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Genetic diversity of 18 male and 18 female accessions of Jojoba [Simmondsia chinensis (link) Schneider] using EST-SSRs
Meta Gene 19 (2019) 134–141
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Genetic diversity of 18 male and 18 female accessions of Jojoba [Simmondsia chinensis (link) Schneider] using EST-SSRs Swati Agarwal, Suphiya Khan
T
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Department of Bioscience and Biotechnology, Banasthali University, P.O. Banasthali, Vidyapith, Rajasthan 304022, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Simmondsia chinensis EST-SSRs Crop improvement Operational Taxonomic Unit Breeding programmes
Jojoba [Simmondsia chinensis (Link) Schneider] is native shrub of northern Mexico and the southwestern United States, commercially utilized for the liquid wax stored in their seeds. It has polyploidy, dioecious, perennial producing, highly heterozygous individual seeds. That is a fact which creates a great mixing of genes when the pollens are blown long distances for fertilization. Genetic diversity is the main source of variability in any crop improvement program. The present study is aimed at evaluating the genetic variation of 18 male and 18 female jojoba accessions using 22 genome-wide SSR markers. Genotyping of 36 jojoba accessions produced a total of 31 alleles in male and 28 alleles in female accessions with an average value of 0.344 and 0.311 for male and female accessions, respectively. Primer BA00213364 showed highest polymorphism information content (PIC) and BA00213368 showed the highest resolving power (Rp) for both male and female accessions in comparison to other primers. Two accessions “879-154 and 40” showed diverse genetic makeup and are placed in a separate Operational Taxonomic Unit (OTU) in comparison to others. The resultant diverse accessions and polymorphic EST-SSRs in the present study will be used for the identification of economically important traits to be utilized in future molecular breeding programmes of jojoba.
1. Introduction Jojoba, Simmondsia chinensis (Link) Schneider, belongs to family Simmondsiaceae, is a native shrub of Northern Mexico, USA and Baja California (Sherbrooke and Haase, 1974). It is a perennial, low-maintenance, drought-resistant and long-lived plant with a deep rooting system, and therefore it can be used in roadside plantings and highways. The plant has great commercial value as its seed store liquid wax (40–60% by dry weight) (Agrawal et al., 2007). Jojoba oil, commonly known as liquid wax, is colorless and odorless with unique physical and chemical properties (Benzioni and Vaknin, 2002). It is similar to sperm whale oil and can be substituted for it in many applications (Kumar et al., 2010). Besides being known for its lubricating properties, jojoba has also attracted interest towards the cosmetics, pharmaceuticals, animal feeding and landscape as a soil conservation plant (AlHamamre, 2013). Unlike most other vegetable seed oils which are triglycerides, jojoba oil is made of wax esters composed of long-chain fatty acids and fatty alcohols with no side branching (Tobares et al., 2003). This unique chemical configuration accords jojoba special characteristics unparalleled in the plant kingdom (Fig. 1). Jojoba is a polyploid, dioecious, perennial shrub producing highly heterozygous individual seeds (Baldwin, 1988). Jojoba plant is single⁎
sex in nature; either female or male, hermaphrodite plants are rarely found. Fertilization occurs with the help of wind pollination. The pollen grains are blown long distance to fertilize the female plant, causes great mixing of genes in jojoba plants (Baldwin, 1988). Jojoba adapts to harsh desert environments with a broad genetic diversity. The sex ratio of female and male plants is unequal; males are outnumbering females by more than five to one. Consequently, each jojoba seed, even from the same female, has an entirely different combination of genes which are phenotypically expressed in a wide variation of plant size, shape, density and wax yield (Wisniak, 1988). The hybrid breeding method takes about 6 years to grow and evaluate one generation of jojoba seedlings. With the help of molecular marker approach, we can find the most diverse species in short duration and further grow them to produce high-performing accessions. Crop species production mainly depends on the understanding of the organization and extent of genetic variations. Genetic diversity assessment is very necessary for two reasons, first for the better understanding of the genetic relationships among different accessions (Li and Nelson, 2001) and second for the selection of the accession in a more effective and systemic fashion (Paterson et al., 1991). A variety of markers are in trend, to understand the relationships among different accessions such as hybridization-based markers and PCR-based
Corresponding author. E-mail address: [email protected] (S. Khan).
https://doi.org/10.1016/j.mgene.2018.11.010 Received 1 July 2018; Received in revised form 8 November 2018; Accepted 20 November 2018 Available online 22 November 2018 2214-5400/ © 2018 Elsevier B.V. All rights reserved.
Meta Gene 19 (2019) 134–141
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Fig. 1. Mature male (left side) and female (right side) flower of S, chinensis (jojoba).
village called Dhand near Jaipur, Rajasthan India (Geographical coordinates: 26.88° N, 75.76°). The leaves samples were dry cleaned, shade dry and stored for the further experiments (Yadav and Agarwala, 2011).
markers. Major attention has been towards the PCR-based markers where random or specific primers such as RAPD- random amplified polymorphic DNA (Arya et al., 2016), AFLP- amplified fragment length polymorphism (Agarwal et al., 2011), ISSR- inter simple sequence repeats (Heikrujam et al., 2014; Agarwal and Khan, 2018), SSR- simple sequence repeats (Kazminska et al., 2017) and SNP- single nucleotide polymorphism (Liu et al., 2012) are used to amplify genomic DNA. Among PCR-based markers, SSR markers have found widespread application because first, they are co-dominant (Lian et al., 2006), second they are multi-allelic (Wurschum et al., 2013) and lastly they are more informative (Powell et al., 1996) than the dominant type of markers (RAPD and ISSR). SSR markers have high reproducibility, hypervariability, multiallelism, codominant inheritance, extensive genome coverage, chromosome-specific location (Agarwal et al., 2008), and easy automated detection by PCR (Castillo et al., 2010). Therefore, SSR markers are useful and powerful tools for population studies (Adam-Blondon et al., 2004). SSR markers can also facilitate better cross-genome comparisons because they target protein-coding regions that are more likely to be conserved between related species (Scott et al., 2000). Expressed sequence tags (ESTs) based on Sanger's sequencing technology have become increasingly abundant in public DNA databases and are being used for genetic analyses, comparative mapping, DNA fingerprinting, diversity analysis and evolutionary studies (Varshney et al., 2002). In the past few years, several investigations have been carried out on sex-linked marker in jojoba (Agrawal et al., 2007; Sharma et al., 2008; Ince et al., 2010, 2011; Agarwal et al., 2011; Hosseini et al., 2011; Heikrujam et al., 2014a,b) however, there is scant information regarding molecular diversity among different genotypes (Sharma et al., 2009; Bhardwaj et al., 2010; Heikrujam et al., 2015; Arya et al., 2016; Agarwal and Khan, 2018). In the present study, the genetic diversity of 36 accessions (18 male and 18 female) of jojoba are analyzed using 22 EST-SSR markers. The objectives of the study are to evaluate the genetic diversity between male and female accessions and among themselves for a future management and its utilization.
2.2. Chemicals and reagents Molecular grade reagents CTAB (Cetyl Trimethyl Ammonium Bromide), dNTP mix, Agarose, taq DNA polymerase, low range DNA ruler, lambda DNA/EcoRI+HindIII marker, SSR primers were purchased from Banglore Genei Pvt. Ltd. All the analytical grade reagents like tris base, isoamylalcohol, isopropanol, boric acid, EDTA (Ethylene Diamine Tetraacetic Acid), ethidium bromide, were purchased from Himedia AR grade.
3. Methods 3.1. DNA extraction Genomic DNA was extracted using a modified CTAB method (Khan and Sharma, 2010). 1 g of dry leaves of jojoba was homogenized using liquid nitrogen. This homogenized mixture was then transferred in a centrifuge tube each containing 5 ml of pre-warmed CTAB extraction buffer (preheated to 65 °C) and the tube was gently swirled to mix the contents. It was incubated at 65 °C for 1 h in a hot water bath. The tube was allowed to cool for sometime then an equal amount of chloroform:isoamylalcohol (24:1) was added to each tube and was gently but thoroughly mixed for 15 min. The tube was centrifuged for 10 min and upper aqueous layer were separated. 1.5 ml NaCl was added and mixed gently then 0.6 volume of chilled isopropanol was added, mixed and left for 2 h at room temperature. Again the tubes were centrifuged for 10 min at 1000 rpm. The supernatant was discarded carefully and the pellet was washed with 70% ethanol. Finally, the pellet was dissolved in 100–200 μl of TE buffer in each tube. Extracted DNA (10 μl) was mixed with 5 μl of gel loading dye and loaded into (0.8%) agarose gel containing 2 μl of ethidium bromide (1 mg ml−1) in 0.5× TBE buffer and run at constant voltage (50 V) for about 2 h. A λ DNA double digest was included on one side of the gel as a molecular standard. The gel was visualized on UV transilluminator (Bio-Rad, USA), photographed and analyzed through Kodak gel documentation system (Model EAS 290) using Kodak ID Image analysis software.
