Genetic Characterisation of Chestnut Cultivars in Crete

(1) Background and objectives: Cretan chestnut belongs to sweet chestnut (Castanea sativa Mill.) and has been historically associated with the lifestyle of rural communities with great economic importance. However, chestnut genetic resources in Crete have rarely been studied and assessed, while chestnuts are threatened by several anthropogenic factors. This study assessed the genetic variability of the Cretan sweet chestnut using 59 trees corresponding to the four best-known chestnut cultivars (Strovliani, Rogdiani, Koutsakera and Katharokastania). (2) Materials and Methods: The trees were evaluated using seven simple sequence repeat markers (SSRs): three nSSRs and four EST-SSRs. (3) Results: Genomic SSR results revealed notable genetic diversity in terms of expected heterozygosity, level of polymorphism and effective number of alleles. Moreover, in the four chestnut cultivars, twenty-two unique genotypes were identified, deeming each cultivar to be in fact a multiclonal variety. Genetic differentiation among cultivars was relatively low, though highly significant. Four different groups of synonymies were found: two homonymy groups in Katharokastania and Strovliani, six in Rogdiani and eight in Koutsakera. The cluster analysis and PCoA results reveal two main clusters, one corresponding to the Rogdiani cultivar and the other to Katharokastania, while the other two could not be assigned to a particular group. (4) Conclusions: The null hypothesis of single-clone genotype-to-cultivar correspondence was tested and could not be accepted.

Development of Genomic SSR for the Subtropical Hardwood Tree Dalbergia hupeana and Assessment of Their Transferability to Other Related Species

Dalbergia hupeana Hance (D. hupeana) is a precious hardwood tree of the genus Dalbergia. It is one of the few species widely distributed within subtropical areas and is important for timber production and forest restoration. At present, there is little published genetic information on D. hupeana. Therefore, we performed a genome survey using next generation sequencing (NGS) and developed a set of novel genomic SSR (gSSR) markers from the assembled data, and assessed the transferability of these markers to other Dalbergia species in Asia. The results of the genome survey show the genome size of D. hupeana to be about 664 Mb and highly heterozygous. The assembly of sequencing data produced 2,431,997 contigs, and the initial assembly of the NGS data alone resulted in contig N50 of 393 kb with a total of 720 Mb. A total of 127,742 perfect SSR markers were found in the assembled contigs. A total of 37 highly polymorphic and easily genotyped gSSR markers were developed in D. hupeana, while the majority of gSSR markers could be successfully transferred to nine other Dalbergia species in Asia. The transferability rate of gSSR markers was highest in D. balansae, which is more closely related to D. hupeana. Seven gSSR markers were able to be amplified in all tested species. In addition, a preliminary assessment of the genetic diversity of three tree species in the Dalbergia genus suggested a high level of genetic diversity within populations distributed in the subtropical area in China. However, the determination of the global status of their genetic variation still requires further and more comprehensive assessment. Our findings will enable further studies on the genetic diversity, phylogenetics, germplasm characterization, and taxonomy of various Dalbergia species.

Assessment of Genetic Diversity and Population Genetic Structure of Santalum album L. in India by Genic and Genomic SSR markers

Sandalwood (Santalum album L.) is highly valued aromatic tropical tree. It is known for its high quality heartwood and oil. In this study 39 genic and genomic SSR markers were used to analyze the genetic diversity and population structure of 177 S. album accessions from 14 populations of three states in India. High genetic diversity was observed in terms of number of alleles 127 expected heterozygosity (He) ranged from 0.63-0.87 and the average PIC was 0.85. The selected population had relatively high genetic diversity with Shannons information index (I) >1.0. 0.02 mean coefficient of genetic differentiation (FST) and 10.55 gene flow were observed. AMOVA revealed that 92% of the variation observed within individuals. Based on cluster and Structure result individuals were not clustered as per their geographical origin. Furthermore the clusters were clearly distinguished by principal component analysis analysis and the result revealed that PC1 reflected the moderate contribution in genetic variation (6%) followed by PC2 (5.5%). From this study, high genetic diversity and genetic differentiation was found in S. album populations. The genetic diversity information of S. album populations can be used for selection of superior genotypes and germplasm conservation to promote the tree improvement of S. album populations.

