Genetic Diversity and Differentiation of Eleven Medicago Species from Campania Region Revealed by Nuclear and Chloroplast Microsatellites Markers

The species belonging to the genus Medicago are considered a very important genetic resource at global level both for planet’s food security and for sustainable rangelands management. The checklist of the Italian flora (2021) includes a total number of 40 Medicago species for Italy, and 27 for Campania region, with a number of doubtful records or related to species no more found in the wild. In this study, 10 Medicago species native to Campania region, and one archaeophyte (M. sativa), identified by means of morphological diagnostic characters, were analyzed in a blind test to assay the efficacy of nine microsatellite markers (five cp-SSRs and four n-SSRs). A total number of 33 individuals from 6 locations were sampled and genotyped. All markers were polymorphic, 40 alleles were obtained with n-SSRs ranging from 8–12 alleles per locus with an average of 10 alleles per marker, PIC values ranged from 0.672 to 0.847, and the most polymorphic SSR was MTIC 564. The cp-SSRs markers were highly polymorphic too; PIC values ranged from 0.644 to 0.891 with an average of 0.776, the most polymorphic cp-SSR was CCMP10. 56 alleles were obtained with cp-SSRs ranging from 7 to 17 alleles per locus with an average of 11. AMOVA analysis with n-SSR markers highlighted a great level of genetic differentiation among the 11 species, with a statistically significant fixation index (FST). UPGMA clustering and Bayesian-based population structure analysis assigned these 11 species to two main clusters, but the distribution of species within clusters was not the same for the two analyses. In conclusion, our results demonstrated that the combination of the used SSRs well distinguished the 11 Medicago species. Moreover, our results demonstrated that the use of a limited number of SSRs might be considered for further genetic studies on other Medicago species.

Biodiversity of some fig cultivars in Southern Syria

This research has been conducted in the Biotechnology Laboratory in Damascus University_ Damascus, Syria where the genetic diversity of fourteen cultivars of figs (Ficus carica L.) growing in Swaida governorate which located in the south region of Syria were investigated using (Simple Sequence Repeats) SSRs technique, where 4 cultivars of them were genotypes scattered in the local fields without any scientific taxonomy and farmers did not classify them properly. Eight pairs of SSRs markers were used depend on their ability to separate between fig cultivars as mentioned in previous studies, six pairs of them gave amplified products in the polymerase chain reaction (PCR), while MFC3 and MFC6 primers did not give any amplification products. A total of 17 alleles were detected at six SSRs loci. The alleles number per locus ranged from 2 to 4 with an average of 2.83 alleles/locus. The observed heterozygosity (Ho) was 0.33, while the expected heterozygosity (He) was 0.17. The mean value of genetic similarity was 0.69 where fig cultivars has separated into two clusters in Cluster Analysis, which confirms a significant genetic similarity between most of the cultivars. MFC1 and MFC2 loci gave about 0.67 and 0.61 PIC (Polymorphism Information Content) values respectively, which confirms their ability to study the genetic diversity of fig cultivars more than other loci. F1 and F2 cultivars greatly affect the quality of the fruits as paternity traits as Caprifigs. Tammozi cultivar has low values of its genetic similarity with the rest of the cultivars which reflect great difference between it and other cultivars. Generally, the challenges in this study were in characterizing unrecognized fig cultivars in southern Syria to distinguish between them as they were not certified in agriculture ministry in Syria because the lack in studies related to the same topic as these cultivars were not studied before, thus, we managed to make a significant contribution in certifying fig cultivars in Syria using SSR technique.

Development and characterization of Simple Sequence Repeats (SSRs) markers in durian kura-kura (Durio testudinarius Becc.) using NGS data

Durian Kura-kura (Durio testudinarius Becc.) belongs to the Malvaceae family and is an endemic species of Borneo. Recently, genomic-based next-generation sequencing (NGS) approaches have been carried out for germplasm conservation and plant breeding programs. The NGS technologies allow plant genomes to be sequenced quickly and inexpensively and enable the efficient development of SSR markers through the in-silico approaches. This study aimed to develop and characterize simple sequence repeats (SSRs) from the assembled genome. The 1203929 scaffolds of the assembled genome were produced from the Ray assembler. The SSRs were identified and extracted using the MISA program produced 4315 sequences containing SSRs. The six motif repeats of SSRs were identified; consist of 431 sequences of dinucleotide (the most motif is AT), 3257 sequences of trinucleotide (the most motif is TTA), 516 sequences of tetranucleotide (the most motif is AAAT), 89 sequences of pentanucleotide (the most motif is ATTTT), 18 sequences of hexanucleotide and four sequences of heptanucleotide. The new SSRs markers will be used in further studies of genetic population of D. testudinarius and plant breeding programs.