2. Materials 2.1. Plant material Eighteen male and eighteen female accessions of jojoba leaves (Table 1) were collected in brown paper envelop from jojoba farm named “Association of Rajasthan Jojoba Plantation and Research Project” (AJORP), Rajasthan (India) (Saini, 2008). The collection month was January 2015. AJORP is distributed around 37 ha of area in a 135
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Table 1 Details of accessions of S. chinensis (jojoba) leaf samples. S.R. no.
Accessions name
Collection name
Samples per accession
Origin site
Geographical coordinations
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
879–154 (M) 32 (M) 40 (M) Clone 64 (M) 48–25 (M) Local (M) C8-R23 (M) 82–18 (M) Pipeline 85 (M) Pipeline 96 (M) K-11 (M) Q-104 (M) Q-106 (M) 92 (M) MS (M) 17–22 (M) 58–5 (M) 47–21 (M) 879–154 (F) 32 (F) 40 (F) Clone 64 (F) 48–25 (F) Local (F) C8-R23 (F) 82–18 (F) Pipeline 85 (F) Pipeline 96 (F) K-11 (F) Q-104 (F) Q-106 (F) 92 (F) MS (F) 17–22 (F) 58–5 (F) 47–21 (F)
J1-J7 J8-J15 J16-J22 J23-J27 J28-J33 J34-J41 J42-J47 J48-J55 J56-J59 J60-J68 J69-J73 J74-J79 J80-J83 J84-J91 J92-J97 J98-J105 J106-J110 J111-J116 J117-J123 J124-J128 J129-J136 J137-J141 J142-J149 J150-J159 J160-J165 J166-J171 J172-J179 J180-J83 J184-J196 J197-J203 J204-J209 J210-J216 J217-J221 J222-J227 J228-J236 J237-J243
7 8 7 5 6 8 6 8 5 9 5 6 4 8 6 8 5 6 7 5 8 5 8 10 6 6 8 4 12 7 6 7 5 6 9 7
Israel Israel Israel Israel Israel USA Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel USA Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel
31.04° 31.04° 31.04° 31.04° 31.04° 37.09° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 37.09° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04°
N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N,
34.85° 34.85° 34.85° 34.85° 34.85° 95.71° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 95.71° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85°
E E E E E W E E E E E E E E E E E E E E E E E W E E E E E E E E E E E E
Note: These jojoba accessions having ancestral origin from Israel and USA, and their seeds were collected and used to grow in different parts of India like Jaipur, Bhavnagar and Jodhpur. Further from these places they are collected and maintain at jojoba farm AJORP (Association of Rajasthan for Jojoba Plantation and Research Project) Jaipur Rajasthan, India.
3.2. Sequence resources, microsatellite mining and primer designing
3.4. Statistical analysis
SSR containing EST sequences of jojoba were identified and downloaded from EST database of NCBI. After identification, flanking regions were designed using PRIMER3 software with the help of PERL5 interface module (Rozen and Skaletsky, 2011). Primers were designed with an optimal melting temperature of 60 °C, size at least 30 bp and %GC ≥ 40 and ≤ 80 was also considered (Thiel et al., 2003). A total of 22 pairs of SSR primers were purchased from Sigma Life Sciences, India (Table S1). Primers were tested to standardize for their melting temperature (Tm).
The reproducible bands on agarose gel were scored on the basis of the size (bp) of all individual samples from the tested populations. Matrix was prepared on the basis of reproducible band size in bp and was analyzed by using POPGENE software packages version 1.32 (Yeh et al., 2002). PopGen32 software was used to calculate observed (Na) and effective (Ne) number of alleles (Tanya et al., 2011), Shannon's information index (I) (Lewontin, 1972), observed (Ho) and expected (He) heterozygosity and gene flow (Nm) (Nei, 1973) for all SSR loci. Nei's Genetic Distance matrix was constructed using PopGen32 software for each pair of the population (Nei, 1978). Resolving power (Rp) for each primer was calculated using following formula Rp = 1 – [2 × (0.5 – p)]. The PIC value (polymorphism information content) for all polymorphic SSR loci was calculated using this formula: PIC = 1 – Pi2 (Anderson et al., 1993). On the basis of the prepared matrix, a dendrogram was prepared using POPGENE software packages version 1.32 (Rohlf, 2000). Principal coordinate analysis (PCoA) was performed to check the individual sample distribution in the form of a scatter-plot diagram by using GenAlEx 6.501 software (Peakal and Smouse, 2012).
3.3. PCR amplification for SSR analysis Twent two pairs of SSR markers were used for the characterization of 36 accessions of jojoba plant samples (Table S1). PCR was performed in a 25-μl reaction volume containing 2.5 μl Taq-buffer, 0.2 mM of each dNTP, 0.4 μM of primers, 1.5 U Taq DNA polymerase, and approximately 10 ng DNA. Thermocycler gradient (Primus96) was used for DNA amplification. The PCR was performed with the following program: 94 °C for 4 min, 40 cycles of 94 °C for 1 min, 1 min at particular Tm, 72 °C for 2 min and a final extension step at 72 °C for 10 min. Amplified PCR products were analyzed by using 2.0% agarose gel electrophoresis and visualized with Gel Documentation System (Alphaimager MINI ProteinSimple, Japan), photographed and analyzed through Computar (Model, H6Z0812) using Alpha View 3.4.0.0 analysis tool software.
4. Results 4.1. Primer designing and validation A total of 22 pairs of EST-SSR primers having GC content > 40% 136
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expected heterozygosity (He) was 0.623 and 0.597 for male and female jojoba accessions, respectively (Table 3). The expected heterozygosity (He) and Shannon's information index (I) were found lowest for female (0.597 and 0.999, respectively) and the highest for male (0.623 and 1.074, respectively) jojoba accessions. The detected heterozygosity was found to be higher and Shannon's information index was found similar to that in Heikrujam et al. (2015), where SSR markers also were used to estimate the genetic diversity in jojoba germplasm. The mean observed numbers of alleles (Na) were found 3.44 and 3.11 for male and female jojoba accessions respectively. The mean effective number of alleles (Ne) was 2.75 and 2.66 for male and female jojoba accessions respectively. This shows the similar pattern for the above values found for heterozygosity and Shannon's information index (Table 3). SSR data were used to make pair-wise comparison of the accessions to construct a genetic distance matrix with POPGENE software packages version 1.32. Distance matrix differentiates all the jojoba accessions and indicated a fair range of variability for male (0.32–0.99) and female (0.28–0.97), suggesting a wide genetic base of 18 male and female accessions investigated in the present study. Male jojoba accessions 879–154 and 47–21 were found most diverse to each other (0.99) and accessions C8-R23 and pipeline-85 (0.32) were least diverse (Table S2). Likewise, female jojoba accessions 879–154 and 47–21 were found most diverse (0.97) and accessions 879–154 and 40 were least diverse to each other (Table S3).
were successfully designed using Primer 3.0 software. Approximately 40.9% (09/22) of these pairs were successfully amplified for the 243 samples of 18 male and 18 female accessions of jojoba plants. 4.2. Characteristics of EST-SSRs The 9 pairs of EST-SSRs produced a total of 498 bands (Table S1). Out of the 22 pairs of primers, 2 primers (BA00213396 and BA00213404) did not generated any amplification product, 11 primers generated monomorphic and 9 primers generated polymorphic products. The failure of polymorphism may be due to the unfavorable primer location or the problem in their sequence at the time of synthesis (Nicot et al., 2004). The monomorphic amplification showed by 11 pairs of EST-SSRs may be due to the highly conserved region of the sequences used for the primer design. Polymorphism was shown only by 9 pairs of primers, produced > 2 alleles per pair of primer. The number of alleles produced was varied from 2 to 6 with an average of 3.44 for male and 2–5 with an average of 3.11 for female accessions. The 9 EST-SSRs pairs altogether generated 31 alleles for male and 28 alleles for female accessions. The primer pair named BA00213394 gave a maximum number of 6 alleles. The major allele frequency varied from 0.028 to 0.611 with an average of 0.290 for male and 0.056 to 0.639 with an average of 0.321 for female accessions (Fig. 2). PIC was calculated for each EST-SSR separately for male and female accessions. PIC values ranged as 0.49–0.87 with mean 0.73 (male accessions) and 0.15–0.83 with mean 0.64 (female accessions). The above 9 EST-SSRs were more informative. The higher value of Rp represents the greater capacity to separate different accessions (Prevost and Wilkinson, 1999). The mean Rp value was found for all SSR loci 1.46 and 1.58 for male and female jojoba accessions, respectively. EST-SSR BA00213364 showed highest PIC and BA00213368 showed highest Rp value for both male and female accessions in comparison to other primers (Table 2).
