Simple Sequence Repeat Markers Research Articles

Empirical phenotyping and genome-wide association study reveal the association of panicle architecture with yield in Chenopodium quinoa.

Chenopodium quinoa manifests adaptability to grow under varying agro-climatic scenarios. Assessing quinoa germplasm's phenotypic and genetic variability is a prerequisite for introducing it as a potential candidate in cropping systems. Adaptability is the basic outcome of ecological genomics of crop plants. Adaptive variation predicted with a genome-wide association study provides a valuable basis for marker-assisted breeding. Hence, a panel of 72 quinoa plants was phenotyped for agro morphological attributes and association-mapping for distinct imperative agronomic traits. Inter simple sequence repeat (ISSR) markers were employed to assess genetic relatedness and population structure. Heatmap analysis showed three genotypes were early maturing, and six genotypes were attributed for highest yield. The SD-121-07 exhibited highest yield per plant possessing green, glomerulate shaped, compact density panicle with less leaves. However, SJrecm-03 yielded less exhibiting pink, intermediate shape, intermediate density panicles with less leaves. The phenotyping revealed strong correlation of panicle architecture with yield in quinoa. A genome-wide association study unraveled the associations between ISSR makers and agro-morphological traits. Mixed linear modes analysis yielded nine markers associated with eight traits at p ≤ 0.01. Moreover, ISSR markers significantly associated with panicle shape and leafiness were also associated with yield per plant. These findings contribute to the provision of authenticity for marker-assisted selection that ultimately would support quinoa breeding programs.

Genetic Diversity and Population Structure of Maize (Zea mays L.) Inbred Lines in Association with Phenotypic and Grain Qualitative Traits Using SSR Genotyping

Maize (Zea mays L.) is an important cereal and is affected by climate change. Therefore, the production of climate-smart maize is urgently needed by preserving diverse genetic backgrounds through the exploration of their genetic diversity. To achieve this, 96 maize inbred lines were used to screen for phenotypic yield-associated traits and grain quality parameters. These traits were studied across two different environments (Anand and Godhra) and polymorphic simple sequence repeat (SSR) markers were employed to investigate the genetic diversity, population structure, and trait-linked association. Genotype–environment interaction (GEI) reveals that most of the phenotypic traits were governed by the genotype itself across the environments, except for plant and ear height, which largely interact with the environment. The genotypic correlation was found to be positive and significant among protein, lysine and tryptophan content. Similarly, yield-attributing traits like ear girth, kernel rows ear−1, kernels row−1 and number of kernels ear−1 were strongly correlated to each other. Pair-wise genetic distance ranged from 0.0983 (1820194/T1 and 1820192/4-20) to 0.7377 (IGI-1101 and 1820168/T1). The SSRs can discriminate the maize population into three distinct groups and shortlisted two genotypes (IGI-1101 and 1820168/T1) as highly diverse lines. Out of the studied 136 SSRs, 61 were polymorphic to amplify a total of 131 alleles (2–3 per loci) with 0.46 average gene diversity. The Polymorphism Information Content (PIC) ranged from 0.24 (umc1578) to 0.58 (umc2252). Similarly, population structure analysis revealed three distinct groups with 19.79% admixture among the genotypes. Genome-wide scanning through a mixed linear model identifies the stable association of the markers umc2038, umc2050 and umc2296 with protein, umc2296 and umc2252 with tryptophan, and umc1535 and umc1303 with total soluble sugar. The obtained maize lines and SSRs can be utilized in future maize breeding programs in relation to other trait characterizations, developments, and subsequent molecular breeding performances for trait introgression into elite genotypes.

