RAPD Analysis Of Youxi Kumquat (Fortunella Crassifolia) And In-vitro Conservation Of Citrus Germplasm In Fujian Province

Citrus belonging to Citreae, Acrantioideae,Rutaceae plants, was a tropical fruit of economic ally important value. China was one of the world centers of its origin, with a cultivation history of 4000 years. After thousands of years of evolutionary development, a wide range of complex and rich citrus germplasm resources had formed. Fujian was rich in citrus germplasm resources,and Youxi kumquat (Fortunella crassifolia) was one local famous citrus fruit and a National Geographic Logo Product with 370-year history in cultivation. Youxi kumquat was propagated by seedling propagation, which resulted in great variance, so the genetic resources were rich. For this reason, genetic diversity of Youxi kumquat was firstly analyzed by the RAPD molecular marker, then the in vitro conservation of Youxi kumquat resources and the main citrus germplasm resources in Fujian were conducted, and further cloned Mn-SOD gene from the leaf of kumquat plantlets, which laid the foundation for the study of SOD mechanism during citrus plantlets conservation. The main results were as follows:1. RAPD analysis of genetic diversity in Youxi kumquatThe leaves of 122 accessions of materials of Youxi kumquat, including 20 selected superior strains and 5 control plants around(No.1-120) through collaboration with Youxi Breeding Farm, together with two mother plants (No.121 and 122) were used as materials for RAPD analysis.The result showed that similarity coefficient between two tested materials was averaged up to 0.947, which indicated that the tested 122 kumquat resources had closer genetic relationship.All materials onⅠ(D1=13.5) district could be distinctively divided into two groups,with group 1 containing 102 individuals and group 2 containing 20 individuals.Group 2 atⅡ(D2=10.5) district could be divided into two subgroups, sub-group 1 includes 19 plants atⅢ(D3=8.5) district that could be divided into two sub-groups, with small group1 including of 3 single plant and small group 2 including 16 single plants; only one mother plant was separated from subgroup 2.Group 1 atⅣ(D4=4.5) district could be divided into 4 small groups, with small group 1 having 94 single plants, the second and the third including 2 respectively, and the fourth containing 6.The 20 plants of the second group at D1=13.5 district were precocious strains, which showed that the selected primers could be clearly distinguished as the kumquat's precocious and non-precocious characteristics At D2=10.5 district could slip the precocious kumquat into slippery peel and non-slippery peel.Overall, the mother plant and the corresponding five contrasting plants around had variation to some extent unevenly,only a few mother plants and the corresponding five contrasting plants around were of no variation(Mother plant No.97, 91, 1, 49, and the corresponding five s contrasting plants around).The code of mother plants which had slight differences with the surrounding five plants were No.55, No.61, No.67, No.85, No.37, No.43, No.31 and No.7. The code of mother plants which had quite differences with the surrounding five plants were No.55, No.61, No.67, No.85, No.37, No.43 and No.31. The code of mother plants which have maximum differences with the surrounding five plants was No.73.The genetic diversity was the basis of the germplasm recourses diversity in Youxi kumquat, which fully showed the great potential of breeding.2. In-Vitro Conservation of Citrus Germplasm in FujianThe nucellar embryos were used as the main test materials,and were inoculated on MS medium without any hormone, with illumination intensity of 1000Lx, temperature of 25±1℃and illumination time of 12h/d.The growth of 16 accessions of different resources and the preservation status of 28 accessions of citrus in-vitro conserved plantlets were observed in detail.For the leaf,16 accessions of in-vitro conserved materials during 18-month conservation, Lugan had maintained the largest number of leaves from 1 month to 18 months all the time and Huyou had the least number of leaves;however,because Sugan had leaf abscission phenomenon after conservation 10 months, which resulted in a variety of the least number of leaves after conservation 12 months.In general, Lugan, mandarin and Ougan had more leaves,Kumquat and Huyou had less leaves.For taproot length, Shatangju had the longest taproot during conservation process all the time,Lugan was the next.The remaining species of root growth rates were fluctuating at different conservation stages;Youxi kumquat No.2 had the shortest taproot all the time during conservation process from 2 months to 18 months. In general, the taproot of kumquat was shorter than other resources in length.According to the survival analysis of 18-month plantlets, the number of leaves and the length of taproot were of no causal relationship with survival. The best conservation materials were Youxi kumquat No.1, No.2 and Huyou, survival rates were 58.3%, 61.6%, 57.6%. Sugan had the least survival rate, and 15-month survival rate was only 11.9% and 18-month survial was only 9.8%, which seemed that MS medium was not suitable for the conservation of Sugan.Many kinds of citrus could be conserved well on MS medium without subculture for a long time.For example,in Gudong Yangchunju,Fuzhou Xuecheng,Fuju and Chongqing Hongju, conservation time was up to 51 months, and the next was Sijiju for 50 months and Fushouju for 49 months, and these citrus plantlets were possible to be conserved for longer time.3. Mn-SOD gene cloning from the plantlet leaf of Youxi kumquatThe young leaves of Youxi kumquat in-vitro plantlets were used as materials for cloning the Mn-SOD gene.The result showed that the full sequence of Mn-SOD gene was 1097bp.It was registered in GenBank, and accession numbers was GU233744.1. The Mn-SOD gene contained 66bp 5'UTR, 347bp 3'UTR, and 3'-end involved 13 poly(A)tails. The results showed the sequence of this gene was highly homologous with that of other plants reported in GenBank.The cDNA contained a 684bp open reading frame, encoded 228 amino acids, and ATG was the initiation codon and TAA was the stop codon.The predict of protein's physical and chemical properties shows that, the molecular weight of protein Mn-SOD was 25159.7 Da;the theoretical isoelectric point(pI) was 4.92;the atomic composition was C1140H1769N303O328S6, the total number of atoms was 3546; the total number of negatively charged residues(Asp+ Glu) was 20 and the total number of positively charged residues(Arg+Lys) was 21; the protein including 20 amino acids,which had more abundant of Leu,Ala,Gly,Ly,Val and Ser,were 24(11.4%), 20(8.8%), 20(8.8%), 17(7.5 %), 17(7.5 %)and 14(6.1 %), Cys was the lowest with only 1(0.4 %).Secondary structure prediction showed that the Youxi kumquat Mn-SOD protein was with 51.32%α-spiral, 14.04% extending chain and 30.07% irregular curl. Looking at the overall structure of the protein, the main structural elements of Youxi kumquat Mn-SOD protein wereα-spiral and irregular curl, and wich were spreaded to the whole protein.Three-dimensional structure prediction showed that Youxi kumquat Mn-SOD protein was surrounded with 3β-fold and 6α-spiral, and the 3β-fold were surrounded by the 6α-spiral. These studies were laid the important foundation for the study of SOD mechanism during citrus plantlets conservation.