Molecular Mapping of a Gene Conferring Fusarium Wilt Resistance in Lentil (Lens culinaris Medikus subsp. culinaris) using Bulked-segregant Analysis

Background: Vascular wilt caused by Fusarium oxysporum f.sp. lentis Vasu. and Srini. is a serious disease of lentil (Lens culinaris Medikus), causes severe yield losses worldwide. For effective disease resistance breeding the inheritance and mapping of wilt resistance gene (s) is necessary. Therefore, the present investigation was focused on study the mode of inheritance and tag/map gene (s) for fusarium wilt resistance in lentil. Methods: Bulked segregant analysis (BSA) approach was used to identify markers that were tightly linked to Fusarium wilt resistance gene. The inheritance and mapping of wilt-resistance gene (s) in lentil was investigated in F2 and F2:3 populations derived from L9-12×ILL10965 cross, whereas L9-12 and ILL10965 were susceptible and resistant parents, respectively. Result: More than two hundreds SSRs markers were surveyed for the parental polymorphism, of which twenty nine were found polymorphic. These polymorphic SSRs were used for the bulked-segregant analysis (BSA) using both parents and its respective resistant and susceptible bulks, and three SSRs viz. PBALC233, PBALC1409 and PBALC203 could distinguish the respective bulks. Linkage analysis showed two SSR markers, PBALC203 and PBALC1409 flanking the wilt resistance gene at 8.2 cM and 9.4 cM distance, respectively. Further, PBLAC233 was also found present on the same linkage group at a distance of 10.2 cM from PBLAC1409.

Diversity of Purple Rice (Oryza sativa L.) Landraces in Northern Thailand

Purple rice is a functional food with health benefits and industrial potentials. In northern Thailand, purple rice landraces are grown either as wetland or upland rice, in the lowlands and highlands, in small amounts along with the staple rice. This study examined diversity of 37 accessions of purple rice collected from farmers with InDel cytoplasm markers for subspecies differentiation, 16 SSRs markers for within and between accessions variation, and selected grain quality features, namely, anthocyanin, iron, zinc, and gamma oryzanol content, when grown together as wetland rice. Most of the purple rice, originally grown as upland rice in both the lowlands and highlands, were identified as tropical japonica, except the wetland accessions from the lowlands that almost all belonged to the indica group. A high degree of genetic differentiation was found between the upland and wetland ecotypes, but none between those from the lowlands and highlands. A highland origin of the purple upland rice populations in the lowlands, possibly with adaptation to the upland rice cultivation of the tropical japonica, is suggested by the close genetic affinity between the highland and lowland populations of the upland ecotype. Grown in a much smaller area than the staple unpigmented rice, purple rice landraces are also less diverse genetically. Identification of purple rice landrace populations with exceptional anthocyanin and gamma oryzanol contents demonstrates that purple rice landraces can be a source of agronomically useful traits, while being an important cultural heritage, and contributing to the genetic diversity of the local rice germplasm.

IMPROVED SSRs-BASED GENETIC DIVERSITY ASSESSMENT OF COCONUTS (COCOS NUCIFERA L) ALONG THE COAST OF KENYA

ABSTRACTCoconut is the most important cash crop along the Coast of Kenya, yet its genetic diversity has not been fully established. A genetic diversity study of 48 coconut genotypes that were collected along the Coast of Kenya was conducted with 13 polymorphic short sequence repeats (SSRs) markers. SSR analysis was performed using GeneMapper while data analysis was done with PowerMarker and DARwin softwares.The results revealed a total of 68 alleles ranging from 2 to 11 per locus with a mean of 5.23 per marker. Gene diversity and polymorphic information content (PIC) ranged between 0.41 to 0.83 and 0.33 to 0.79, respectively. A neighbour-joining dendrogram grouped the genotypes into three major clusters containing distinct sub-clusters. This study underscored that capillary electrophoresis is a more accurate and informative technique for SSRs allele scoring than agarose gels, which was reported in a previous study with the same SSRs markers and coconut genotypes in Kenya. The clusters observed forms the basis to isolate conservation blocks, which are the key to establishing a genebank, since there is no documented coconut genebank for ex-situ conservation in Kenya.

Assessment of Microsatellite Markers (SSRs) For Genetic Diversity in Asparagus Officinalis L. And Allied Species

The present study was hypothesized to evaluate a set of SSRs for the assessment of genetic variations in Asparagus officinalis L. and their allied species. Nine genic SSR markers were especially developed for Asparagus genome and employed for DNA profiling studies of Asparagus species. These SSRs markers have revealed the allelic polymorphism ranging from 1.0 to 2.0. Allele frequency was found highest for psbD-trnL (1.0), petB (1.0) and AG7 (1.0), while it was lowest for ZHD1 (0.1). Polymorphism Information content (PIC) was highest for TC7 (0.9) while it was lowest (0.0) for psbD-trnL, petB and AG7 respectively. The genetic similarity coefficients were found to range from 0.42 to 1.0. The UPGMA clustering algorithm based on SSRs data have clustered Asparagus species into 4 groups (I, II, III & IV) indicating Asparagus officinalis (L.) cultivars and allied species in the first clade, while Asparagus officinalis (L.) ‘Gersengum’, Asparagus densiflorus (Kunth) Jessop, and Asparagus racemosus willd. were clustered in separate clades respectively. The present study has endorsed the origin status of Asparagus officinalis and their allied species. A. officinalis cultivars and other allied Asparagus species are clustered in separate clades, and it was revealed that they have monophyletic origin. It was established that SSRs markers could be informative markers for the differentiation of Asparagus officinalis cultivars, and their allied Asparagus species. Bangladesh J. Bot. 50(3): 595-602, 2021 (September)

