Genetic Diversity and Population Structure of Job’s Tears(Coix lacryma-jobi L.) Germplasm Based on ISSR Marker

11 ABSTRCT 12 Background: Job's （ Coix lachryma-jobi L ） is a minor cereal and important food 13 item in some parts of Asia It has also been used in the traditional medicine for 14 relieving various ailments, therefore, it plays an important role Lack of 15 excellent new varieties hinders the development of coix as a sustainable crop, and urgent 16 to provide new with Coix industry 17 Results: ISSR markers were used to assess the genetic and population 18 structure of 8 populations of Job’s tears in China A genotyping analysis that ten ISSR 19 primer pairs resulted in the production of 116 bands, of which 98 polymorphic The 20 Guizhou population (PPB = 8190%, h = 03113, I = 04589) was the genetically diverse, 21 while the lowest was observed in the Hebei population (PPB = 4655%, h = 01842, I = 22 02701) Genetic differentiation analyses including GST and AMOVA that genetic 23 variation was most prevalent within populations while only minor were observed 24 among populations Genetic distance coefficients ranged from 00095 to 00948 for the 8 25 populations; the genetic relationship between the Guizhou and Chongqing populations was 26 the closest, while the most distant genetic relationship occurred between the and 27 populations The of an UPGMA cluster analysis that genetic diversity 28 among the were consistent with the distance results The STRUCTURE analysis suggested that 94 Job’s tears accessions could be grouped into two 30 subpopulations Moreover, according to a cluster analysis based on the UPGMA for 31 individuals of Job’s tears, accessions were divided into two major clusters The results of the 32 Bayesian cluster and UPGMA cluster analyses were largely consistent despite minor 33 differences There was no significant correlation between genetic distance and geographic 34 distance (r = 0055, p = 0782) 35 Conclusions: Our study was undertaken to systematically analyze genetic diversity and 36 population structure in 94 Job’s tears accessions using ISSR markers And this study provides 37 us with valuable information pertaining to germplasm collection, genetic improvement, and 38 systematic utilization of Job’s tears