4.4. Cluster analysis Male accessions of jojoba were grouped into two major clusters (Fig. 4a). The first cluster was again divided into two sub-clusters ‘1a’ and ‘1b’. Sub-cluster 1a contains three accessions (879–154, 32, 40), sub-cluster 1b having highest number (thirteen) of accessions which were named, Clone-64, Local, C8-R23, Pipeline 85, 82–18, 58–5, Pipeline 96, 47–21, MS, 40, K-11, Q-104, Q-106 and 17–22. Cluster 2 having only two accessions 48–25 and 92. Out of 18 accessions, only 1 accession was originated from the USA and other 17 accessions were from the Israel. Female accessions of jojoba were also grouped into two major clusters (Fig. 4b). The first cluster was divided into two subclusters ‘1a’ and ‘1b’. Sub-cluster 1a contains three accessions (879–154, Pipeline 96, 40), while sub-cluster 1b contains nine accessions (32, K-11, 47–21, Clone-64, Local, C8-R23, Pipeline 85, 92, 58–5). Cluster 2 has 6 accessions, named as 48–25, Q-104, 82–18, Q-106, MS and 17–22. Two accessions named “879-154 and 40” out of 18 jojoba accessions (male and female) were grouped in separate OTU (Operational Taxonomic Unit) which was sub-cluster 1a, showing less similarity with other accessions. Hence, these accessions (879–154 and 40) had the most distinct and possibly diverse genetic makeup. Diverse genetic makeup helps the breeders to improve existing variety or create a new
4.3. Genetic diversity Different diversity indices (Nm, Fst, Nei, He and Ho) were calculated for the determination of genetic variability in different jojoba accessions (Table 2). The coefficient of genetic differentiation (Fst) was 0.28 and 0.95 for the marker BA00213362 and BA00213370, respectively, with the mean value of 0.65 for male jojoba accessions. For female jojoba accessions, the value of Fst was 0.16 and 0.94 for marker BA00213380 and BA00213370, respectively, with the mean value of 0.58. Fst value which was > 0.5 showed strong genetic drift or population subdivision. The mean value of gene flow (Nm) is 0.12 and 0.18 for male and female jojoba accessions, respectively (Fig. 3). The mean observed heterozygosity (Ho) was found 0.426 and 0.500 for male and female jojoba accessions, respectively, whereas the
Fig. 2. Major allele frequencies of 9 EST-SSRs for (a) male and (b) female accessions; ssr1 = BA00213362, ssr2 = BA00213364, ssr4 = BA00213368, ssr5 = BA00213370, ssr9 = BA00213378, ssr10 = BA00213380, ssr11 = BA00213382, ssr12 = BA00213384 and ssr17 = BA00213394. 137
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Table 2 Primer characterizations of 9 SSR loci of male and female S. chinensis (jojoba) based on a sample of 36 individuals. Male aAccessions Locus
PIC
Rp
Nei
Fst
Nm
PIC
Rp
Nei
Fst
Nm
BA00213362 BA00213364 BA00213368 BA00213370 BA00213378 BA00213380 BA00213382 BA00213384 BA00213394 Mean
0.5833 0.8726 0.6712 0.8672 0.7109 0.4984 0.7818 0.7921 0.8635 0.7379
1.8333 1.2777 2.1111 1.0555 1.6111 1.3888 1.2222 1.2222 1.5 1.4691
0.6235 0.6713 0.6929 0.6373 0.6373 0.4753 0.5679 0.5633 0.7361 0.6228
0.2871 0.7931 0.4788 0.9564 0.6077 0.5325 0.8043 0.8027 0.6604 0.658
0.6207 0.0652 0.2721 0.0114 0.1614 0.2195 0.0608 0.0614 0.1286 0.1299
0.7191 0.8379 0.628 0.7206 0.7232 0.1589 0.4182 0.788 0.7981 0.6436
1.3888 1.4444 2.1111 1.0555 1.5 1.8333 1.5 1.3333 2.0555 1.5802
0.5262 0.6836 0.6466 0.4985 0.6481 0.4985 0.4614 0.6343 0.7762 0.5971
0.6305 0.6749 0.3986 0.9443 0.5714 0.1641 0.4582 0.7372 0.6064 0.5813
0.1465 0.1204 0.3772 0.0148 0.1875 0.2736 0.2956 0.0891 0.1623 0.1801
Where, Nei = Nei's 1973 expected heterozygosity; Fst = Subpopulation within the total population; Nm = Gene flow estimated from Fst = 0.25 (1 – Fst)/Fst; PIC=; Rp = Resolving power 1-(Pi)2.
grouping obtained through the dendrogram (Fig. 5). Spatial representation of the relative genetic distances among the individuals was provided by correspondence analysis, which also determined the consistency of differentiation among populations defined by the cluster analysis. The scattering of male and female jojoba accessions was found similar to the cluster analysis results. PCoA provides a field representation of the variability in 2D or 3D set of axes. The analysis was very useful for inspecting visually the sample similarity since less similar samples will appear distant than highly similar samples. 5. Discussion Jojoba has broad genetic diversity and the main reason behind this is the great mixing of genes. The pollens of the male jojoba trees are scattered for miles by the wind. Only female trees produce seeds (Gentry, 1958). This outbreeding has resulted in highly heterogeneous seeds that provide a wide range of hybrid vigor and fertility. Ironically, the extreme genetic variation that was a major cause of failure in the seed-planted fields of the early jojoba pioneers will also be a key step for developing high yields in the future (Purcell et al., 2000). Several studies have been carried out for genetic diversity analysis in different jojoba cultivars using various marker systems, such as RAPD (Amarger and Mercier, 1995; Arya et al., 2016), ISSR (Sharma et al., 2008; Al- Soqueer Motawei et al., 2012; Agarwal and Khan, 2018), both RAPD and ISSR (Bhardwaj et al., 2012) and SCoT and CBDP (Heikrujam et al., 2015) markers to determine the genetic diversity among different jojoba accessions. The present investigation used ESTSSRs for genetic diversity analysis of different jojoba accessions. Application of specific markers has advantages over the random markers (RAPD and ISSR) as they measure genetic diversity from the genic regions, i.e. functional diversity present in any species (Paliwal et al., 2013). The existing variations in the nature of accessions can be identified using a specific statistical method or combination of methods (Kubik et al., 2009). The estimation of genetic diversity using EST-SSRs was also reported in different oil crops like Jatropha curcas, Glycine max, Brassica napus, Helianthus annuus etc. (Wen et al. 2009; Sun et al., 2008; Diwan and Cregan, 1997; Brown-Guedira et al., 2000; Wang et al., 2006; Hasan et al., 2006; Mandel et al., 2011; Pashley et al., 2006). But there was no previous report present on the use of EST-SSRs on Indian accessions of jojoba for diversity analysis. In this study, a set of 22 highly polymorphic, nuclear, single-locus, and co-dominant SSR markers were selected for jojoba accessions in order to better assess their genetic diversity. 9 pairs of EST-SSRs showed high polymorphism but some of the markers (BA00213366, BA00213372, BA00213386, BA00213388, BA00213396, BA00213400, and BA00213404) showed low polymorphism. This may be due to the relatively lower susceptibility of EST-SSRs containing genic regions for
Fig. 3. UPGMA dendrogram of 36 male and female accessions of S. chinensis (jojoba) calculated from polymorphic SSR data. Table 3 Summary of mean genetic diversity indices of 36 male and female accessions of S. chinensis using 9 EST-SSRs. N
Na
Ne
I
Ho
He
uHe
F
Male accessions Mean 18.000 SE 0.000
3.444 0.377
2.752 0.188
1.074 0.079
0.426 0.088
0.623 0.026
0.642 0.027
0.316 0.137
Female accessions Mean 18.000 SE 0.000
3.111 0.351
2.666 0.274
0.999 0.102
0.500 0.079
0.597 0.035
0.614 0.036
0.152 0.147
Where, N: Sample size; Na: No of allels; Ne: Effective no of alleles; I: Information index; Ho: Observed heterozygosity; He: Expected heterozygosity; uHe: Unbiased expected heterozygosity; F: Fixation index.
variety. Genetic diversity also reduces the incidence of unfavorable inherited traits. The more the parents are diverse; the higher chances of getting a new genetic combination (Whitley et al., 1990). From the combined dendrogram of male and female accessions this separate OTU was also observed (Fig. 4). The above two accessions (879–154 and 40) were also placed distinctly from remaining accessions. The principal coordinate analysis (PCoA) was used to verify the 138
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Fig. 4. UPGMA dendrogram of (a) 18 male and (b) 18 female accessions of S. chinensis (jojoba) calculated from polymorphic SSR data.
Fig. 5. Principal co-ordinate analysis showing spatial distribution of (a) 18 male and (b) 18 female accessions of S. chinensis (jojoba); 1 = 879–154, 2 = 32, 3 = 40, 4 = Clone 64, 5 = 48–25, 6 = Local, 7 = C8-R23, 8 = 82–18, 9 = Pipeline 85, 10 = Pipeline 96, 11 = K-11, 12 = Q-104, 13 = Q-106, 14 = 92, 15 = MS, 16 = 17–22, 17 = 58–5 and 18 = 47–21.