Genetic variability and association study of SSR markers for yield and powdery mildew disease in pea (Pisum sativum)

Makhyatmubi, one of the most popular local varieties of field pea (Pisum sativum L.) in Manipur has morpho- physiological bottlenecks such as tall nature and high susceptibility to fungus Erysiphe pisi causing powdery mildew disease. Therefore, an experiment was conducted during winter (rabi) seasons of 2018–19 and 2020–21 at College of Post-Graduate Studies in Agricultural Sciences (Central Agricultural University, Imphal, Manipur), Umiam, Meghalaya to generate genetic variability by hybridization of Makhyatmubi with Prakash variety which is a well-known powdery mildew resistant variety in India and possesses several contrasting characters. Fruitful heterosis was estimated with highest record in number of pods per plant (114.75%) and seed yield per plant (108.04%). These two characters also showed high PCV, GCV, heritability and GAM. Number of pods per plant (0.95) was found to have highest positive significant correlation with seed yield per plant. MP-5 and MP-119 recorded superior performance in several characters under study. For the association study of simple sequence repeats (SSR) markers, easily scorable polymorphic markers were selected for surveying association with the traits. Results revealed that SSR marker AA122 may be associated with seed yield and number of pods per plant while marker AA136 with number of seeds per pod. A5 marker was found to be associated with the powdery mildew disease. Hence, it can be employed for marker assisted selection (MAS) to identify and select pea genotypes exhibit resistance to powdery mildew disease.

Assessment of phenotypic and genotypic diversity in rice for salinity tolerance at reproductive stage

Soil salinity has turned out to be an important global concern, which affects tenable rice production in many agricultural countries like Bangladesh. A cross was made between Binadhan-7 with FL-478 for developing salt tolerant advance lines to assess phenotypic and genotypic diversity in rice for salinity tolerance. Twenty advance rice lines (Binadhan-7/FL-478) along with check varieties were screened for salinity tolerance at reproductive stage using sea site saline water having electrical conductivity (EC) 10 dS m-1. Among the twenty rice lines, seven lines were tolerant, three were moderately tolerant, six were susceptible, four were highly susceptible on the basis of phenotypic evaluation. Seven simple sequence repeat (SSR) markers linked with salt tolerance quantitative trait loci were used for investigation of salt tolerant rice lines. The result revealed that an average number of 6.286 alleles per locus were detected, with polymorphism information content (PIC) values ranging from 0.672 (RM490) to 0.838 (RM562). The highest gene diversity value (0.812) was found in RM562 and the lowest (0.684) was in RM490. A dendrogram constructed from the genetic distance of the genotypes produced four distinct clusters of twenty rice genotypes. Considering both phenotypic and genotypic observation, seven genotypes viz., Binadhan-10, FL-478, SL-51, SL-56, SL-77, EFSD-59 and IZSD-45 were identified as salt-tolerant; on the other hand, EFSD-21, SL-28, SL-32, SL-10, BRRI dhan28, and Binadhan-7 were identified as salt-susceptible. The identified salt-tolerant rice genotypes could be used in the improvement of rice breeding programs. Bangladesh J. Nuclear Agric, 37(2): 1-18, 2023

Genetic linkage map construction and QTL analysis for plant height in proso millet (Panicum miliaceum L.).

A genetic linkage map representing proso millet genome was constructed with SSR markers, and a major QTL corresponding to plant height was mapped on chromosome 14 of this map. Proso millet (Panicum miliaceum L.) has the lowest water requirements of all cultivated cereal crops. However, the lack of a genetic map and the paucity of genomic resources for this species have limited the utility of proso millet for detailed genetic studies and hampered genetic improvement programs. In this study, 97,317 simple sequence repeat (SSR) markers were developed based on the genome sequence of the proso millet landrace Longmi 4. Using some of these markers in conjunction with previously identified SSRs, an SSR-based linkage map for proso millet was successfully constructed using a large mapping population (316 F2 offspring). In total, 186 SSR markers were assigned to 18 linkage groups corresponding to the haploid chromosomes. The constructed map had a total length of 3033.42 centimorgan (cM) covering 78.17% of the assembled reference genome. The length of the 18 linkage groups ranged from 88.89cM (Chr. 15) to 274.82cM (Chr. 16), with an average size of 168.17cM. To our knowledge, this is the first genetic linkage map for proso millet based on SSR markers. Plant height is one of the most important traits in crop improvement. A major QTL was repeatedly detected in different environments, explaining 8.70-24.50% of the plant height variations. A candidate gene affecting auxin biosynthesis and transport, and ROS homeostasis regulation was predicted. Thus, the linkage map and QTL analysis provided herein will promote the development of gene mining and molecular breeding in proso millet.

Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

Application of SSR markers for assessment of genetic similarity and genotype identification in local winter wheat breeding program

Background. Simple sequence repeat (SSR) markers are widely used for genetic analysis in plant breeding, allowing for the investigation of genetic divergence and similarity of genotypes, identification of unique alleles and determination of levels of genetic diversity. Materials and Methods. Analysis of 42 wheat cultivars and lines from the bree­ding program of Poltava State Agrarian University was carried out using 11 SSR mar­kers located on different chromosomes. A set of 11 microsatellite single locus primer pairs was used in this study (Xgwm 11, Xgwm 44, Xgwm 46, Xgwm 135, Xgwm 174, Xgwm 186, Xgwm 194, Xgwm 219, Xgwm 312, Xgwm 372, Xgwm 389). Amplification of 11 loci was performed using the Kapa2G FastHotStart PCR Kit (Kapa Biosystems, Boston, USA). The mixture for PCR amplification contained 1.5 x Kapa2G buffer, 0.5 mM dNTP mix, 0.5 μM of each primer (Sigma-Aldrich), 1 unit of Kapa2G FastHotStart DNA Polymerase and 11.8 ng of template DNA in a volume of 25 μl. Fragment lengths were determined using GeneMapper 4.0 software (Applied Biosystems). Dendrogram was constructed using UPGMA (unweighted pair-group method with arithmetic average) in DarWin 6.0 software (Perrier and Jacquemoud-Collet 2006) for clustering analysis. Results and Discussion. The number of alleles detected per locus varied from 5 (Xgwm 11, Xgwm 135, Xgwm 219) to 12 (Xgwm 174). A total of 80 alleles were identified for the 11 loci studied. Among these, 25 unique alleles were found, each of which was present in only one genotype. The polimorphism information content (PIC) values ranged from 0.48 to 0.87. The markers Xgwm 174 (PIC = 0.87), Xgwm 389 (PIC = 0.84) and Xgwm 372 (PIC = 0.83) were the most polymorphic in our study. We obtained a distribution of cultivars and lines by genetic similarity into five clusters. Conclusion. The use of SSR markers made it possible to identify rare alleles within the varieties presented. The study of the genetic similarity of the presented geno­types showed their relationship according to their origin. It was shown that unique alleles tended to occur in certain local breeding genotypes. This study has shown that genotypes representing the local Ukrainian breeding program often have the same allelic variants and at the same time some genotypes have unique allelic variants. The results obtained from the study of 42 winter wheat genotypes based on 11 SSR markers showed that molecular markers can be very useful in assessing genetic similarity and identifying genotypes in the local breeding program.

Identification and transfer of resistance to Fusarium head blight from Elymus repens chromosome arm 7StL into wheat

Fusarium head blight (FHB), mainly caused by Fusarium graminearum (Fg), is one of the most devastating fungal diseases in wheat production worldwide. Elymus repens (2n=6x=42, StStStStHH) is a wild relative of wheat with many biotic and abiotic stress resistance traits. To transfer and apply the wild germplasm's resistance gene (s) for wheat breeding, we identified a new translocation line K140-7 with high resistance to FHB, developed from the derivative progenies of E. repens crossed with common wheat cultivars. Cytogenetic analyses based on genomic in situ hybridization (GISH), non-denaturing fluorescence in situ hybridization (ND-FISH), oligonucleotide-FISH painting (Oligo-FISH painting), and single-gene FISH revealed that K140-7 had 40 wheat chromosomes and two 7DS·7StL translocated chromosomes. Wheat 55K SNP array analysis confirmed that the translocated breakpoint (340.8~342.5 Mb) was close to the centromere region of chromosome 7D (336.3~341.7 Mb), supporting the 7DS·7StL translocation event. Based on the diploid reference St genome of Pseudoroegneria libanotica, we developed 21 simple sequence repeats (SSR) markers, specific for chromosome arm 7StL. Genotyping and phenotyping analysis of the 7DS·7StL translocation in different wheat backgrounds demonstrated that the chromosome arm 7StL confers FHB resistance and possesses the dominant FHB resistance locus (s) named QFhb.Er-7StL. We further transferred QFhb.Er-7StL into three different wheat cultivars, their second 7DS·7StL translocation line-generations showed improved agronomic traits, representing new germplasms that could be used in wheat FHB-resistant breeding programs.