INTRODUCTION
Job's tears (Coix lachryma-jobi L ) belongs to the Coix genus, the Andropogononeae 41 tribe, and the Gramineae family [1] Job's tears, also known as coixseed, tear grass, hato mugi, 42 and adlay, is a tall grain-bearing tropical plant [2] This crop is predominantly planted in 43 Southeast Asian countries including China, Vietnam, Laos, Japan, and Korea [3] Job's tears 44 seeds are sweet to taste and are widely used in both culinary and medicinal practices; they are 45 considered an ideal health food and effective medicine Indeed, the seeds have been shown to 46 reduce fever, invigorate the spleen, while also exhibiting diuretic, anti-cancer, hypolipidemic, 47 hypoglycemic, antioxidant, anti-inflammatory, and anti-allergic properties [4][5][6] Job's tears 48 seeds are nutritious and are commonly processed into products including Job's tears seed tea, 49 Job's tears seed powder, Job's tears seed biscuits, Job's tears seed alcoholic beverages and 50 vinegar [7][8] Until now, research conducted on Job's tears has predominantly focused on 51 agronomic characteristics, inherent chemical constituents and associated pharmacological 52 functions There is currently a lack of international peer-reviewed articles pertaining to 53 breeding programs associated with Job's tears Evaluation of genetic diversity and genetic 54 relationships in the germplasm can provide useful information for breeding programs 55 Morphological or phenotypic descriptors have traditionally been used to distinguish one 56 accession from another Although this type of agronomical characterization provides useful 57 information, these investigations are subject to environmental influences; they are also 58 time-consuming and must be assessed during a fixed vegetative phase of the crop life cycle [9] Molecular markers have significant advantages over morphological and isozyme markers 60 because they are not influenced by growth and environmental conditions and can be applied 61 during any growth phase Compared with strategies that analyze other molecular markers, 62 analyses investigating inter-simple sequence repeats (ISSR) are attractive methods because 63 they do not require DNA sequence information These methods facilitate the detection of 64 highly variable sequences; they are also reproducible and cost effective ISSR analysis has 65 been widely used to study the genetic diversity of various plants [10] (Table 2) 113 PCR amplification 114 The ISSR reaction mixture (total volume = 25 μL) contained 2 5 μL of 10 × buffer (0 1 115 M of Tris-HCl, pH 8 3, 0 5 M KCl), 1 μL of DNA template (50 ng), 2 μL of each primer (10 116 μM concentration), 0 5 μL of Taq DNA polymerase (2 5 U/μL), 1 μL of MgCl2 (2 mM), 2 μL 117 of dNTPs (2 5 µM) and 16 μL of ddH2O The amplifications were performed in an Eppendorf 118 Mastercycler Gradient PCR machine (Eppendorf, Germany) using the following program conditions: an initial denaturation step of 5 min at 94°C; 35 cycles of 94°C for 30 s, 46-60°C   120   (depending on primer pair chosen) for 30 s, 72°C for 30 s; and a final extension at 72°C for 5   121   min The amplification products were electrophoresed at 100 V for 45 min on a horizontal gel   122 apparatus (Bio-Rad, SA) using a 1 5% agarose gel in 1 × TAE (pH 8 0) The gels were stained 123 with 0 8 μg/mL ethidium bromide for approximately 30 min, and then photographed under 124 UV light using the UVP-GDS8000 Gel Documentation System (UVP, USA) [6] 125 Data analysis 126 The discernible and reproducible DNA bands ranging from 250 to 2000 bp were scored 127 1 for presence and 0 for absence These bands were used to construct the binary data matrix 128 for statistical analysis The number of polymorphic bands (NPB), the percentage of 129 polymorphic bands (PPB), the observed number of alleles (Na), Shannon's Information index 130 (I), effective number of alleles (Ne), Nei's gene diversity (h), genetic differentiation, genetic 131 identity and genetic distance were calculated using PopGene 1 32 [13] AMOVA was used to 132 analyze genetic diversity using ARLEQUIN version 3 01 [14] Based on the Nei's genetic 133 distance, cluster analysis was performed to generate a dendrogram; the dendrogram exhibited 134 the genetic relationships among populations and was assimilated using the un-weighted 135 pair-group method with arithmetic mean (UPGMA) using the MEGA 4 v 4 1 software [15] . 136 The population structure was also estimated using a Bayesian assignment test as 137 implemented using STRUCTURE v 2 2 software K (number of populations) values were set 138 from 1 to 16, 6 repetitions The posterior probabilities were estimated using the Markov Chain 139 Monte Carlo (MCMC) method Ninety-four Job's tear individuals were clustered using the 140 admixture model program and the genetic clustering number (K) was inferred; burn-in was 141 100000 and run-length was 1000000 The value of ΔK was calculated from the values of Ln P 142 (D) corresponding to different values of K [16] , and the possible genetic structures were 143 analyzed Accessions were assigned to a subpopulation if the probability of membership was 144 greater than 70% [17] If membership was ≤ 70%, the accessions were assigned to the mixed 145 subpopulation 146 The genetic distance matrix was calculated using the NTSYS-pc version 2 1 software 147 package [18] The UPGMA [19] tree was constructed based on the genetic distance matrix using 148 MEGA 4 1 software [15] The geographical distance was obtained by the actual latitude and longitude of each 150 population, and the genetic distance was calculated by POPGENE 3 2 software The Mantel 151 correlation test [20] in the TFPGA software [21] was used to analyze the correlation between 152 geographical distance and genetic distance for the different populations 153

Figure 2
Inter simple sequence repeat (ISSR) pro les for 24 Job's tears genotypes following ampli cation with UBC857 primers and agarose gel electrophoresis.

Figure 3
Inter simple sequence repeat (ISSR) pro les for 24 Job's tears genotypes following ampli cation with UBC857 primers and agarose gel electrophoresis.

Figure 4
Inter simple sequence repeat (ISSR) pro les for 22 Job's tears genotypes following ampli cation with UBC857 primers and agarose gel electrophoresis.

Figure 5
Dendrogram of populations of Job's tears generated following UPGMA cluster analysis according to Nei's genetic distance.

Figure 9
MANTEL test plots of the genetic distance and geographical distance. The vertical axis represents the genetic distance, and the abscissa axis represents the geographical distance.