Mechanistic Association of Quantitative Trait Locus with Malate Secretion in Lentil (Lens culinaris Medikus) Seedlings under Aluminium Stress

Aluminium (Al) toxicity acts as a major delimiting factor in the productivity of many crops including lentil. To alleviate its effect, plants have evolved with Al exclusion and inclusion mechanisms. The former involves the exudation of organic acid to restrict the entry of Al3+ to the root cells while latter involves detoxification of entered Al3+ by organic acids. Al-induced secretion of organic acids from roots is a well-documented mechanism that chelates and neutralizes Al3+ toxicity. In this study, F6 recombinant inbred lines (RILs) derived from a cross between L-7903 (Al-resistant) and BM-4 (Al-sensitive) were phenotyped to assess variation in secretion levels of malate and was combined with genotypic data obtained from 10 Al-resistance linked simple sequence repeat (SSRs) markers. A major quantitative trait loci (QTL) was mapped for malate (qAlt_ma) secretion with a logarithm of odd (LOD) value of 7.7 and phenotypic variation of 60.2%.Validated SSRs associated with this major QTL will be useful in marker assisted selection programmes for improving Al resistance in lentil.

Development of Maize Hybrids With Enhanced Vitamin-E, Vitamin-A, Lysine, and Tryptophan Through Molecular Breeding

Malnutrition is a widespread problem that affects human health, society, and the economy. Traditional maize that serves as an important source of human nutrition is deficient in vitamin-E, vitamin-A, lysine, and tryptophan. Here, favorable alleles of vte4 (α-tocopherol methyl transferase), crtRB1 (β-carotene hydroxylase), lcyE (lycopene ε-cyclase), and o2 (opaque2) genes were combined in parental lines of four popular hybrids using marker-assisted selection (MAS). BC1F1, BC2F1, and BC2F2 populations were genotyped using gene-based markers of vte4, crtRB1, lcyE, and o2. Background selection using 81–103 simple sequence repeats (SSRs) markers led to the recovery of recurrent parent genome (RPG) up to 95.45%. Alpha (α)-tocopherol was significantly enhanced among introgressed progenies (16.13 μg/g) as compared to original inbreds (7.90 μg/g). Provitamin-A (proA) (10.42 μg/g), lysine (0.352%), and tryptophan (0.086%) were also high in the introgressed progenies. The reconstituted hybrids showed a 2-fold enhancement in α-tocopherol (16.83 μg/g) over original hybrids (8.06 μg/g). Improved hybrids also possessed high proA (11.48 μg/g), lysine (0.367%), and tryptophan (0.084%) when compared with traditional hybrids. The reconstituted hybrids recorded the mean grain yield of 8,066 kg/ha, which was at par with original hybrids (mean: 7,846 kg/ha). The MAS-derived genotypes resembled their corresponding original hybrids for the majority of agronomic and yield-related traits, besides characteristics related to distinctness, uniformity, and stability (DUS). This is the first report for the development of maize with enhanced vitamin-E, vitamin-A, lysine, and tryptophan.

Diversity and Relationships among Neglected Apricot (Prunus armeniaca L.) Landraces Using Morphological Traits and SSR Markers: Implications for Agro-Biodiversity Conservation

Apricot (Prunus armeniaca L.) is an economically important tree species globally cultivated in temperate areas. Italy has an ample number of traditional varieties, but numerous landraces are abandoned and at risk of extinction because of increasing urbanization, agricultural intensification, and varietal renewal. In this work, we investigated the morphological and genetic diversity present in an ex-situ collection of 28 neglected varieties belonging to the so-called “Vesuvian apricot”. Our aim was to understand the level of diversity and the possible link between the promotion of specific fruit types (e.g., by public policies) and the intraspecific variation in apricot. The combination of five continuous and seven categorical traits allowed us to phenotypically distinguish the varieties; while fruit quality-related attributes displayed high variation, both apricot size and skin colour were more uniform. The twelve fluorescent-based Simple Sequence Repeats (SSRs) markers identified cultivar-specific molecular profiles and revealed a high molecular diversity, which poorly correlated with that described by the morphological analysis. Our results highlighted the complementary information provided by the two sets of descriptors and that DNA markers are necessary to separate morphologically related apricot landraces. The observed morphological and genetic differences suggest a loss of diversity influenced by maintenance breeding of specific pomological traits (e.g., skin colour and size). Finally, our study provided evidence to recommend complementary strategies to avoid the loss of diversity in apricot. Actions should pivot on both the promotion of easily identified premium products and more inclusive biodiversity-centred on-farm strategies.