HUBUNGAN POSISI APOKOL DALAM PERKECAMBAHAN AREN (Arenga pinnata Merr.) DENGAN PERTUMBUHAN KECAMBAH DAN KERAGAMAN GENETIK

Sugar palm seed germination does indicate by the appearance of a tissue that extends like a sponge called apokol which is penetrating the hard seed coat and can occur in several positions in germinated sugar palm seeds. No research reports discuss the relationship between this apokol position and the phenology of sugar palm seed germination. This research aims to study the relationship between apokol position and the growth of sugar palm seedlings and their genetic diversity. The research applies Experiment in Completely Random Design with four treatments, each repeated five times. The four treatments are the apokol position, namely A: Apokol in right-center, B: Apokol in left-center, C: Apokol in left-bottom, and D: Apokol in right-bottom. The results showed that the fastest time for the coleoptile emergence was 40 days after transplanting in the right-center apokol position. Morphologically, there was no significant difference in the sugar palm seedling growth with each apokol position. On the other hand, we found that there were genetic diversities among them revealed by Simple Sequence Repeat (SSR) markers. The palm seeds with the right-center apokol position included the first cluster, and the second cluster consisted of left-center apokol, right- bottom apokol, and left-bottom apokol.

Association mapping analysis for cultivated and weedy types of Perilla crop collected from South Korea using morphological characteristics and SSR markers

There are two cultivated and weedy types of Perilla crop (TCWTPC), and they are widely distributed and cultivated in East Asia, especially in South Korea and Japan. The objective of this study is to create simple sequence repeat (SSR) markers linked to morphological traits that show differences between accessions of the TCWTPC using recently designed SSR primer sets in Perilla crop. Genetic diversity within 52 accessions of the TCWTPC, gathered from South Korea, was assessed using 28 novel Perilla SSR primer sets. Based on the assessment, a collection of 28 Perilla SSR primer sets were shown to exhibit polymorphism and yielded a total of 142 alleles across the 52 accessions of the TCWTPC. Through inspection of a phylogenetic tree and population structure, the 52 accessions of the TCWTPC were classified into three major groups. Although most accessions of the TCWTPC were relatively clearly distinguished, SSR markers failed to distinguish several accessions belonging to the two weedy types of the Perilla crop. By using an association mapping analysis (AMA) of the 28 Perilla SSR markers and seven morphological characteristics in the 52 TCWTPC accessions, we detected that three of the Perilla SSR markers (KNUPF134, KNUPF137, KNUPF149) were associated with plant and seed characteristics. The novel SSR primer sets developed in Perilla crop should be useful in AMA for assessing genetic diversity and relationships between and within TCWTPC accessions, and this information will be helpful for genetic mapping in breeding programs for Perilla crop.

Unravelling the novel genetic diversity and marker-trait associations of corn leaf aphid resistance in wheat using microsatellite markers.

