Genetic Diversity Of Germplasm Resource Of Casuarina Equisetifolia

Casuarina equisetifolia ssp. equisetifolia(L), nitrogen fixation trees, natural distributed in subtropical and tropical coastal areas from northern Queensland and the Northern Territory in Australia, throughout southern Thailand, Malaysia, Indonesia, the Philippines, Melanesia, Polynesia and Guam, etc. This species was commonly used in agroforestry systems and coastal protection and rehabilitation, at same time, its wood was used as a fuel, also pulp and paper industry. It was the earliest introduction species of Casuarina and has the largest cultivated area in China. This research investigated the genetic diversity level of natural provenances and introduced provenances of C. equisetifolia from phenotype, physiological and DNA molecular markers, by using the seedlings growth characteristic, element content and photosynthetic differentiation and EST-SSR technology. The geneic diversity pattern and genetic structure has been analysised, for the hope to offer the information for germplasm collection and protection, the selection of excellent provenances, and genetic improvement and breeding project establishment of C. equisetifolia. The results about this reaearch as followings:(1)At 9 months old seedlings of C. equisetifolia, height and diameter have significant differentiation between regions and between provenances. Similar growth trend of different regions of C. equisetifolia can be found at both nurseries, height and diameter of seedlings from Oceania natural provenances were minimal, while growth condition of seedlings from Asia natural and Asia introduced provenances were better, the growth of seedlings from Africa introduced provenances taken the second place. In general, variation coefficient of height and diameter between provenances were both more than 25%, provenances heritability of height and diameter were 24.17% and 13.61%(at DD), 38.41% and 20.06%(at CH), heritability of height were higher than of diameter at both nurseries. Among the provenances, AU1 has the worst growth condition, height of this provenance were 13.18 cm(at DD). TH2 has the best growth condition, height of this provenance reached 3.4 times of AU1.(2)At 9 months old seedlings of C. equisetifolia, 8 traits(thickness of branchlet(Thk), length of first oder branchlet(Len1), length of second oder branchlet(Len2), density of branchlet(Den), branchlet angle(Ang), nodes of firest oder branchlet(Node), teethes(Teeth)and color of branchlet(Col))have significant difference between regions and between regions. At DD nursesies, mean of Node, Teeth, Thk, Len1 and Len2 were 25.39, 6.79, 0.73 mm, 9.39 cm and 4.23 cm, respectively; change range of these trait were 15.06-40.78, 5.69-7.91, 0.53-0.90 mm, 4.68-14.74 cm and 2.15 cm-5.86 cm, respectively; phenotype variation coefficient of thesr trait were 22.29%, 10.61%, 22.35%, 31.69% and 32.72%, respectively. Ang and Den of difference provenances have great change. According to Ang’s change, two types could be found, namely upward and horizontal branchlet anglet. Also, density types and pole density types of branchlet density were existed as the change of Den. Provenance heritability of the 8 traits were 55.46%、34.22%、29.71%、45.32%、50.11%、48.64%、38.59% and 37.75%,respectively. Genetic variation coefficient of Den(24.92%), Len1(20.63%), Len2(19.86%),and Ang(18.00%)were higher.(3)Among 9 months old seedlings of C. equisetifolia, N content(F = 7.24**), P content(F = 3.47**), K content(F = 4.10**) and B content(F = 4.71**)have significantdifferentiation. Among 1.5 years old young trees of C. equisetifolia, the net photosynthetic rate(Photo)(F = 4.77**)and transpiration rate(Tr)(F = 2.70**)have significant differentiation.Variation coefficient of N content, P content, K content and B content were 2.90%, 11.16%,16.66% and 31.82%. Mean of Photo was 14.13 μmol CO2 m-2s-1, change range was 6.70-19.57 μmol CO2 m-2s-1; mean of Tr was 5.38 mmol H2 O m-2 s-1, changes range was 3.27-7.27 mmol H2 O m-2 s-1. Variation coefficient of Photo and Tr were 13.23% and 16.08%.(4)The 34,752 EST sequences of casuarina trees can be download from the NCBI website. The 12,063 non repetitive sequences(Uni Gene) can be obtained after pre-treatment and moving redundant by software, the redundant rate of EST sequences of casuarinae was 65.29%. The 367 SSR loci can be identified from the 12,063 Uni Gene, these SSR loci were distribution in the 352 EST sequences, and the frequency of EST sequence that contained SSR loci was 2.92%, which was lower compared with other tropical tree species, e.g., oil palm(4.45%). Among the SSR, dinucleotide repeat motif AG/CT had the largest number 199, accounting for 93.87% of the dinucleotide repeat. AAG/CTT had the largest number 56 among the trimucleotide repeat.(5)The 13 pairs of EST-SSR primers with ampliticant stability, band clear and polymorphism were obtained for gentic diversity analysis of C.equisetifolia through selection. The 308 alleles can be identified from the 13 SSR loci, average alleles number per loci was 23.69, change range of alleles number was 11-48. Change range of effective alleles number, Shannon’s index, observed heterozygosity and effective heterozygosity were 1.533-7.029,0.691-2.139,0.270-0.655 and 0.393-0.858, respectively. According Shannon’s index, the order of genetic diversity level from high to low of the 5 regions was: Afican introduced(AF) >Asia natural(AN) >Oceania natural(OP)> Central American introduced(CI) >Asia introduced(AI);the order of genetic diversity level of 29 provenances was: KE2>AU1>MY1>TH1>MY2>PH1>CU2>SB>MU>CN2>CN1>IN2>IN1>PH3>BJ>IN4> VU > LK > VN > AU2 > EG > KE1 > TH4 > GU > TO > CU1 > PG > BD > PH2. Hardy-Weingerg test showed that highly significant positive deviation was existed in the 23 provenances, these provenances indicated deficit heterozygous or excessive homozygous. On the region level, individuals from the 5 regions(OP, AN, AI, AF and CI) had significant positive deviation on the 11 SSR loci, characterized by deficit heterozygous or excessive homozygous. The results implied that serious inbreeding between populations were occured during the whole distribution of C. equisetifolia, which would be very helpful to activities to lay down breeding strategy, to select breeding material and to set seed orchards, and also indicate that it should be paid more attentions to inbreeding among populations in these activities.(6)The main variation of C.equisetifolia populations were from the individuals within provenances, which accounting for 70.12% of total variance. On regions level, the order of variance was: AN(81.15%) > AI(74.58%) >CI(72.29%) >AF(68.43%) >OP(61.45%). Results showed that family selection among provenance should be the focus of C.equisetifolia breeding. Meanwhile, though variation from provenances accounted for only 25.42 to 38.49% of the total variantion, given the serious inbreeding that identified in the population of C. equisetifolia, provenance selection of C. equisetifolia should attach great importancec in future.(7)According to clustering based on seedling phenotypic traits and Nei’s genetic distance UPGMA clustering of EST-SSR genetic diversity analysis, introducted provenances of China should be from Asia natural provenances, while introduced provenances of Kenya, and India and Veitnam might from Oceania natural provenances.