Sivaprakash et al. (2004), the ability of a marker system to resolve genetic variation is directly related to the degree of polymorphism. Presently the relatively very high mean value of PIC (0.73 for male and 0.64) confirmed the indicative and discriminating nature of the selected 9 EST-SSRs. Similar results showing high PIC value (0.80) were reported by Agarwal and Khan (2018) but some other groups, Bhardwaj et al. (2010) and Heikrujam et al. (2015) showed very less value of PIC (0.40 and 0.18, respectively) for different jojoba accessions. It is generally assumed that markers with PICs of > 0.5 are efficient in genotype discrimination and extremely useful for measuring the degree of polymorphism at a given locus (DeWoody et al., 1995). The Nm represents the number of individuals entering into the
mutations. The low mutation rate in genic regions due to the vitality of the function of the gene could be the reason for showing low polymorphism by some genomic markers. The other reasons for showing low polymorphism by markers are the selective forces which made the locus conserved (Eckert and Hile, 2009). Marker BA00213404 showed the highest number of alleles (6 for male and 5 for female) in comparison to other markers. This particular marker “BA00213404” could be useful in assessing genetic diversity and species identification within jojoba accessions. Polymorphic Information Content (PIC), Gene Flow (Nm) and Resolving Power (Rp) values of a primer help in determining their effectiveness in the study of genetic diversity of any plant. According to 139
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To succeed, we must clearly understand the genetic diversity in seedplanted fields is the major contributor to the wide variation in yields and growth habit. Thus, from the results which indicates high genetic diversity of the accessions can be utilized in the parental line selection and hybrid development by utilizing the natural genetic variation that exists in the population.
population in a generation. From the results, the mean Nm was found to be very low (0.12 for male and 0.18 for female jojoba accessions), which suggests that the jojoba accessions have low dispersal or mobility i.e. jojoba populations have high genetic divergence. Male accessions of jojoba show greater divergence in comparison to female accessions. Different markers showed different resolving power (Rp), which could be due to differences in the resolution of targeted different regions of the genome (Souframanien and Gopalakrishna, 2004; Gajera et al., 2010). Previously, several studies were reported on the use of EST-SSRs for genetic diversity analysis in oil crops. Wen et al. (2010) reported a high level of genetic diversity (0.55) and on the other hand Sun et al. (2008) reported very low polymorphism using EST-SSRs for 45 and 58 accessions of J. curcas, respectively. A high level of genetic diversity in jojoba had been reported earlier by Sharma et al. (2009), Bhardwaj et al. (2010) and Heikrujam et al. (2015). The mean expected heterozygosity (He) 0.62 and 0.59, for male and female accessions, respectively whereas, the mean Fst found to be 0.65 and 0.58 for male and female, respectively (Table 3). But the high value of Nm in female accessions ensures that more number of female individuals was exchanged among individuals per generation as the species maintains predominant outbreeding behavior as means of reproduction. The heterozygosity (H) reflects diversity and differentiation among the germplasm collections while Shannon's information index (I) reflects the genetic diversity within and between the populations (Que et al., 2014). The higher the indices, the greater are the genetic diversity. High genetic diversity (Nei) was found in male accessions (0.62) of jojoba in comparison to female (0.59). From all the above genetic differentiation parameters, Fst (population differentiation), Nm (gene flow), Nei (genetic distance) and He (expected heterozygosity) male accessions of jojoba proved to be more genetically diverse as compared to the female accessions of jojoba. One of the other dioceous plant named Pistacia atlantica also showed higher levels of genetic variation in male populations in comparison to female populations due to the higher number and even distribution of male plants than females (Nosrati et al., 2012). The similar interpretation could be drawn for jojoba plant due to its natural male-biased population (5 male: 1 female). On the basis of genetic diversity data, the UPGMA dendrogram was constructed. Dendrogram showed the mixed population of male and female accessions of jojoba (Fig. 4). This showed that there is no such separate cluster formation for the male and female individuals. The separate dendrograms for male and female accessions were prepared on the basis of their individual genetic distance matrix. From Fig. 5, it was clear that male accessions have less genetic diversity in comparison to female ones. For crop improvement through plant breeding program, the study of genetic diversity is very essential. The first step of this procedure involves the screening of germplasm to study its genetic diversity. With increased knowledge on genome, molecular markers could be used with some other markers like morphological, biochemical and quantitative trait for further genetic characterization. There was no previous report available on the development of genome-derived SSR markers for jojoba. Hence, there is an urgent need to develop efficient genetic markers to enrich the marker pool of jojoba, which can be utilized in genetic improvement program through molecular breeding.
Acknowledgment The authors acknowledge the UGC “42-208/2013 (SR)” for providing funding under the major research project titled “Comparative molecular and phytochemical investigation of commercially oil yielding desert plant Simmondsia chinensis (Jojoba),” for this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mgene.2018.11.010. References Adam-Blondon, A.F., Roux, C., Claux, D., Butterlin, G., Merdinoglu, D., This, P., 2004. Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics. Theor. Appl. Genet. 109 (5), 1017–1027. Agarwal, S., Khan, S., 2018. Comparative fatty acid and trace elemental analysis identified the best raw material of jojoba (Simmondsia chinensis) for commercial applications. A. O. A. S. 63 (1), 37–45. Agarwal, M., Shrivastava, N., Padh, H., 2008. Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep. 27 (4), 617–631. Agarwal, M., Shrivastava, N., Padh, H., 2011. Development of sex-linked AFLP markers in Simmondsia chinensis. Plant Breed. 130 (1), 114–116. Agrawal, V., Sharma, K., Gupta, S., Prasad, M., 2007. Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers. Plant Biotechnol. Rep. 1, 207–210. Al- Soqueer Motawei, M.I., Al-Dakhil, M., El-Mergawi, R., Al-Khalifah, N., 2012. Genetic variation and chemical traits of selected new jojoba (Simmondsia chinensis (link) Schneider) genotypes. J. Am. Oil Chem. Soc. 89, 1455–1461. Al-Hamamre, Z., 2013. Jojoba is a possible alternative green fuel for Jordan. Energ. Source. Part B. 8, 1–10. Amarger, V., Mercier, L., 1995. Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider). Biochimie 77 (12), 931–936. Anderson, D.J., Reeve, J., Gomez, J.E.M., Weathers, W.W., Hutson, S., Cunningham, H.V., Bird, D.M., 1993. Sexual size dimorphism and food requirements of nestling birds. Can. J. Zool. 71, 2541–2545. Arya, D., Agarwal, S., Khan, S., 2016. Authentication of different accessions of Simmondsia chinensis (link) Schneider (Jojoba) by DNA fingerprinting and chromatography of its oil. Ind. Crop. Prod. 94, 376–384. Baldwin, A.R., 1988. Propagation of jojoba by stem cuttings. In: Proceedings of 7th International Conference on Jojoba and Its Uses, pp. 80–101. Benzioni, A., Vaknin, Y., 2002. Effect of female and male genotypes and environment on wax composition in jojoba. J. Am. Oil Chem. Soc. 79 (3), 297–302. Bhardwaj, M., Uppal, S., Jain, S., Kharb, P., Dhillon, R., Jain, R.K., 2010. Comparative assessment of ISSR and RAPD marker assays for genetic diversity analysis in jojoba [Simmondsia chinensis (link) Schneider]. J. Plant Biochem. Biotechnol. 19 (2), 255–258. Brown-Guedira, G.L., Thompson, J.A., Nelson, R.L., Warburton, M.L., 2000. Evaluation of genetic diversity of soybean introductions and north American ancestors using RAPD and SSR markers. Crop Sci. 40 (3), 815–823. Castillo, A., Budak, H., Martín, A.C., Dorado, G., Börner, A., Röder, M., Hernandez, P., 2010. Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markers. Annal. Appl. Biol. 156 (3), 347–356. DeWoody, J.A., Honeycutt, R.L., Skow, L.C., 1995. Microsatellite markers in white-tailed deer. J. Hered. 86 (4), 317–319. Diwan, N., Cregan, P.B., 1997. Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean. Theor. Appl. Genet. 95 (5–6), 723–733. Eckert, K.A., Hile, S.E., 2009. Every microsatellite is different: Intrinsic DNA features dictate mutagenesis of common microsatellites present in the human genome. Mol. Carcinog. 48 (4), 379–388. Gajera, B.B., Kumar, N., Singh, A.S., Punvar, B.S., Ravikiran, R., Subhash, N., Jadeja, G.C., 2010. Assessment of genetic diversity in castor (Ricinus communis L.) using RAPD and ISSR markers. Indus. Crops Prod. 32 (3), 491–498. Gentry, H.S., 1958. The natural history of jojoba (Simmondsia chinensis) and its cultural aspects. Econ. Bot. 12, 261–291. Hasan, M., Seyis, F., Badani, A.G., Pons-Kühnemann, J., Friedt, W., Lühs, W., Snowdon, R.J., 2006. Analysis of genetic diversity in the Brassica napus L. gene pool using SSR markers. Genet. Resour. Crop Ev. 53 (4), 793–802. Heikrujam, M., Kumar, J., Agrawal, V., 2015. Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon
6. Conclusion Based on the above results, we identified high genetic variation among male jojoba accessions. Two accessions named “879-154 and 40” formed a separate OTU, showed the most distinct and diverse genetic makeup. The high number of average alleles per locus and the expected gene heterozygosity has indicated the broad genetic base of the collection. The success of jojoba growers and indeed of entire jojoba industry depends on our ability to increase yield to a profitable level. We can attain this level by planting accessions of high-yielding plants. 140
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Genetic diversity of 18 male and 18 female accessions of Jojoba [Simmondsia chinensis (link) Schneider] using EST-SSRs Swati Agarwal, Suphiya Khan
T
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Department of Bioscience and Biotechnology, Banasthali University, P.O. Banasthali, Vidyapith, Rajasthan 304022, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Simmondsia chinensis EST-SSRs Crop improvement Operational Taxonomic Unit Breeding programmes
Jojoba [Simmondsia chinensis (Link) Schneider] is native shrub of northern Mexico and the southwestern United States, commercially utilized for the liquid wax stored in their seeds. It has polyploidy, dioecious, perennial producing, highly heterozygous individual seeds. That is a fact which creates a great mixing of genes when the pollens are blown long distances for fertilization. Genetic diversity is the main source of variability in any crop improvement program. The present study is aimed at evaluating the genetic variation of 18 male and 18 female jojoba accessions using 22 genome-wide SSR markers. Genotyping of 36 jojoba accessions produced a total of 31 alleles in male and 28 alleles in female accessions with an average value of 0.344 and 0.311 for male and female accessions, respectively. Primer BA00213364 showed highest polymorphism information content (PIC) and BA00213368 showed the highest resolving power (Rp) for both male and female accessions in comparison to other primers. Two accessions “879-154 and 40” showed diverse genetic makeup and are placed in a separate Operational Taxonomic Unit (OTU) in comparison to others. The resultant diverse accessions and polymorphic EST-SSRs in the present study will be used for the identification of economically important traits to be utilized in future molecular breeding programmes of jojoba.