The study was conducted to identify novel simple sequence repeat (SSR) markers associated with resistance to corn aphid (CLA), Rhopalosiphum maidis L. in 48 selected bread wheat (Triticum aestivum L.) and wild wheat (Aegilops spp. & T. dicoccoides) genotypes during two consecutive cropping seasons (2018-19 and 2019-20). A total of 51 polymorphic markers containing 143 alleles were used for the analysis. The frequency of the major allele ranged from 0.552 (Xgwm113) to 0.938 (Xcfd45, Xgwm194 and Xgwm526), with a mean of 0.731. Gene diversity ranged from 0.116 (Xgwm526) to 0.489 (Xgwm113), with a mean of 0.354. The polymorphic information content (PIC) value for the SSR markers ranged from 0.107 (Xgwm526) to 0.370 (Xgwm113) with a mean of 0.282. The results of the STRUCTURE analysis revealed the presence of four main subgroups in the populations. Analysis of molecular variance (AMOVA) showed that the between-group difference was around 37 per cent of the total variation contributed to the diversity by the whole germplasm, while 63 per cent of the variation was attributed between individuals within the group. A general linear model (GLM) was used to identify marker-trait associations, which detected a total of 23 and 27 significant new marker-trait associations (MTAs) at the p < 0.01 significance level during the 2018-19 and 2019-20 crop seasons, respectively. The findings of this study have important implications for the identification of molecular markers associated with CLA resistance. These markers can increase the accuracy and efficiency of aphid-resistant germplasm selection, ultimately facilitating the transfer of resistance traits to desirable wheat genotypes.

New linkage maps for the Cupressaceae species Chamaecyparis obtusa and Thujopsis dolabrata var. hondae and comparison with the linkage map of Cryptomeria japonica

ABSTRACT Comparative mapping studies, which can determine whether the genome structure differs among genera, have yet to be conducted in Cupressaceae. This study analyzed three important forestry species in Japan: Cryptomeria japonica, Chamaecyparis obtusa, and Thujopsis dolabrata var. hondae. To date, linkage maps have only been constructed for C. japonica. We constructed the first linkage maps for C. obtusa and T. dolabrata var. hondae using double digest restriction-site associated DNA sequencing (ddRAD-Seq) data, expressed sequence tag-derived simple sequence repeat (EST-SSR) markers, and single-nucleotide polymorphism (SNP) markers designed from homologous unigenes obtained from RNA-seq data. The lengths of the linkage maps were 1,835 cM (1,981 markers) for C. obtusa and 1,833 cM (1,295 markers) for T. dolabrata var. hondae. A total of 11 distinct linkage groups were identified in these maps, corresponding to the haploid number of Cupressaceae. Using four published linkage maps for C. japonica, we then constructed a composite linkage map with 7,784 markers. Finally, the three linkage maps were compared using 207 SNP markers designed from homologous unigenes. Although most markers were mapped to the same LG despite representing different species, indicating strong synteny. The high level of interspecific and intergeneric synteny and macro-collinearity detected in Cupressaceae were similar to those of other trees. We also detected three major chromosomal rearrangements between but not within subfamilies, which may have been caused by the taxonomic distance. This is the first example of comparative mapping for Cupressaceae species.

Unravelling genetic diversity and population structure of Sarocladium oryzae causing sheath rot disease in rice using hyper-variable SSR markers

Rice sheath rot, caused by the fungus Sarocladium oryzae, poses a major threat to rice crops. This pathogen exhibits substantial variability in the field, yet limited information on its genetic diversity and population structure hampers the development of effective disease management strategies. In this study, hyper-variable Simple Sequence Repeats (HV-SSR) markers were employed for the first time to assess the genetic diversity of Sarocladium oryzae, collected from various rice cultivating regions in Odisha, Eastern India. The isolates were identified using both morphological and molecular criteria. A pathogenicity test was performed for all the isolates. A total of 2558 HV-SSRs with more than 10 repeat units were identified in the genome of S. oryzae. The most prevalent types of repeats were dinucleotides (26.8%), followed by trinucleotides (22.8%), tetranucleotides (8.67%), pentanucleotides (2.83%), and hexanucleotides (1.83%). A total of 150 HV-SSR primers were designed and 24 were randomly selected and validated, of which ten of them demonstrating polymorphism in the 87 isolates. These ten polymorphic hyper-variable SSR markers were subsequently used to assess the genetic diversity and population structure of Sarocladium oryzae isolates. The markers revealed a total of 32 alleles, with the number of alleles per locus varying from 2 to 4. Gene diversity ranged from 0.409 to 0.641, while the major allele frequency varied from 0.448 to 0.736. The Polymorphic Information Content (PIC) values ranged from 0.325 to 0.569, with an average of 0.457. Cluster analysis categorized the sheath rot isolates into three distinct major clusters. Principal coordinate analysis and structure analysis identified two genetic clusters within S. oryzae isolates. Analysis of molecular variance indicated that there was more genetic variation within populations (55%) and less variation among populations (45%). The findings of this study provide insights into the genetic diversity of S. oryzae in the rice-cultivating regions of Odisha, which can be used to develop more effective disease management strategies.

