Kalmegh (Andrographis paniculata (Burm. F.) Nees) is one of the most important medicinal plants and has been widely explored as traditional medicine. To exploit its natural genetic diversity and initiations of molecular breeding to develop novel cultivars or varieties, developments of genomic resources are essential. Four microsatellite-enriched genomic libraries—(CT)₁₄, (GT)₁₂, (AG)₁₅ and (AAC)₈—were constructed using the genomic DNA of A. paniculata. Initially, 183 recombinant colonies were screened for the presence of CT, GT, AG, and AAC microsatellite repeats, out of which 47 clones found positive for the desired simple sequence repeats (SSRs). It was found that few colonies had more than one desirable SSR. Thus, a sum of 67 SSRs were designed and synthesized for their validation among 42 A. paniculata accessions. Out of the 67 SSRs used for genotyping, only 41 were found to be polymorphic. The developed set of g-SSR markers showed substantial genetic variability among the selected A. paniculata accessions, with an average polymorphic information content (PIC) value of 0.32. Neighbor-joining tree analysis, population structure analysis, analysis of molecular variance (AMOVA), and principal coordinate analysis (PCoA) illustrated the considerable genetic diversity among them. The novel g-SSR markers developed in the present study could be important genomic resources for future applications in A. paniculata.

Guava (Psidium guajava L.) is one of the economically major fruit crops, abundant in nutrients and found growing in tropical-subtropical regions around the world. Ensuring sufficient genomic resources is crucial for crop species to enhance breeding efficiency and facilitate molecular breeding. However, genomic resources, especially microsatellite or simple sequence repeat (SSR) markers, are limited in guava. Therefore, novel genome-wide SSR markers were developed by utilizing chromosome assembly (GCA_016432845.1) of the “New Age” cultivar through GMATA, a comprehensive software. The software evaluated about 397.8 million base pairs (Mbp) of the guava genome sequence, where 87,372 SSR loci were utilized to design primers, ultimately creating 75,084 new SSR markers. After in silico analysis, a total of 75 g-SSR markers were chosen to screen 35 guava genotypes, encompassing wild Psidium species and five jamun genotypes. Of the 72 amplified novel g-SSR markers (FHTGSSRs), 53 showed polymorphism, suggesting significant genetic variation among the guava genotypes, including wild species. The 53 polymorphic g-SSR markers had an average of 3.04 alleles per locus for 35 selected guava genotypes. Besides, in this study, the mean values recorded for major allele frequency, gene diversity, observed heterozygosity, and polymorphism information content were 0.73, 0.38, 0.13, and 0.33, respectively. Among the wild Psidium species studied, the transferability of these novel g-SSR loci across different species was found to be 45.83% to 90.28%. Furthermore, 17 novel g-SSR markers were successfully amplified in all the selected Syzygium genotypes, of which only four markers could differentiate between two Syzygium species. A neighbour-joining (N-J) tree was constructed using 53 polymorphic g-SSR markers and classified 35 guava genotypes into four clades and one outlier, emphasizing the genetic uniqueness of wild Psidium species compared to cultivated genotypes. Model-based structure analysis divided the guava genotypes into two distinct genetic groups, a classification that was strongly supported by Principal Coordinate Analysis (PCoA). In addition, the AMOVA and PCoA analyses also indicated substantial genetic diversity among the selected guava genotypes, including wild Psidium species. Hence, the developed novel genome-wide genomic SSRs could enhance the availability of genomic resources and assist in the molecular breeding of guava.

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant’s leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.

