North China Mountain Walnut (NCMW) is one of the ancestors of extant cultivated species, and a valuable gene resource for resistance breeding of walnut in China. Inter-Simple Sequence Repeat (ISSR) primers were designed to evaluate the level and pattern of genetic diversity in eight populations of NCMW. Nine ISSR primers yielded 91 amplification products with different sizes, of which 84 (92.31%) were polymorphic. A high species-level genetic diversity was detected with Nei’s ( H = 0.2592) and Shannon’s diversity ( I = 0.4003). In contrast, the population-level genetic diversity was relatively lower (PPB = 43.27%, H = 0.1347, I = 0.1862). Coefficient of populations differentiation (GST) was 0.5066, indicating that inter-population and intra-population variation contributed 50.66% and 49.34% respectively to the total genetic variability. This relative level of variation was further supported by AMOVA analysis. Limited gene flow ( Nm = 0.5133.), habitat fragmentation and geographical isolation might be responsible for the population structure of NCMW. UPGMA cluster analysis classified the eight populations into three groups which showed no significant relationship between the genetic similarity coefficient and geographic origin but showed remarkable association with morpho-physiological characters, particularly nut traits. The results of the study provide species-level and population-level genetic profiles for further exploitation and conservation of genetic diversity of NCMW.
Several walnut (Juglans spp.) species are important dual purpose fruit and timber economic forest tree species, of these the globally most important is “Common Walnut” (J. regia). China is the one of origin centre of wal- nuts and possesses abundant resources. There are five main species of the Juglans genus available as walnut breeding resources in China. These are J. regia L., J. Sigillata Dode, J. mandshuria Maxim, J. openensis Hu, and J. nigra L. [
In China, North China Mountain Walnuts are mainly distributed in the foothills of the North China Mountains, which spread across the northern latitudes from 35˚ to 40˚, and the western longitudes from 112˚ to 119˚. The area is a rocky, mountainous region with serious soil erosion, a warm temperate climate, an annual precipitation from 400 - 700 mm, and slightly calcareous cinnamon soils [
In the past few years, a quantity of molecular markers, such as RFLP (Restriction Fragment-Length Poly- morphism) [
It is reported for the first time to assess genetic diversity and genetic structure of North China Mountain Wal- nut sampled out of eight significant natural populations in China by ISSR. We evaluate the population-level and species-level genetic diversity of eight North China Mountain Walnut populations throughout their known dis- tribution by ISSR analysis to give some references for conservation measures and germplasm resource devel- opment and utilization.
In this study, a total of 138 individuals of North China Mountain Walnut, representing eight populations (GX, FY, LY, YX, LC, SX, ZQ, and LZ), were sampled across three provinces in China, including Shanxi, Hebei and Henan along the Taihang and Lvliang Mountains (
Population | Location | Longitude (E)/Latitude (N) | Elevation (m) | Sample sizes |
---|---|---|---|---|
GX | Gu County, Linfen City, Shanxi Province | 36˚32'N 112˚04'E | 1430 | 15 |
FY | Fengyang County, Luliang City, Shanxi Province | 37˚15'N 111˚37'E | 1047 | 15 |
ZQ | Zunquan County, Jinzhong City, Shanxi Province | 36˚51'N 113˚27'E | 821 | 23 |
YX | Yu County, Yangquan City, Shanxi Province | 38˚23'N 113˚21'E | 653 | 20 |
LC | Licheng County, Changzhi City, Shanxi Province | 36˚48'N 113˚27'E | 757 | 25 |
SX | Shexian County, Handan City, Hebei Province | 36˚41'N 113˚27'E | 562 | 11 |
LY | Laiyuan County, Baoding City, Hebei Province | 39˚53'N 118˚53'E | 45 | 8 |
HN | Linzhou County, Anyang City, Henna Province | 36˚16'N 113˚48'E | 420 | 4 |
Genomic DNA was extracted from storage leaves of walnut by using the optimized Cetyl Trimethyl Ammonium Bromide (CTAB) method [