The sacred deer conflict of management after a 1000‐year history: Hunting in the name of conservation or loss of their genetic identity

AbstractHuman–wildlife conflict involves diverse stakeholders with conflicting values. Resolving such conflicts necessitates the development of management plans rooted in scientific knowledge and establishing of social consensus. In Nara City, situated within the Japanese Archipelago, wild sika deer (Cervus nippon) hold sacred significance due to religious reasons and have been protected for over a millennium, resulting in a distinct genetic identity. However, the escalating deer population has caused significant agricultural damage in the areas surrounding the sanctuary. Consequently, a debate has arisen regarding the advantages and disadvantages of implementing lethal measures to address individuals that might be considered sacred deer but are regarded as pests in the vicinity of the sanctuary. Here, we used mitochondrial DNA (mtDNA) control region and nuclear simple sequence repeat (SSR) markers to detect the origin of deer in the areas neighboring the sanctuary (management areas). As a result, two genetic populations of deer were detected in Nara City. In the sanctuary, we detected only one specific mtDNA haplotype (S4). On the other hand, seven haplotypes, including S4, were detected in the management area. SSR analysis also suggested that the sika deer in the management area may be an admixed population of multiple origins from the sanctuary and out of Nara City. Interbreeding populations may expand into the sanctuary, and unique genetic populations for more than 1000 years may disappear. This study suggests that ordinary deer could soon replace the deer revered and protected by the people of Nara. Additionally, the proximity of sanctuary deer to tourists worldwide and the interaction with wild deer in other areas pose a potential risk of spreading zoonotic diseases. Urgent decisions are required to determine whether to advocate for extermination in managed areas for ‘conservation’ purposes or risk losing the genetic identity of the sacred deer.

Molecular Profiling for Identification of Zea mays L. True Hybrids

Background: Zea mays (L.), commonly known as maize or corn, is a diploid (2n= 2x= 20) grain crop which belongs to the family Poaceae. It is important to protect and prevent unauthorized commercial usage of hybrids at large scale, which can be best done through molecular profiling. The present study was operated with the purpose to obtain specific simple sequence repeat (SSR) markers which can identify maize hybrids from their parental lines. Methods: For this work, seven maize hybrids viz. HM 5, HM 8, HM 9, HM 10, HM 11, HM 12 and HM 13 along with their ten respective parents were used. A total of 45 SSR primers, well distributed on all the 10 chromosomes of maize were used for polymerase chain reaction amplification. Result: Molecular study using SSRs resulted in 12 polymorphic and 24 monomorphic markers. Three SSR primers viz. pumc2246, pumc1020 and pumc1040 could clearly distinguish different hybrids and their parents. The study will be useful in detecting unwanted seed mixing with the hybrid seeds.