1. Introduction Jojoba, Simmondsia chinensis (Link) Schneider, belongs to family Simmondsiaceae, is a native shrub of Northern Mexico, USA and Baja California (Sherbrooke and Haase, 1974). It is a perennial, low-maintenance, drought-resistant and long-lived plant with a deep rooting system, and therefore it can be used in roadside plantings and highways. The plant has great commercial value as its seed store liquid wax (40–60% by dry weight) (Agrawal et al., 2007). Jojoba oil, commonly known as liquid wax, is colorless and odorless with unique physical and chemical properties (Benzioni and Vaknin, 2002). It is similar to sperm whale oil and can be substituted for it in many applications (Kumar et al., 2010). Besides being known for its lubricating properties, jojoba has also attracted interest towards the cosmetics, pharmaceuticals, animal feeding and landscape as a soil conservation plant (AlHamamre, 2013). Unlike most other vegetable seed oils which are triglycerides, jojoba oil is made of wax esters composed of long-chain fatty acids and fatty alcohols with no side branching (Tobares et al., 2003). This unique chemical configuration accords jojoba special characteristics unparalleled in the plant kingdom (Fig. 1). Jojoba is a polyploid, dioecious, perennial shrub producing highly heterozygous individual seeds (Baldwin, 1988). Jojoba plant is single⁎
sex in nature; either female or male, hermaphrodite plants are rarely found. Fertilization occurs with the help of wind pollination. The pollen grains are blown long distance to fertilize the female plant, causes great mixing of genes in jojoba plants (Baldwin, 1988). Jojoba adapts to harsh desert environments with a broad genetic diversity. The sex ratio of female and male plants is unequal; males are outnumbering females by more than five to one. Consequently, each jojoba seed, even from the same female, has an entirely different combination of genes which are phenotypically expressed in a wide variation of plant size, shape, density and wax yield (Wisniak, 1988). The hybrid breeding method takes about 6 years to grow and evaluate one generation of jojoba seedlings. With the help of molecular marker approach, we can find the most diverse species in short duration and further grow them to produce high-performing accessions. Crop species production mainly depends on the understanding of the organization and extent of genetic variations. Genetic diversity assessment is very necessary for two reasons, first for the better understanding of the genetic relationships among different accessions (Li and Nelson, 2001) and second for the selection of the accession in a more effective and systemic fashion (Paterson et al., 1991). A variety of markers are in trend, to understand the relationships among different accessions such as hybridization-based markers and PCR-based
Corresponding author. E-mail address: [email protected] (S. Khan).
https://doi.org/10.1016/j.mgene.2018.11.010 Received 1 July 2018; Received in revised form 8 November 2018; Accepted 20 November 2018 Available online 22 November 2018 2214-5400/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Mature male (left side) and female (right side) flower of S, chinensis (jojoba).
village called Dhand near Jaipur, Rajasthan India (Geographical coordinates: 26.88° N, 75.76°). The leaves samples were dry cleaned, shade dry and stored for the further experiments (Yadav and Agarwala, 2011).
markers. Major attention has been towards the PCR-based markers where random or specific primers such as RAPD- random amplified polymorphic DNA (Arya et al., 2016), AFLP- amplified fragment length polymorphism (Agarwal et al., 2011), ISSR- inter simple sequence repeats (Heikrujam et al., 2014; Agarwal and Khan, 2018), SSR- simple sequence repeats (Kazminska et al., 2017) and SNP- single nucleotide polymorphism (Liu et al., 2012) are used to amplify genomic DNA. Among PCR-based markers, SSR markers have found widespread application because first, they are co-dominant (Lian et al., 2006), second they are multi-allelic (Wurschum et al., 2013) and lastly they are more informative (Powell et al., 1996) than the dominant type of markers (RAPD and ISSR). SSR markers have high reproducibility, hypervariability, multiallelism, codominant inheritance, extensive genome coverage, chromosome-specific location (Agarwal et al., 2008), and easy automated detection by PCR (Castillo et al., 2010). Therefore, SSR markers are useful and powerful tools for population studies (Adam-Blondon et al., 2004). SSR markers can also facilitate better cross-genome comparisons because they target protein-coding regions that are more likely to be conserved between related species (Scott et al., 2000). Expressed sequence tags (ESTs) based on Sanger's sequencing technology have become increasingly abundant in public DNA databases and are being used for genetic analyses, comparative mapping, DNA fingerprinting, diversity analysis and evolutionary studies (Varshney et al., 2002). In the past few years, several investigations have been carried out on sex-linked marker in jojoba (Agrawal et al., 2007; Sharma et al., 2008; Ince et al., 2010, 2011; Agarwal et al., 2011; Hosseini et al., 2011; Heikrujam et al., 2014a,b) however, there is scant information regarding molecular diversity among different genotypes (Sharma et al., 2009; Bhardwaj et al., 2010; Heikrujam et al., 2015; Arya et al., 2016; Agarwal and Khan, 2018). In the present study, the genetic diversity of 36 accessions (18 male and 18 female) of jojoba are analyzed using 22 EST-SSR markers. The objectives of the study are to evaluate the genetic diversity between male and female accessions and among themselves for a future management and its utilization.
2.2. Chemicals and reagents Molecular grade reagents CTAB (Cetyl Trimethyl Ammonium Bromide), dNTP mix, Agarose, taq DNA polymerase, low range DNA ruler, lambda DNA/EcoRI+HindIII marker, SSR primers were purchased from Banglore Genei Pvt. Ltd. All the analytical grade reagents like tris base, isoamylalcohol, isopropanol, boric acid, EDTA (Ethylene Diamine Tetraacetic Acid), ethidium bromide, were purchased from Himedia AR grade.
3. Methods 3.1. DNA extraction Genomic DNA was extracted using a modified CTAB method (Khan and Sharma, 2010). 1 g of dry leaves of jojoba was homogenized using liquid nitrogen. This homogenized mixture was then transferred in a centrifuge tube each containing 5 ml of pre-warmed CTAB extraction buffer (preheated to 65 °C) and the tube was gently swirled to mix the contents. It was incubated at 65 °C for 1 h in a hot water bath. The tube was allowed to cool for sometime then an equal amount of chloroform:isoamylalcohol (24:1) was added to each tube and was gently but thoroughly mixed for 15 min. The tube was centrifuged for 10 min and upper aqueous layer were separated. 1.5 ml NaCl was added and mixed gently then 0.6 volume of chilled isopropanol was added, mixed and left for 2 h at room temperature. Again the tubes were centrifuged for 10 min at 1000 rpm. The supernatant was discarded carefully and the pellet was washed with 70% ethanol. Finally, the pellet was dissolved in 100–200 μl of TE buffer in each tube. Extracted DNA (10 μl) was mixed with 5 μl of gel loading dye and loaded into (0.8%) agarose gel containing 2 μl of ethidium bromide (1 mg ml−1) in 0.5× TBE buffer and run at constant voltage (50 V) for about 2 h. A λ DNA double digest was included on one side of the gel as a molecular standard. The gel was visualized on UV transilluminator (Bio-Rad, USA), photographed and analyzed through Kodak gel documentation system (Model EAS 290) using Kodak ID Image analysis software.
2. Materials 2.1. Plant material Eighteen male and eighteen female accessions of jojoba leaves (Table 1) were collected in brown paper envelop from jojoba farm named “Association of Rajasthan Jojoba Plantation and Research Project” (AJORP), Rajasthan (India) (Saini, 2008). The collection month was January 2015. AJORP is distributed around 37 ha of area in a 135
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Table 1 Details of accessions of S. chinensis (jojoba) leaf samples. S.R. no.