Background: Guava ( Psidium guajava L.) is a major pantropical fruit crop, valued for its nutritional composition, wider adaptability, and economic significance. Since its introduction in India during the 17 ^th century, guava cultivars have exhibited a narrow genetic base, with most existing genotypes arising from open-pollinated selections or crosses involving a limited number of genotypes. Furthermore, much of the available germplasm has been named based on fruit characteristics or place/regions of origin, leading to confusion in classification and hindering the precise identification and utilization of genetic resources in guava breeding programs. To address this, 49 morpho-biochemical parameters were assessed in 51 diverse Psidium genotypes, including cultivars, varieties, hybrids, related wild species, and exotic/USDA introductions. Results: Substantial variability was observed among globally collected guava germplasm for fruit traits, with fruit weight ranging from 11.67 g ( Psidium molle ) to 379 g (S. N90-53), seed number from 8 (S. N11-3) to 512 (S. N15-3), leaf length from 5.57 to 17.17 cm, leaf width from 2.07 to 7.90 cm, and petiole length from 0.17 to 0.77 cm. Germplasm also differed in qualitative descriptors, including branch orientation, bark colour, leaf morphology, tip structure, and fruit surface traits, with wild species forming distinct morphological groups. Additionally, biochemical traits varied widely, including the contents of total soluble solid (6.8–13.8 °Brix), acidity (0.25–1.60%), total sugars (3.62–9.21 g/100 g FW), ascorbic acid (88.36–302.46 mg/100 g FW), ferric reducing antioxidants power (7.74–20.69 µM TE/g FW), free radical scavenging activity (2,2-diphenyl-1-picrylhydrazyl assay)(85.72–98.47%), lycopene (0.05–8.91 mg/100 g FW), and total carotenoids (0.05–2.76 mg/100 g FW). Besides, principal component analysis indicated PC1 (22.1%) related to fruit size and PC2 (15.2%) to biochemical traits. Furthermore, cluster analysis grouped 51 guava genotypes into three clusters, distinguishing wild from cultivated types. Conclusions: The study confirms the presence of wide morpho-biochemical variability in Psidium germplasm, offering valuable genetic resources for genetic improvement programs, aimed at enhanced fruit quality with rich antioxidants, bio-fortification, and the tailoring of novel guava genotypes with reduced seed content, enhanced seed mellowness, and tenet branching, fitting in high-density planting and improved yield efficiency. Graphical

Guava ( Psidium guajava L.) is an important commercial fruit crop valued for its exceptional nutraceutical value and pharmacological properties. Growing awareness about guava’s health benefits is driving demand for higher-quality, nutraceutical-rich cultivars. Varietal development and genetic improvement in guava involve crossing between heterozygous elite parents to generate segregating full-sib populations for future selection. In this study, hybridization between Allahabad Safeda and Arka Kiran, was attempted, and hybrid progenies were raised. A total of 50 hybrid progenies were confirmed as hybrid with three SSRs (FHTGSSR-3.4, FHTGSSR-7.5, and FHTGSSR-3.6). These confirmed hybrid progenies exhibited wide phenotypic variability with both intermediate and transgressive segregation across tree, leaf, fruit physical and biochemical parameters. Fruit parameters varied markedly in size (length: 5.59–10.13 cm; weight: 79.33–406.33 g) while qualitative variation was evident in leaf shape, fruit shape, peel, and pulp colour. Biochemical profiling revealed high variability in respect of TSS (10.30–14.80°Brix), ascorbic acid (65.00–302.00 mg/100 g), total carotenoids (0.78–9.55 mg/100 g), lycopene (0.53–9.03 mg/100 g) and antioxidant activities (DPPH: 86–95%; FRAP: 8.9–22.0 µmol Trolox/g). MGIDI analysis identified seven guava hybrids as superior multi-trait performers amongst the studied guava hybrids. Multivariate analyses helped characterize the underlying trait variability, and hierarchical clustering grouped progenies into distinct clusters. Additionally, transgressive segregation for key nutraceutical traits underscores the potential for selecting superior recombinants. Overall, this study highlights the effectiveness of hybridization between heterozygous guava genotypes in generating genetic variability, while establishing SSR markers are robust and reliable tools for confirming hybridity in crop improvement programs.