Spatial Population Structuring and Genetic Analysis of Exotic Grevillea robusta in Northwestern India

Abstract The introduction of exotics is common in forestry, and majorly important species, like eucalypts, poplar, and Casuarina sp., occupy vast areas in the Indian subcontinent. Importantly, with the latest cost-effective sequencing techniques, genetic diversity research through molecular marker approaches on major exotics provides novel information for genetic improvement in economic traits with climatic adaptation. The study was carried out on Grevillea robusta to assess genetic relatedness and diversity among 228 genotypes belonging to five northwestern states in India. These genotypes were assayed using twelve simple sequence repeat (SSR) markers. A population structure analysis using structure software identified four major gene pool subgroups with clear-cut differences from each other. Principal coordinate analysis also supports the clustering patterns of the unweighted pair group method with arithmetic mean analysis. In the case of genetic diversity assessment, a total of seventy-three alleles were detected from twelve SSRs, with an average of 6.08 ± 0.71 per locus. Polymorphism information content ranged from 0.17 to 0.67 with a mean of 0.44 ± 0.045, indicating high levels of polymorphism across the genotypes. All the evaluated SSRs demonstrated moderate genetic diversity (observed heterozygosity = 0.31 ± 0.03; expected heterozygosity = 0.32 ± 0.03; and genetic differentiation = 0.295) among the sampled genotypes. These findings indicate significant genetic variability in the germplasm to warrant selection and have potential for a long-term tree improvement program of G. robusta in India. Study Implications: The study on population genetics of Grevillea robusta in exotic environments assessed probable gene pools, provenances, and genetic diversity in geographical distribution range of the species in the Indian scenario. The basic population genetic measures revealed the high diversity areas and probable seed zones of G. robusta in its exotic range. Importantly, the study will be helpful in the selection of candidate plus trees of G. robusta to further strengthen the genotype × environment interaction study for future breeding programs. Further, studying the genetic makeup for moderately diverse species offers valuable insights into evolutionary history, population dynamics, and ecological interactions, which may lead to conservation efforts and guidance for the management of trees such as G. robusta in their native and introduced regions.

Development of novel SSR markers and validation by assessing the genetic diversity of endangered Deccan mahseer, Tor khudree.

The Deccan mahseer, Tor khudree (Sykes, 1839) is a potential game and food fish species belonging to the family cyprinidae and is categorized as endangered. Its distribution is restricted to southern part of India, specifically to Peninsular Rivers. This study is first to assess the genetic diversity and differentiation in Tor khudree by developing novel simple sequence repeat (SSR) markers. Low depth next generation sequencing followed by sequence analysis in MISA software identified 187,649 SSRs. The novel fourteen validated SSR loci were used for population genetic analysis. All of the SSR loci were highly informative with mean PIC > 0.5. High mean allelic richness (9.29) observed heterozygosity (0.98) and expected heterozygosity (0.79) were observed across the loci. However, genetic differentiation was low but significant (0.052). Negative FIS values were observed in both locus-wise and populations indicating the presence of high heterozygosity. Intrapopulation variation was found to be high (96.29%). The population structure revealed two genetic stocks. The results from the present study including the highly polymorphic markers developed would be a useful resource for further research on population genetics and conservation genetics of the species.

Cross-Species Transferability of SSR Markers for Analyzing Genetic Diversity of Different Vicia species Collections

Legumes play an essential role in sustainable agriculture due to their ability to fix nitrogen and high protein content. Vicia is a relevant genus of the Fabaceae family that includes important crop species, such as V. faba and V. sativa, but also other species considered minor crops. They are mainly used as animal feed and usually cope resiliently with extreme conditions; therefore, they could play an essential role in sustainable agriculture under the present scenario of climate change and growing population. However, the scarcity of commercial cultivars limits their use. The Spanish National Plant Genetic Resources Center has collections of several species of the genus Vicia, including international landraces, which could be an essential source of biodiversity for breeding programs. These seed resources are underutilized due to the limited availability of characterization data, including the lack of molecular markers for these species. In this study, we analyzed the cross-transferability of SSR (simple sequence repeat) molecular markers from V. sativa and V. ervilia to distinct species of the genus Vicia. We also used heterologous validated markers for the genotypic characterization and genetic diversity analysis of almost 500 accessions of three undercharacterized Vicia collections: V. articulata (one-flower vetch), V. ervilia (bitter vetch), and V. narbonensis (narbon bean or French vetch). Subsequently, these molecular data were integrated with passport and agromorphological data to select representative varieties from these Vicia collections and establish core collections, with minimum loss of genetic diversity versus the Spanish total collections. The characterization of these legume collections is an essential step from an economic and ecological point of view to obtain selected Vicia varieties to be used in sustainable agriculture.