Accessions name
Collection name
Samples per accession
Origin site
Geographical coordinations
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
879–154 (M) 32 (M) 40 (M) Clone 64 (M) 48–25 (M) Local (M) C8-R23 (M) 82–18 (M) Pipeline 85 (M) Pipeline 96 (M) K-11 (M) Q-104 (M) Q-106 (M) 92 (M) MS (M) 17–22 (M) 58–5 (M) 47–21 (M) 879–154 (F) 32 (F) 40 (F) Clone 64 (F) 48–25 (F) Local (F) C8-R23 (F) 82–18 (F) Pipeline 85 (F) Pipeline 96 (F) K-11 (F) Q-104 (F) Q-106 (F) 92 (F) MS (F) 17–22 (F) 58–5 (F) 47–21 (F)
J1-J7 J8-J15 J16-J22 J23-J27 J28-J33 J34-J41 J42-J47 J48-J55 J56-J59 J60-J68 J69-J73 J74-J79 J80-J83 J84-J91 J92-J97 J98-J105 J106-J110 J111-J116 J117-J123 J124-J128 J129-J136 J137-J141 J142-J149 J150-J159 J160-J165 J166-J171 J172-J179 J180-J83 J184-J196 J197-J203 J204-J209 J210-J216 J217-J221 J222-J227 J228-J236 J237-J243
7 8 7 5 6 8 6 8 5 9 5 6 4 8 6 8 5 6 7 5 8 5 8 10 6 6 8 4 12 7 6 7 5 6 9 7
Israel Israel Israel Israel Israel USA Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel USA Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel Israel
31.04° 31.04° 31.04° 31.04° 31.04° 37.09° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 37.09° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04° 31.04°
N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N,
34.85° 34.85° 34.85° 34.85° 34.85° 95.71° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 95.71° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85° 34.85°
E E E E E W E E E E E E E E E E E E E E E E E W E E E E E E E E E E E E
Note: These jojoba accessions having ancestral origin from Israel and USA, and their seeds were collected and used to grow in different parts of India like Jaipur, Bhavnagar and Jodhpur. Further from these places they are collected and maintain at jojoba farm AJORP (Association of Rajasthan for Jojoba Plantation and Research Project) Jaipur Rajasthan, India.
3.2. Sequence resources, microsatellite mining and primer designing
3.4. Statistical analysis
SSR containing EST sequences of jojoba were identified and downloaded from EST database of NCBI. After identification, flanking regions were designed using PRIMER3 software with the help of PERL5 interface module (Rozen and Skaletsky, 2011). Primers were designed with an optimal melting temperature of 60 °C, size at least 30 bp and %GC ≥ 40 and ≤ 80 was also considered (Thiel et al., 2003). A total of 22 pairs of SSR primers were purchased from Sigma Life Sciences, India (Table S1). Primers were tested to standardize for their melting temperature (Tm).
The reproducible bands on agarose gel were scored on the basis of the size (bp) of all individual samples from the tested populations. Matrix was prepared on the basis of reproducible band size in bp and was analyzed by using POPGENE software packages version 1.32 (Yeh et al., 2002). PopGen32 software was used to calculate observed (Na) and effective (Ne) number of alleles (Tanya et al., 2011), Shannon's information index (I) (Lewontin, 1972), observed (Ho) and expected (He) heterozygosity and gene flow (Nm) (Nei, 1973) for all SSR loci. Nei's Genetic Distance matrix was constructed using PopGen32 software for each pair of the population (Nei, 1978). Resolving power (Rp) for each primer was calculated using following formula Rp = 1 – [2 × (0.5 – p)]. The PIC value (polymorphism information content) for all polymorphic SSR loci was calculated using this formula: PIC = 1 – Pi2 (Anderson et al., 1993). On the basis of the prepared matrix, a dendrogram was prepared using POPGENE software packages version 1.32 (Rohlf, 2000). Principal coordinate analysis (PCoA) was performed to check the individual sample distribution in the form of a scatter-plot diagram by using GenAlEx 6.501 software (Peakal and Smouse, 2012).
3.3. PCR amplification for SSR analysis Twent two pairs of SSR markers were used for the characterization of 36 accessions of jojoba plant samples (Table S1). PCR was performed in a 25-μl reaction volume containing 2.5 μl Taq-buffer, 0.2 mM of each dNTP, 0.4 μM of primers, 1.5 U Taq DNA polymerase, and approximately 10 ng DNA. Thermocycler gradient (Primus96) was used for DNA amplification. The PCR was performed with the following program: 94 °C for 4 min, 40 cycles of 94 °C for 1 min, 1 min at particular Tm, 72 °C for 2 min and a final extension step at 72 °C for 10 min. Amplified PCR products were analyzed by using 2.0% agarose gel electrophoresis and visualized with Gel Documentation System (Alphaimager MINI ProteinSimple, Japan), photographed and analyzed through Computar (Model, H6Z0812) using Alpha View 3.4.0.0 analysis tool software.
4. Results 4.1. Primer designing and validation A total of 22 pairs of EST-SSR primers having GC content > 40% 136
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expected heterozygosity (He) was 0.623 and 0.597 for male and female jojoba accessions, respectively (Table 3). The expected heterozygosity (He) and Shannon's information index (I) were found lowest for female (0.597 and 0.999, respectively) and the highest for male (0.623 and 1.074, respectively) jojoba accessions. The detected heterozygosity was found to be higher and Shannon's information index was found similar to that in Heikrujam et al. (2015), where SSR markers also were used to estimate the genetic diversity in jojoba germplasm. The mean observed numbers of alleles (Na) were found 3.44 and 3.11 for male and female jojoba accessions respectively. The mean effective number of alleles (Ne) was 2.75 and 2.66 for male and female jojoba accessions respectively. This shows the similar pattern for the above values found for heterozygosity and Shannon's information index (Table 3). SSR data were used to make pair-wise comparison of the accessions to construct a genetic distance matrix with POPGENE software packages version 1.32. Distance matrix differentiates all the jojoba accessions and indicated a fair range of variability for male (0.32–0.99) and female (0.28–0.97), suggesting a wide genetic base of 18 male and female accessions investigated in the present study. Male jojoba accessions 879–154 and 47–21 were found most diverse to each other (0.99) and accessions C8-R23 and pipeline-85 (0.32) were least diverse (Table S2). Likewise, female jojoba accessions 879–154 and 47–21 were found most diverse (0.97) and accessions 879–154 and 40 were least diverse to each other (Table S3).
were successfully designed using Primer 3.0 software. Approximately 40.9% (09/22) of these pairs were successfully amplified for the 243 samples of 18 male and 18 female accessions of jojoba plants. 4.2. Characteristics of EST-SSRs The 9 pairs of EST-SSRs produced a total of 498 bands (Table S1). Out of the 22 pairs of primers, 2 primers (BA00213396 and BA00213404) did not generated any amplification product, 11 primers generated monomorphic and 9 primers generated polymorphic products. The failure of polymorphism may be due to the unfavorable primer location or the problem in their sequence at the time of synthesis (Nicot et al., 2004). The monomorphic amplification showed by 11 pairs of EST-SSRs may be due to the highly conserved region of the sequences used for the primer design. Polymorphism was shown only by 9 pairs of primers, produced > 2 alleles per pair of primer. The number of alleles produced was varied from 2 to 6 with an average of 3.44 for male and 2–5 with an average of 3.11 for female accessions. The 9 EST-SSRs pairs altogether generated 31 alleles for male and 28 alleles for female accessions. The primer pair named BA00213394 gave a maximum number of 6 alleles. The major allele frequency varied from 0.028 to 0.611 with an average of 0.290 for male and 0.056 to 0.639 with an average of 0.321 for female accessions (Fig. 2). PIC was calculated for each EST-SSR separately for male and female accessions. PIC values ranged as 0.49–0.87 with mean 0.73 (male accessions) and 0.15–0.83 with mean 0.64 (female accessions). The above 9 EST-SSRs were more informative. The higher value of Rp represents the greater capacity to separate different accessions (Prevost and Wilkinson, 1999). The mean Rp value was found for all SSR loci 1.46 and 1.58 for male and female jojoba accessions, respectively. EST-SSR BA00213364 showed highest PIC and BA00213368 showed highest Rp value for both male and female accessions in comparison to other primers (Table 2).
