nSSR showed that 21 materials could be divided into 8 groups at the similarity coefficient of 0.74.Red tangerine, ‘Bendiguangju'‘Ruju'and‘Nianju'were classified into group Ⅰ, Red limonia, Newhall navel orange, fragrant citrus (CQ7) and‘Ziyangxiangcheng'group Ⅱ, ‘Goutoucheng', Hongkong kumquat, and trifoliate orange group Ⅲ, Ⅵ and Ⅶ separately. Sour pummelo, ‘Wusuanyou'‘Shuijingmiyou'‘Guanximiyou', and Ichange papeda were divided into group Ⅳ, Citron (YN 5) and Citron (YN 11) group Ⅴ. The remaining group Ⅷ was consisted of Citrange, Citrumelo and‘Zhi Que'.‘Zhi Que'clustered together with hybrid varieties with paternal genetic background of trifoliate orange and existed certain genetic distance with trifoliate orange.【Conclusion】Combining with morphological data and molecular evidence of cpSSR and nSSR analysis, we speculated that the maternal parent of‘Zhi Que'would be pummelo or a hybrid citrus variety with pummelo maternal genetic background and the maternal parent was a hybrid citrus variety with paternal genetic background of trifoliate orange.