Effect of Cucurbit Host, Production Region, and Season on the Population Structure of Pseudoperonospora cubensis in Florida.

Pseudoperonospora cubensis, the causal agent of Cucurbit downy mildew (CDM), is one of the most important diseases affecting cucurbit production in the United States. This disease is especially damaging to Florida production areas, as the state is a top producer of many cucurbit species. In addition, winter production in central and south Florida likely serves as a likely source of P. cubensis inoculum for spring and summer cucurbit production throughout the eastern United States, where CDM is unable to overwinter in the absence of a living host. Over 2 years (2017 and 2018) and four seasons (spring 2017, spring 2018, fall 2017, and fall 2018), 274 P. cubensis isolates were collected from cucurbit hosts at production sites in south, central, and north Florida. The isolates were analyzed with 10 simple sequence repeat (SSR) markers to establish population structure and genetic diversity and further assigned to a clade based on a qPCR assay. Results of population structure and genetic diversity analyses differentiated isolates based on cucurbit host and clade (1 or 2). Of the isolates assigned to clade by qPCR, butternut squash, watermelon, and zucchini were dominated by clade 1 isolates, whereas cucumber isolates were split 34 and 59% between clades 1 and 2, respectively. Clade assignments agreed with isolate clustering observed within discriminant analysis of principal components (DAPC) based on SSR markers, although watermelon isolates formed a group distinct from the other clade 1 isolates. For seasonal collections from cucumber at each location, isolates were typically skewed to one clade or the other and varied across locations and seasons within each year of the study. This variable population structure of cucumber isolates could have consequences for regional disease management. This is the first study to characterize P. cubensis populations in Florida and evaluate the effect of cucurbit host and clade-type on isolate diversity and population structure, with implications for CDM management in Florida and other United States cucurbit production areas.

Development and application of SSR markers of Saposhnikovia divaricata based on transcriptome

Transcriptome sequencing was employed to mine the simple sequence repeat(SSR) locus information of Saposhnikovia divaricata and design specific primers, which aimed to provide a basis for the research on the genetic diversity of S. divaricata germplasm resources. The seed purity, 1 000-seed weight, germination rate, and seed vigor were determined. MISA was used to obtain the SSR locus information from 12 606 unigene longer than 1 kb in the transcriptome database. Forty-three pairs of SSR primers designed in Primer 3 were used to analyze the polymorphism of 28 S. divaricata samples of different sources. The results showed that there were differences in the seed purity, 1 000-seed weight, germination rate, vigor, and seed length and width among S. divaricata samples of different sources. Particularly, the germination rate and seed vigor had significant differences, and HB-ZJK1, NMG-CF4, NMG-BT, NMG-HLE1, and NMG-CF2 had significantly higher 1 000-seed weight, germination rate, and seed vigor than the samples of other sources. Among the 86 233 unigene, 12 606(14.62%) unigene contained 15 958 SSR loci, with one SSR locus every 5 009 bp on average. The SSR loci were mainly single nucleotide and dinucleotide repeats, which were dominated by G/C and TC/AG, respectively. All the primers were screened by using 28 S. divaricata sample from different habitats, and the primers corresponding to the amplification products with clear bands and stable polymorphism were obtained. The clustering results of the biological characteristics and genetic diversity of the 28 S. divaricata samples were basically consistent, and the samples of the same origin(HB-AG1, HB-AG2, HB-ZJK1, and HB-ZJK2) generally gathered together and had close genetic relationship. The SSRs in S. divaricata transcriptome has high frequency, rich types, and high polymorphism, which provides candidate molecular markers for the germplasm identification, genetic map construction, and molecular-assisted breeding.