4.4. Cluster analysis Male accessions of jojoba were grouped into two major clusters (Fig. 4a). The first cluster was again divided into two sub-clusters ‘1a’ and ‘1b’. Sub-cluster 1a contains three accessions (879–154, 32, 40), sub-cluster 1b having highest number (thirteen) of accessions which were named, Clone-64, Local, C8-R23, Pipeline 85, 82–18, 58–5, Pipeline 96, 47–21, MS, 40, K-11, Q-104, Q-106 and 17–22. Cluster 2 having only two accessions 48–25 and 92. Out of 18 accessions, only 1 accession was originated from the USA and other 17 accessions were from the Israel. Female accessions of jojoba were also grouped into two major clusters (Fig. 4b). The first cluster was divided into two subclusters ‘1a’ and ‘1b’. Sub-cluster 1a contains three accessions (879–154, Pipeline 96, 40), while sub-cluster 1b contains nine accessions (32, K-11, 47–21, Clone-64, Local, C8-R23, Pipeline 85, 92, 58–5). Cluster 2 has 6 accessions, named as 48–25, Q-104, 82–18, Q-106, MS and 17–22. Two accessions named “879-154 and 40” out of 18 jojoba accessions (male and female) were grouped in separate OTU (Operational Taxonomic Unit) which was sub-cluster 1a, showing less similarity with other accessions. Hence, these accessions (879–154 and 40) had the most distinct and possibly diverse genetic makeup. Diverse genetic makeup helps the breeders to improve existing variety or create a new
4.3. Genetic diversity Different diversity indices (Nm, Fst, Nei, He and Ho) were calculated for the determination of genetic variability in different jojoba accessions (Table 2). The coefficient of genetic differentiation (Fst) was 0.28 and 0.95 for the marker BA00213362 and BA00213370, respectively, with the mean value of 0.65 for male jojoba accessions. For female jojoba accessions, the value of Fst was 0.16 and 0.94 for marker BA00213380 and BA00213370, respectively, with the mean value of 0.58. Fst value which was > 0.5 showed strong genetic drift or population subdivision. The mean value of gene flow (Nm) is 0.12 and 0.18 for male and female jojoba accessions, respectively (Fig. 3). The mean observed heterozygosity (Ho) was found 0.426 and 0.500 for male and female jojoba accessions, respectively, whereas the
Fig. 2. Major allele frequencies of 9 EST-SSRs for (a) male and (b) female accessions; ssr1 = BA00213362, ssr2 = BA00213364, ssr4 = BA00213368, ssr5 = BA00213370, ssr9 = BA00213378, ssr10 = BA00213380, ssr11 = BA00213382, ssr12 = BA00213384 and ssr17 = BA00213394. 137
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Table 2 Primer characterizations of 9 SSR loci of male and female S. chinensis (jojoba) based on a sample of 36 individuals. Male aAccessions Locus
PIC
Rp
Nei
Fst
Nm
PIC
Rp
Nei
Fst
Nm
BA00213362 BA00213364 BA00213368 BA00213370 BA00213378 BA00213380 BA00213382 BA00213384 BA00213394 Mean
0.5833 0.8726 0.6712 0.8672 0.7109 0.4984 0.7818 0.7921 0.8635 0.7379
1.8333 1.2777 2.1111 1.0555 1.6111 1.3888 1.2222 1.2222 1.5 1.4691
0.6235 0.6713 0.6929 0.6373 0.6373 0.4753 0.5679 0.5633 0.7361 0.6228
0.2871 0.7931 0.4788 0.9564 0.6077 0.5325 0.8043 0.8027 0.6604 0.658
0.6207 0.0652 0.2721 0.0114 0.1614 0.2195 0.0608 0.0614 0.1286 0.1299
0.7191 0.8379 0.628 0.7206 0.7232 0.1589 0.4182 0.788 0.7981 0.6436
1.3888 1.4444 2.1111 1.0555 1.5 1.8333 1.5 1.3333 2.0555 1.5802
0.5262 0.6836 0.6466 0.4985 0.6481 0.4985 0.4614 0.6343 0.7762 0.5971
0.6305 0.6749 0.3986 0.9443 0.5714 0.1641 0.4582 0.7372 0.6064 0.5813
0.1465 0.1204 0.3772 0.0148 0.1875 0.2736 0.2956 0.0891 0.1623 0.1801
Where, Nei = Nei's 1973 expected heterozygosity; Fst = Subpopulation within the total population; Nm = Gene flow estimated from Fst = 0.25 (1 – Fst)/Fst; PIC=; Rp = Resolving power 1-(Pi)2.
grouping obtained through the dendrogram (Fig. 5). Spatial representation of the relative genetic distances among the individuals was provided by correspondence analysis, which also determined the consistency of differentiation among populations defined by the cluster analysis. The scattering of male and female jojoba accessions was found similar to the cluster analysis results. PCoA provides a field representation of the variability in 2D or 3D set of axes. The analysis was very useful for inspecting visually the sample similarity since less similar samples will appear distant than highly similar samples. 5. Discussion Jojoba has broad genetic diversity and the main reason behind this is the great mixing of genes. The pollens of the male jojoba trees are scattered for miles by the wind. Only female trees produce seeds (Gentry, 1958). This outbreeding has resulted in highly heterogeneous seeds that provide a wide range of hybrid vigor and fertility. Ironically, the extreme genetic variation that was a major cause of failure in the seed-planted fields of the early jojoba pioneers will also be a key step for developing high yields in the future (Purcell et al., 2000). Several studies have been carried out for genetic diversity analysis in different jojoba cultivars using various marker systems, such as RAPD (Amarger and Mercier, 1995; Arya et al., 2016), ISSR (Sharma et al., 2008; Al- Soqueer Motawei et al., 2012; Agarwal and Khan, 2018), both RAPD and ISSR (Bhardwaj et al., 2012) and SCoT and CBDP (Heikrujam et al., 2015) markers to determine the genetic diversity among different jojoba accessions. The present investigation used ESTSSRs for genetic diversity analysis of different jojoba accessions. Application of specific markers has advantages over the random markers (RAPD and ISSR) as they measure genetic diversity from the genic regions, i.e. functional diversity present in any species (Paliwal et al., 2013). The existing variations in the nature of accessions can be identified using a specific statistical method or combination of methods (Kubik et al., 2009). The estimation of genetic diversity using EST-SSRs was also reported in different oil crops like Jatropha curcas, Glycine max, Brassica napus, Helianthus annuus etc. (Wen et al. 2009; Sun et al., 2008; Diwan and Cregan, 1997; Brown-Guedira et al., 2000; Wang et al., 2006; Hasan et al., 2006; Mandel et al., 2011; Pashley et al., 2006). But there was no previous report present on the use of EST-SSRs on Indian accessions of jojoba for diversity analysis. In this study, a set of 22 highly polymorphic, nuclear, single-locus, and co-dominant SSR markers were selected for jojoba accessions in order to better assess their genetic diversity. 9 pairs of EST-SSRs showed high polymorphism but some of the markers (BA00213366, BA00213372, BA00213386, BA00213388, BA00213396, BA00213400, and BA00213404) showed low polymorphism. This may be due to the relatively lower susceptibility of EST-SSRs containing genic regions for
Fig. 3. UPGMA dendrogram of 36 male and female accessions of S. chinensis (jojoba) calculated from polymorphic SSR data. Table 3 Summary of mean genetic diversity indices of 36 male and female accessions of S. chinensis using 9 EST-SSRs. N
Na
Ne
I
Ho
He
uHe
F
Male accessions Mean 18.000 SE 0.000
3.444 0.377
2.752 0.188
1.074 0.079
0.426 0.088
0.623 0.026
0.642 0.027
0.316 0.137
Female accessions Mean 18.000 SE 0.000
3.111 0.351
2.666 0.274
0.999 0.102
0.500 0.079
0.597 0.035
0.614 0.036
0.152 0.147
Where, N: Sample size; Na: No of allels; Ne: Effective no of alleles; I: Information index; Ho: Observed heterozygosity; He: Expected heterozygosity; uHe: Unbiased expected heterozygosity; F: Fixation index.
variety. Genetic diversity also reduces the incidence of unfavorable inherited traits. The more the parents are diverse; the higher chances of getting a new genetic combination (Whitley et al., 1990). From the combined dendrogram of male and female accessions this separate OTU was also observed (Fig. 4). The above two accessions (879–154 and 40) were also placed distinctly from remaining accessions. The principal coordinate analysis (PCoA) was used to verify the 138
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Fig. 4. UPGMA dendrogram of (a) 18 male and (b) 18 female accessions of S. chinensis (jojoba) calculated from polymorphic SSR data.
Fig. 5. Principal co-ordinate analysis showing spatial distribution of (a) 18 male and (b) 18 female accessions of S. chinensis (jojoba); 1 = 879–154, 2 = 32, 3 = 40, 4 = Clone 64, 5 = 48–25, 6 = Local, 7 = C8-R23, 8 = 82–18, 9 = Pipeline 85, 10 = Pipeline 96, 11 = K-11, 12 = Q-104, 13 = Q-106, 14 = 92, 15 = MS, 16 = 17–22, 17 = 58–5 and 18 = 47–21.