A total of one hundred ISSR primers were used. The primers were designed according to public sequences provided by the University of British Columbia, Canada and biosynthesised by the Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). They were sifted firstly grounded on one sam- ple from each population. Nine primers, which yielded clear, polymorphic, and reproducible bands, were used for the further genetic diversity analysis of all 138 DNA individuals (
Polymorphic bands were scored visually as either present “ 1” or absent “ 0” to form a binary matrix. On the supposition of Hardy-Weinberg equilibrium, POPGENE versions 1.31 [
91 bands in total were generated from nine screened primers for the 138 individual samples from the eight pop- ulations, of which 84 (92.13%) were polymorphic bands. The size of the amplified band ranged from 300 bp to 1200 bp. The percentage of polymorphic band (PPB) of the 9 ISSR primers differed from 66.67% (UBC855) to 100% (UBC815, UBC836, UBC840 and UBC853), with an average of 92.31%. The number of bands presented by each primer also differed from 6 (UBC855) to 11 (UBC840) with an average of 10.11 (
Estimates of genetic diversity in eight populations of North China Mountain Walnut are summarized in
Primer | Sequencea | Tmb/˚C | Number of band | Number of polymorphic band | PPB/% |
---|---|---|---|---|---|
UBC811 | (GA)8C | 54.7 | 11 | 9 | 81.82 |
UBC815 | (CT)8A | 59.1 | 10 | 10 | 100 |
UBC836 | (AG)8YA | 52.5 | 8 | 8 | 100 |
UBC840 | (GA)8YT | 50.6 | 15 | 15 | 100 |
UBC843 | (CT)8RC | 54.3 | 10 | 8 | 80 |
UBC853 | (TC)8RT | 55.2 | 11 | 11 | 100 |
UBC855 | (AC)8YT | 51.8 | 6 | 4 | 66.67 |
UBC856 | (AC)8YA | 56.1 | 9 | 9 | 100 |
UBC888 | BDB(CA)7 | 53.4 | 11 | 10 | 90.91 |
Average | 10.11 | 9.33 | 92.31 |
Y = (C,T), R = (A,G), B = (C,G,T), D = (A,G,T). aSequence 5' - 3'; bPCR reaction annealling temperature for tabulated primers (˚C); cPercentage of polymorphic band.
Population | Number of polymorphic loci | PPB (%) | Na | Ne | H | I |
---|---|---|---|---|---|---|
GX | 48 | 52.75 | 1.575 | 1.3024 | 0.1788 | 0.2695 |
YX | 30 | 32.97 | 1.3297 | 1.1505 | 0.0955 | 0.1496 |
LY | 38 | 41.76 | 1.4176 | 1.1795 | 0.1098 | 0.1716 |
FY | 52 | 57.14 | 1.5714 | 1.3163 | 0.1839 | 0.2778 |
LC | 39 | 42.86 | 1.4286 | 1.1769 | 0.1090 | 0.1717 |
SX | 41 | 45.05 | 1.4505 | 1.2764 | 0.1617 | 0.2413 |
ZQ | 49 | 53.85 | 1.5385 | 1.2584 | 0.1569 | 0.2433 |
HN | 18 | 19.78 | 1.1978 | 1.1419 | 0.0818 | 0.1192 |
Average | 39.375 | 43.27 | 1.4386 | 1.2253 | 0.1347 | 0.1862 |
Species | 84 | 92.31 | 1.9231 | 1.4243 | 0.2592 | 0.4003 |
SD | 0.2679 | 0.3335 | 0.1699 | 0.2290 |
PPB, Percentage of polymorphic loci (%); Na, Observed number of alleles; Ne, Effective number of alleles; H, Nei’s genetic diversity; I, Shannon’s information index; SD, Indicates the standard deviation.
At the species level, the effective number of alleles (Ne), Nei’s genetic diversity (H) and the Shannon informa- tion index (I) were 1.4243, 0.2592, 0.4003 respectively. At the population level, PPB ranged from 19.78% for the LZ population to 57.14% for the YX population, with an average of 43.27%, and the average Ne, H, and I were 1.2253, 0.1347, and 0.1862, respectively. The results showed that the genetic diversity of North China Mountain Walnuts from the YX population (Ne = 1.3163, H = 0.1839, I = 0.2778) was the richest in three esti- mates among the eight populations and the lowest diversity was exhibited in the LZ population.
Nei’s coefficient of genetic differentiation (GST) was 0.5066, which showed that 50.66% of the total genetic va- riability is attributed to inter-populations and 49.34% intra-populations. Results of AMOVA also agreed with which genetic differentiation inter-populations is relatively high (FST = 0.5161). At two hierarchical levels, 51.61% of the total genetic variation was partitioned inter-populations, and 48.39% (P < 0.001) intra-populations. The level of gene flow (Nm) was estimated to be only 0.5133, which implied that a low gene flow would not prevent genetic drift thus enabling the gene differentiation between populations.