Genome survey sequencing and identification of genomic SSR markers for Batocera horsfieldi (Coleoptera: Cerambycidae)

The white-striped longhorn beetle Batocera horsfieldi (Coleoptera: Cerambycidae) is a polyphagous wood-boring pest that causes substantial damage to the lumber, fruit and nut industry. Here, next-generation sequencing was used to generate a whole genome survey dataset to provide fundamental information of its genome and develop genome-wide microsatellite markers for it. The genome size of B. horsfieldi was estimated as approximate 520 Mb by using K-mer analyses, and its heterozygosity ratio and repeat sequence ratio were 0.26% and 51.03%, respectively. The assembled genome was 528.56Mb with GC content of 35.40%. A total of 121750 microsatellite motifs were identified. The most frequent repeat motif was mononucleotide with a frequency of 85.84%, followed by 8.08% of dinonucleotide, 5.04% of trinonucleotide, 0.73% of tetranonucleotide, 0.20% of pentanonucleotide and 0.12% of hexanonucleotide motifs. The AT/AT, TA/TAand GA/TC repeats were the most abundant motifs of dinucleotide motifs, and AAT/ATT, TAA/TTA and ATA/TAT were the most abundant motifs of trinucleotide motifs, respectively. ninety six pairs of SSR primers were randomly selected for PCR amplification and agarose gel electrophoresis detection, among which 56 pairs of primers can be effectively amplified to obtain the target fragment. In summary, various candidate microsatellite markers were identified and characterized in this study using genome survey analysis.

Screening of Bush Type French Bean (Phaseolus vulgaris L.) Accessions for Micronutrient Variations and Characterization of Selected Genotypes using Micronutrient Content Linked Markers

Background: French bean or kidney bean is a rich source of protein, fibers and minerals in diet and is used by poor people in several Asian countries as a cheap source of protein and mineral vegetable/seed grain. Thus, the study on micronutrient variations in French bean local genotypes was undertaken to screen for Fe, Zn, Cu and Mn content in comparison to developed varieties. Methods: Selected twenty local genotypes/accessions and four released varieties of French bean were grown in the field. The morphological characterization was carried out as per crop descriptors. The micronutrient analysis for Fe, Zn, Mn and Cu content was carried out using AAS. Further, accessions were screened using fourteen SSR markers linked to Fe and Zn content.Result: The results revealed that accession IC342273 exhibited significantly higher dry pod (58.94 g) and seed yield (809.74 g) per plot. The accession IC538420 (177.31 ppm) exhibited significantly higher mean Fe content and was on par with Arka Suvidha (176 ppm). Accession EC500226 showed significantly higher Zn (26.91 ppm), Mn (15.31 ppm) and Cu (8.55 ppm) content over other accessions and varieties. Among fourteen SSR primers amplified, two genomic SSR markers (BM154 and BM211) exhibited higher polymorphism among the accessions. The SSR markers classified the accessions and varieties into two clusters, which can be used in crop improvement programmes on French bean for micronutrients bio-fortification studies.

Comparative analysis of genomic- and EST-SSRs in European plum (Prunus domestica L.): implications for the diversity analysis of polyploids

Simple sequence repeats (SSRs) are among the most useful DNA markers in plant science. The aim of this study was to compare the features and usefulness of genomic SSR (gSSR) and EST-SSRs in European plum (Prunus domestica L.), an economically important, hexaploid stone fruit crop globally cultivated to produce fleshy fruits and derived foodstuff. The analysis of an ample set of morphologically diverse varieties indicated that gSSRs and EST-SSRs provide different estimates of some of the locus-based indicators of diversity. Moreover, the two classes of SSRs gave different, weakly correlated, estimations of distance-based parameters with gSSRs being more powerful for discriminating purposes. The two SSR classes provide complementary information in European plum, making the contribution of EST-SSRs useful not only as non-neutral markers. The differences between SSR classes are discussed considering the neutral and non-neutral evolution, and the polyploidy and asexual propagation of the cultivated tree varieties.

Genome survey of Zanthoxylum bungeanum and development of genomic-SSR markers in congeneric species

Zanthoxylum bungeanum, a spice and medicinal plant, is cultivated in many parts of China and some countries in Southeast Asia; however, data on its genome are lacking. In the present study, we performed a whole-genome survey and developed novel genomic-SSR markers of Z. bungeanum. Clean data (∼197.16 Gb) were obtained and assembled into 11185221 scaffolds with an N50 of 183 bp. K-mer analysis revealed that Z. bungeanum has an estimated genome size of 3971.92 Mb, and the GC content, heterozygous rate, and repeat sequence rate are 37.21%, 1.73%, and 86.04%, respectively. These results indicate that the genome of Z. bungeanum is complex. Furthermore, 27153 simple sequence repeat (SSR) loci were identified from 57288 scaffolds with a minimum length > 1 kb. Mononucleotide repeats (19706) were the most abundant type, followed by dinucleotide repeats (5154). The most common motifs were A/T, followed by AT/AT; these SSRs accounted for 71.42% and 11.84% of all repeats, respectively. A total of 21243 non-repeating primer pairs were designed, and 100 were randomly selected and validated by PCR analysis using DNA from 10 Z. bungeanum individuals and 5 Zanthoxylum armatum individuals. Finally, 36 polymorphic SSR markers were developed with polymorphism information content (PIC) values ranging from 0.16 to 0.75. Cluster analysis revealed that Z. bungeanum and Z. armatum could be divided into two major clusters, suggesting that these newly developed SSR markers are useful for genetic diversity and germplasm resource identification in Z. bungeanum and Z. armatum.