Sivaprakash et al. (2004), the ability of a marker system to resolve genetic variation is directly related to the degree of polymorphism. Presently the relatively very high mean value of PIC (0.73 for male and 0.64) confirmed the indicative and discriminating nature of the selected 9 EST-SSRs. Similar results showing high PIC value (0.80) were reported by Agarwal and Khan (2018) but some other groups, Bhardwaj et al. (2010) and Heikrujam et al. (2015) showed very less value of PIC (0.40 and 0.18, respectively) for different jojoba accessions. It is generally assumed that markers with PICs of > 0.5 are efficient in genotype discrimination and extremely useful for measuring the degree of polymorphism at a given locus (DeWoody et al., 1995). The Nm represents the number of individuals entering into the
mutations. The low mutation rate in genic regions due to the vitality of the function of the gene could be the reason for showing low polymorphism by some genomic markers. The other reasons for showing low polymorphism by markers are the selective forces which made the locus conserved (Eckert and Hile, 2009). Marker BA00213404 showed the highest number of alleles (6 for male and 5 for female) in comparison to other markers. This particular marker “BA00213404” could be useful in assessing genetic diversity and species identification within jojoba accessions. Polymorphic Information Content (PIC), Gene Flow (Nm) and Resolving Power (Rp) values of a primer help in determining their effectiveness in the study of genetic diversity of any plant. According to 139
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To succeed, we must clearly understand the genetic diversity in seedplanted fields is the major contributor to the wide variation in yields and growth habit. Thus, from the results which indicates high genetic diversity of the accessions can be utilized in the parental line selection and hybrid development by utilizing the natural genetic variation that exists in the population.
population in a generation. From the results, the mean Nm was found to be very low (0.12 for male and 0.18 for female jojoba accessions), which suggests that the jojoba accessions have low dispersal or mobility i.e. jojoba populations have high genetic divergence. Male accessions of jojoba show greater divergence in comparison to female accessions. Different markers showed different resolving power (Rp), which could be due to differences in the resolution of targeted different regions of the genome (Souframanien and Gopalakrishna, 2004; Gajera et al., 2010). Previously, several studies were reported on the use of EST-SSRs for genetic diversity analysis in oil crops. Wen et al. (2010) reported a high level of genetic diversity (0.55) and on the other hand Sun et al. (2008) reported very low polymorphism using EST-SSRs for 45 and 58 accessions of J. curcas, respectively. A high level of genetic diversity in jojoba had been reported earlier by Sharma et al. (2009), Bhardwaj et al. (2010) and Heikrujam et al. (2015). The mean expected heterozygosity (He) 0.62 and 0.59, for male and female accessions, respectively whereas, the mean Fst found to be 0.65 and 0.58 for male and female, respectively (Table 3). But the high value of Nm in female accessions ensures that more number of female individuals was exchanged among individuals per generation as the species maintains predominant outbreeding behavior as means of reproduction. The heterozygosity (H) reflects diversity and differentiation among the germplasm collections while Shannon's information index (I) reflects the genetic diversity within and between the populations (Que et al., 2014). The higher the indices, the greater are the genetic diversity. High genetic diversity (Nei) was found in male accessions (0.62) of jojoba in comparison to female (0.59). From all the above genetic differentiation parameters, Fst (population differentiation), Nm (gene flow), Nei (genetic distance) and He (expected heterozygosity) male accessions of jojoba proved to be more genetically diverse as compared to the female accessions of jojoba. One of the other dioceous plant named Pistacia atlantica also showed higher levels of genetic variation in male populations in comparison to female populations due to the higher number and even distribution of male plants than females (Nosrati et al., 2012). The similar interpretation could be drawn for jojoba plant due to its natural male-biased population (5 male: 1 female). On the basis of genetic diversity data, the UPGMA dendrogram was constructed. Dendrogram showed the mixed population of male and female accessions of jojoba (Fig. 4). This showed that there is no such separate cluster formation for the male and female individuals. The separate dendrograms for male and female accessions were prepared on the basis of their individual genetic distance matrix. From Fig. 5, it was clear that male accessions have less genetic diversity in comparison to female ones. For crop improvement through plant breeding program, the study of genetic diversity is very essential. The first step of this procedure involves the screening of germplasm to study its genetic diversity. With increased knowledge on genome, molecular markers could be used with some other markers like morphological, biochemical and quantitative trait for further genetic characterization. There was no previous report available on the development of genome-derived SSR markers for jojoba. Hence, there is an urgent need to develop efficient genetic markers to enrich the marker pool of jojoba, which can be utilized in genetic improvement program through molecular breeding.
Acknowledgment The authors acknowledge the UGC “42-208/2013 (SR)” for providing funding under the major research project titled “Comparative molecular and phytochemical investigation of commercially oil yielding desert plant Simmondsia chinensis (Jojoba),” for this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mgene.2018.11.010. References Adam-Blondon, A.F., Roux, C., Claux, D., Butterlin, G., Merdinoglu, D., This, P., 2004. Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics. Theor. Appl. Genet. 109 (5), 1017–1027. Agarwal, S., Khan, S., 2018. Comparative fatty acid and trace elemental analysis identified the best raw material of jojoba (Simmondsia chinensis) for commercial applications. A. O. A. S. 63 (1), 37–45. Agarwal, M., Shrivastava, N., Padh, H., 2008. Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep. 27 (4), 617–631. Agarwal, M., Shrivastava, N., Padh, H., 2011. Development of sex-linked AFLP markers in Simmondsia chinensis. Plant Breed. 130 (1), 114–116. Agrawal, V., Sharma, K., Gupta, S., Prasad, M., 2007. Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers. Plant Biotechnol. Rep. 1, 207–210. Al- Soqueer Motawei, M.I., Al-Dakhil, M., El-Mergawi, R., Al-Khalifah, N., 2012. Genetic variation and chemical traits of selected new jojoba (Simmondsia chinensis (link) Schneider) genotypes. J. Am. Oil Chem. Soc. 89, 1455–1461. Al-Hamamre, Z., 2013. Jojoba is a possible alternative green fuel for Jordan. Energ. Source. Part B. 8, 1–10. Amarger, V., Mercier, L., 1995. Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider). Biochimie 77 (12), 931–936. Anderson, D.J., Reeve, J., Gomez, J.E.M., Weathers, W.W., Hutson, S., Cunningham, H.V., Bird, D.M., 1993. Sexual size dimorphism and food requirements of nestling birds. Can. J. Zool. 71, 2541–2545. Arya, D., Agarwal, S., Khan, S., 2016. Authentication of different accessions of Simmondsia chinensis (link) Schneider (Jojoba) by DNA fingerprinting and chromatography of its oil. Ind. Crop. Prod. 94, 376–384. Baldwin, A.R., 1988. Propagation of jojoba by stem cuttings. In: Proceedings of 7th International Conference on Jojoba and Its Uses, pp. 80–101. Benzioni, A., Vaknin, Y., 2002. Effect of female and male genotypes and environment on wax composition in jojoba. J. Am. Oil Chem. Soc. 79 (3), 297–302. Bhardwaj, M., Uppal, S., Jain, S., Kharb, P., Dhillon, R., Jain, R.K., 2010. Comparative assessment of ISSR and RAPD marker assays for genetic diversity analysis in jojoba [Simmondsia chinensis (link) Schneider]. J. Plant Biochem. Biotechnol. 19 (2), 255–258. Brown-Guedira, G.L., Thompson, J.A., Nelson, R.L., Warburton, M.L., 2000. Evaluation of genetic diversity of soybean introductions and north American ancestors using RAPD and SSR markers. Crop Sci. 40 (3), 815–823. Castillo, A., Budak, H., Martín, A.C., Dorado, G., Börner, A., Röder, M., Hernandez, P., 2010. Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markers. Annal. Appl. Biol. 156 (3), 347–356. DeWoody, J.A., Honeycutt, R.L., Skow, L.C., 1995. Microsatellite markers in white-tailed deer. J. Hered. 86 (4), 317–319. Diwan, N., Cregan, P.B., 1997. Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean. Theor. Appl. Genet. 95 (5–6), 723–733. Eckert, K.A., Hile, S.E., 2009. Every microsatellite is different: Intrinsic DNA features dictate mutagenesis of common microsatellites present in the human genome. Mol. Carcinog. 48 (4), 379–388. Gajera, B.B., Kumar, N., Singh, A.S., Punvar, B.S., Ravikiran, R., Subhash, N., Jadeja, G.C., 2010. Assessment of genetic diversity in castor (Ricinus communis L.) using RAPD and ISSR markers. Indus. Crops Prod. 32 (3), 491–498. Gentry, H.S., 1958. The natural history of jojoba (Simmondsia chinensis) and its cultural aspects. Econ. Bot. 12, 261–291. Hasan, M., Seyis, F., Badani, A.G., Pons-Kühnemann, J., Friedt, W., Lühs, W., Snowdon, R.J., 2006. Analysis of genetic diversity in the Brassica napus L. gene pool using SSR markers. Genet. Resour. Crop Ev. 53 (4), 793–802. Heikrujam, M., Kumar, J., Agrawal, V., 2015. Genetic diversity analysis among male and female Jojoba genotypes employing gene targeted molecular markers, start codon
6. Conclusion Based on the above results, we identified high genetic variation among male jojoba accessions. Two accessions named “879-154 and 40” formed a separate OTU, showed the most distinct and diverse genetic makeup. The high number of average alleles per locus and the expected gene heterozygosity has indicated the broad genetic base of the collection. The success of jojoba growers and indeed of entire jojoba industry depends on our ability to increase yield to a profitable level. We can attain this level by planting accessions of high-yielding plants. 140
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