Study On The Genetic Diversity And Photosynthetic Characteristics Of Chinese Pine(Pinus Tabulaeformis)

Chinese pine (Pinus tabulaeformis Carr.) is an endemic coniferous species to China, and it is one of the most important tree species for afforestation in the northern China. It has a fragmented or island-like distribution over its natural range across the northern China. Due to the variations of climatic conditions, habitat conditions, and natural selection pressures, the populations of this species in different areas have shown a clear differentiation in morphology and physiology. It is of great importance to further examine the spatial patterns of the genetic variation, and the adaptation capacity of this species to the environmental factors for the effective protection of its genetic resource, and the proper use of its various provenances in afforestation. In this study, a total of 20 natural populations of the species was used to evaluate the genetic diversity and genetic structure, and genetic relationships among the populations using amplified fragment length polymorphism markers (AFLP) and mitochondrial DNA (mtDNA) sequence markers; while provenances of this species [i.e., the provenance of Qian Mountain (QN) from Liaoning, provenances of Pingquan (PQ), Wuling Mountain (WL) from Hebei, provenances of Iiiliang Mountain (LL), Luya Mountain (LY), Guandi Mountain (GD), Taihang Mountain (TH), Heicha Mountain (HC), Zhongtiao Mountain (ZT), Taiyue Mountain (TY) from Shanxi, provenances of Huangling (HL) and Luonan (LN) from Shaanxi] were selected to test their photosynthetic parameters responses to the changes of light intensity, CO2 concentration, and soil water content using the Li-6400 photosynthesis system for assessment of their capacity of adaptability to the environmental factors. The results were as follows:(1) A total of 445 fragments were detected with 8 pairs of primers for 480 individuals of the 20 populations,379 (85.17%) of which were polymorphic. A relative high level of genetic diversity was detected at the species level (percentage of polymorphic bands PPB=85.17%, Shannon’s information index 7=0.356, Nei’s gene diversity HE=0.271), and at the population level (PPB=41.90%,I=0.219,HE=0.206, respectively). The population Huzhu exhibited the highest genetic diversity (I=0.271 and HE=0.254, respectively), while population Li county displayed the lowest genetic diversity (I=0.175 and HE=0.165, respectively). AMOVA analysis revealed that 69.60% of genetic variation resided within populations and 30.40% existed among populations. The genetic differentiation among population was significant (Fst=0.304, p<0.001). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (Nm=0.572). The Mantel test indicated that there was a significant correlation (r=0.455,p<0.001) between Nei’s genetic distance and geographical distance among all the populations. The 20 populations were classified into two major groups using UPGMA cluster and Bayesian model-based cluster method. The population Kaiyuan from the northeast of China was the only member of Group 1, while the rest 19 populations were clustered into Group 2. The latter were further divided into four subgroups, the seven central populations were clustered into one group.(2) The genetic diversity of 8 natural populations of Chinese pine was explored using the mitochondrial DNA (Nadl intron b/c and matR. introns) sequence marker. The results indicated that a total of 25 haplotypes,134 polymorphic sites, including 34 parsimony informative sites and 100 singleton variable sites were identified from 80 individuals of these populations. The haplotype diversity was high at the species level (the haplotype diversity h=0.844) and at the population level (h=0.681). The population from the east side of the Helan Mountain exhibited the highest haplotype diversity (h=0.911), while the population Ningshan displayed the lowest haplotype diversity (h=0). Of all the haplotypes (from In to h25), In was shared by all the populations, and 7 of the 8 populations had one to four unique haplotypes. Genetic diversity distributed within populations (85.21%) rather than among populations (14.79%). Despite the mountainous barriers, the populations in the north and south sides of the Qin Mountains and in the east and west sides of the Helan Mountains had no significant genetic differentiation. Therefore it was concluded that the genetic structure of Chinese pine was hardly associated with the mountainous barriers over the distribution area.