To further clarify the gene differentiation among different populations, Nei’s pairwise genetic similary coeffi- cients were assessed. Genetic similar coefficients varied from 0.7423 to 0.9298 with an average 0.8159, which showed higher levels of genetic differentiation partitioned among populations of North China Mountain Walnut. The highest similar coefficient was between the ZQ population and the LC population, while difference in simi- lary coefficients between the FY population and the LC population was revealed as the lowest (
In order to further illustrated relationships among populations, a dendrogram generated by UPGMA algo- rithm based on Nei’s genetic similary, which clustered the eight populations into three major groups (
Our data shows that genetic diversity is higher at the species level (PPB = 92.31%, H = 0.2592, I = 0.4003), but
Population | GX | FY | LY | YX | LC | SX | ZQ | HN |
---|---|---|---|---|---|---|---|---|
GX | **** | 0.8567 | 0.7954 | 0.7892 | 0.8077 | 0.8337 | 0.7913 | 0.8024 |
FY | 0.1546 | **** | 0.7658 | 0.7451 | 0.7423 | 0.7562 | 0.7653 | 0.7562 |
LY | 0.2289 | 0.2668 | **** | 0.8751 | 0.8891 | 0.8735 | 0.8740 | 0.8424 |
YX | 0.2367 | 0.2943 | 0.1334 | **** | 0.9011 | 0.8868 | 0.8709 | 0.8350 |
LC | 0.2136 | 0.2979 | 0.1176 | 0.1041 | **** | 0.9102 | 0.9298 | 0.8259 |
SX | 0.1818 | 0.2794 | 0.1353 | 0.1202 | 0.0941 | **** | 0.8993 | 0.8454 |
ZQ | 0.2340 | 0.2675 | 0.1346 | 0.1382 | 0.0728 | 0.1062 | **** | 0.8761 |
HN | 0.2201 | 0.2795 | 0.1715 | 0.1804 | 0.1913 | 0.1679 | 0.1322 | **** |
lower at population level (PPB = 43.27%, H = 0.1347, I = 0.1862), which is alike to the results obtained from same species using different molecular markers [
North China Mountain Walnuts are distributed in different ecological environments and geographic condi- tions, such as having a range of growing elevations from 45 m to 1430 m above sea level. With increasing ele- vation, climate and soil physicochemical properties also show a significant change, which results in greater pop- ulation variation. As observed in our study, the population differentiation parameters such as PPB, Ne, H and I in eight populations showed a similar pattern, the YX population is the highest, and the LZ population is the lowest. This can be interpreted as a consequence of habitat fragmentation and small numbers of individuals. Due to genetic drift, small populations tend to lose genetic variation has been verified in many studies. Therefore, the results need to be further studied and verified by a more extensive investigation.
Compared with other ecotypes, genetic diversity of North China Mountain Walnut was at a moderate level, which was marginally higher than Sinkiang walnut and Qinling-Daba Mountain walnut [
In accordance with the GST value (GST = 0.5066), found in this study a significant amount of genetic differentiation is observed among and within eight populations of North China Mountain Walnut. This point is reinforced by the AMOVA, which indicating that 51.61% of the genetic variation was partitioned inter-populations and 49.34% intra-populations. Significant genetic differentiation was also reported by Wu et al. using RAPD. In contrast, less genetic differentiation was found by Xi [
High genetic differentiation of North China Mountain Walnut is attributed to the following points: Firstly, because of low gene flow of the North China Mountain Walnut habitat (Nm = 0.5133), the present population structure was shaped by genetic drift mainly. Secondly, the Taihang and Lvliang Mountains contribute to the geographical isolation, which severely hindered dispersal of pollen or seeds and exchange of genes between populations, ultimately promoting the large genetic differentiation among populations. Thirdly, in order to adapt to the diverse habitats of the region in the evolutionary process, variants to some extent occurred, which may have been preserved and fixed gradually due to limited gene flow, thereby, genetic differentiation among popu- lations occurred. The GST value and AMOVA analysis results obtained in this study indicated that North China Mountain Walnut has a significant genetic differentiation among populations, which suggests that the species has a strong environmental adaptability. Put briefly, high genetic variation among populations is due to genetic drift and geographical isolation of the populations.
Based on the genetic similarity, eight populations of North China Mountain Walnut were grouped into three clusters, but genetic differentiation did not completely cluster according to geographical distances (
To know about the geographic structure of intraspecific genetic variation is indispensable for the protection of germplasm resources. Our study provides an insight into genetic diversity and genetic differentiation at popula- tion levels of North China Mountain Walnut, and indicates a decrease of genetic diversity caused by genetic drift is unacceptable. Accordingly, so as to secure existing diversity, preservation areas covering large popula-
tions as well as many small populations should be established. Mixing genetically diverse populations of North China Mountain Walnut in situ or ex situ is unacceptable in conservation.
We gratefully thank Professor Chuck Leslie at Department of Plant Sciences, University of California, Davis; and Jihua Tang at Henan Agriculture University, for critically reviewing our manuscript. This work was finan- cially supported by the National Natural Science Foundation of China (31171943), the Key Scientific and Technological Project in Henan province (92101110600), and the Scientific and Technological Project in Henan province (102102110178).