(3) The light and CO2 response curves of photosynthesis for the 12 provenances were studied under natural environmental condition. The results indicated that the light and CO2 response curves of photosynthesis could be simulated by the modified model of rectangular hyperbola and the fitted photosynthesis parameters were close to the measured data. The obtained maximum values of photosynthetic rate under light saturation point (Pmax, μmol·m-2·s-1) had the following order:provenances PQ(17.72)> WL(16.95)> ZT(15.74)> QN(15.42)> LY(15.25)> LL(14.30)> GD(12.68)> TH(12.41) > HC(12.29)> TY(9.57)> LN(4.57)> HL(4.20), with the pattern that northern provenances> central provenances> southern provenances. The light saturation points (LSP) and compensation points (LCP) were high and significantly different among the provenances, with the range from 1629 to 2811μmol·m-2·s-1 for LSP and the range from 54.70 to 230.72μmol·m-2·s-1 for LCP, and with the pattern that southern provenances> northern provenances > central provenances. In addition, the values of apparent quantum efficiency (AQY) for northern provenances were higher than southern provenances. This showed that southern provenances had a weaker shade-toleranance and lower photosynthetic rate. The maximum photosynthetic rates under CO2 saturation point (Jmax, μmol·m-2·s-1) had the following order:provenances LL(39.47)> GD(32.24)> HL(31.75)> HC(31.67)> WL(31.65)> QN(31.21) > LY(30.19)> ZT(28.71)> LN(28.26)> TY(26.88)> TH(25.69)> PQ(22.32). Photosynthetic rate could be enhanced with the increase of CO2 concentration. The CO2 saturation points (CSP) and compensation points (CCP) were also high and significantly different among the provenances, with the range from 1461 to 4565μmol·m-2·s-1 for CSP, and the range from 110 to 145μmol·m-2·s-1 for LCP. Most of the northern provenances had high CCP values, so they probably had high photosynthetic efficiency. The carboxylation efficiency for the 12 provenances had the following order: ZT(0.042)> WL(0.040)> GD(0.039)> HL(0.038)> TH(0.037)> TY(0.033) = HC(0.033)= PQ(0.033)> LY(0.031)> QN(0.030)> LN(0.029)> LL(0.018). The 12 provenances could be divided into four groups by the fuzzy mathematic cluster analysis. The first group with the strongest adaptability to light intensity and CO2 concentration was mainly consisted of the northern provenances (QN, HC, GD, WL, PQ and LY). The second group with the medium adaptability was mainly comprised the central provenances (TH, TY and ZT). The third group with the weakest adaptability was of the southern provenances (HL and LN). The central provenance LL that had stronger adaptability than most provenances, and it was divided into the fourth group.(4) The transplanting experiment for the seedlings of eight provenances of Chinese pine with soil water treatment in a greenhouse showed that compared with normal water condition (T1), severe water stress (T3) had obvious effect on the photosynthetic parameters. A decreasing trend was found for the photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), potential efficiency of primary conversion of light energy of PS Ⅱ (Fv/Fm), potential activity of PSⅡ(Fv/Fo), actual quantum yield of PS Ⅱ electron transport (OPS Ⅱ), apparent photosynthetic electron transport rate (ETR), and photochemical quenching (qP), while an increasing trend was detected for water use efficiency (WUE), and initial fluorescence(Fo). A significant difference was detected in the Pn, Tr, Gs, WUE, φPS Ⅱ 、 qP and ETR under T3 (p<0.05) for the eight provenances. The Pn(μmol·m-2·S-1) for the 8 provenances had the following order:LY(4.83)> QN(4.43)> ZT(4.28) > LL(3.84)> HL(3.54)> WL(3.30)> LN(2.64)= TY(2.63). The higher values of Pn for provenances LY、QN、ZT were detected than provenances LN andTY. While the WUE (mmol·mol-1) for the 8 provenances had the following order:TY(9.17)> ZT(7.96)> QN(5.34)≈LY(5.04)> HL(4.79)≈ WL(4.45)> LN(3.71)> LL(3.19). The higher values of Pn, φPS Ⅱ, qP and ETR were found for provenances QN, LY, ZT than other ones. For these four provenances, the water stress had little effect on their photosynthetic ability, photochemical efficiency of PS Ⅱ and electron transport rate. However, for the provenaces WL and HL, the water stress had serious influence on these parameters. This indicated that provenances QN, LY, ZT had higher drought resistance than provenaces WL and HL.In a word, the high genetic diversity was found for the natural populations of Chinese pine, and the genetic differentiation was significant among the populations. The genetic structure of this species was hardly associated with the mountainous barriers over the distribution area. Provenances QN, LY and ZT had higher adaptation capacity to changes of light intensity, CO2 concentration and water stress.