RAPD primer screening as a preliminary study to analyze the genetic diversity of Citrus spp. in South Sulawesi, Indonesia

The typical citrus germplasm collection in South Sulawesi has not been thoroughly characterized, especially in several citrus development centers, which have begun to be promoted again after the decline in productivity due to CVPD infection. The study of citrus diversity is very important to support future citrus breeding programs. Random amplified polymorphic DNA (RAPD) has been widely used for the analysis of genetic diversity among species in populations. In this study, 23 RAPD primers were used on Citrus cultivated in Selayar and Pangkep Regencies, which are citrus development areas in South Sulawesi. A total of 19 primers (OPA-05, OPA-09, OPA-17, OPC-09, OPC-17, OPE-04, OPH-04, OPH-15, OPN-14, 0PN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51) can form polymorphic bands in randomly selected DNA samples. Monomorphic bands were formed by OPA-12 and OPD-07 primer in 12 samples. The primers OPX-13 and OPX-16 produced unclear bands. These 19 primers can be used to amplify DNA and determine the genetic diversity of Citrus in further analysis.

Modern issues of sugar beet (Beta vulgaris L.) hybrid breeding

High efficiency of the cultivation of unfertilized sugar beet ovules and preparation of haploid regenerants (microclones) of pollinators – maintainers of О-type sterility and MS forms of the RMS 120 hybrid components has been shown. A technological method that accelerates the creation of new uniform starting material is proposed. It speeds up the breeding process two to threefold. The identification of haploid regenerants with sterile cytoplasm in initial populations is of great theoretical and practical importance for breeding, as it facilitates the production of homozygous lines with cytoplasmic male sterility and high-performance hybrids on sterile basis. As shown by molecular analysis, a single-nucleotide polymorphism never reported hitherto is present in the mitochondrial genome of the haploid plant regenerants. It allows identification of microclones as fertile and sterile forms. It has been found that DNA markers of the sugar beet mitochondrial genome belonging to the TR minisatellite family (TR1 and TR3) enable reliable enough identification of haploid microclonal plants as MSor O-type forms. Fragments of 1000 bp in length have been detected in monogenic forms in the analysis of 11 sugar beet plants cultured in vitro by PCR with the OP-S4 random RAPD primer. Testing of the OP-S4 marker’s being in the same linkage group as the genes responsible for expression of the economically valuable trait monogermity demonstrates its relative reliability. By the proposed method, dihaploid lines (DH) of the male-sterile form and the О-type sterility maintainer of the RMS 120 sugar beet hybrid have been obtained in in vitro culture. These lines are highly uniform in biomorphological traits, as proven under field conditions.

Strain Typing of Trichosporon asahii Clinical Isolates by Random Amplification of Polymorphic DNA (RAPD) Analysis

Objective The aim of this study was to identify and isolate Trichosporon asahii (T. asahii) from clinical samples and to assess the genetic relatedness of the most frequently isolated strains of T. asahii using random amplification of polymorphic DNA (RAPD) primers GAC-1 and M13. Methods All the clinical samples that grew Trichosporon species, identified and confirmed by polymerase chain reaction (PCR) using Trichosporon genus-specific primers, were considered for the study. Confirmation of the species T. asahii was carried out by T. asahii-specific PCR. Fingerprinting of the most frequently isolated T. asahii isolates was carried out by RAPD using random primers GAC-1 and M13. Results Among the 72 clinical isolates of Trichosporon sp. confirmed by Trichosporon-specific PCR, 65 were found to be T. asahii as identified by T. asahii-specific PCR. Fingerprinting of the 65 isolates confirmed as T. asahii using GAC-1 RAPD primer yielded 11 different patterns, whereas that of M13 primer produced only 5 patterns. The pattern I was found to be the most predominant type (29.2%) followed by pattern III (16.9%) by GAC-1 primer. Conclusions This study being the first of its kind in India on strain typing of T. asahii isolates by adopting RAPD analysis throws light on genetic diversity among the T. asahii isolates from clinical samples. Fingerprinting by RAPD primer GAC-1 identified more heterogeneity among the T. asahii isolates than M13.

PHÂN TÍCH ĐA DẠNG DI TRUYỀN GIỐNG ỚT XIÊM ĐỊA PHƯƠNG Ở QUẢNG NGÃI BẰNG CHỈ THỊ RAPD

Sự đa dạng di truyền của 15 mẫu giống ớt Xiêm địa phương của Quảng Ngãi và mối quan hệ giữa chúng với 5 giống ớt A Riêu và 5 giống ớt Bay đã được đánh giá dựa trên kết quả của 10 mồi RAPD. Hệ số tương đồng di truyền giữa 25 mẫu ớt dao động từ 0,56 đến 1,0. Hệ số tương đồng di truyền và sơ đồ về cây phát sinh loài đã phân ớt A Riêu của Quảng Nam và ớt Bay của Gia Lai thành các nhóm khác nhau. Ớt Xiêm của Quảng Nam thuộc vào cả 2 nhóm và việc phân chia ớt Xiêm vào các nhóm này có mối liên hệ mật thiết với hình dạng quả, Ớt Xiêm với dạng quả lớn thuộc cùng phân nhóm với ớt A Riêu, ớt Xiêm dạng quả nhỏ đến trung bình thuộc cùng phân nhóm với ớt Bay. ABSTRACT Genetic diversity of 15 Xiem chili accessions of Quang Ngai province and its relationship with 5 A Rieu chili accessions of Quang Nam and 5 Bay chili accessions of Gia Lai was assessed by using 10 PCR - RAPD primer. The genetic dissimilarity of the total of 25 accessions was ranged from 0,56 to 1,0. RAPD analysis combined with the construction of phylogenetic tree revealed that big Xiem accessions had the same cluster to A Rieu, while small to medium Xiem accessions had the same cluster to Bay.

RAPD primer screening for markers development of Jinten Hitam (Nigella sativa L.) authentication

Jinten hitam (Nigella sativa) is one of the medicinal plants in the family Ranunculaceae, native to the Mediterranean region and several regions of Asia. The seed of Nigella sativa is widely cultivated, distributed, and used in pharmaceuticals, cosmetics, and food industries. Due to similar seed morphology to its potential adulterant such as Corchorus spp., N. sativa seeds are susceptible to adulteration and substitution in markets. Molecular markers have become one of the most reliable methods for the identification and authentication of medicinal plants. The objective of this study was to select the random amplified polymorphic DNA (RAPD) primer for generating authentication methods of Jinten hitam (N. sativa). Genomic DNA was extracted from samples of N. sativa seed, Corchorus sp. seed, and a mixture of both samples. Forty-two random RAPD primers were used in this study. A total of 227 DNA fragments were produced from 37 amplified RAPD primers, out of which 65% were polymorphic. Primer OPK-4 and OPC-12 generated specific fragments in N. sativa, meanwhile, Primer OPB-1, OPL-5, OPM-3, OPD-5, and OPC-12 generated specific fragments for Corchorus. RAPD molecular marker was able to authentication Jinten hitam (N. sativa) and Corchorus sp. using a selected primer. This research was the first report on RAPD primer screening for Nigella sativa authentication from its potential adulterant (Corchorus spp.).

Determination of a sex-related RAPD marker in Carob (Ceratonia siliqua L.)

Molecular markers are used in the characterization and breeding of organisms. Carob (Ceratonia siliqua L.) is a species with both dioecious and hermaphrodite flower forms. The determination of sex at an early stage of growth in this species, whose juvenility period is long, is important in breeding studies. The objective of this study was to identify the sex-related markers using RAPD method. Ten genotypes were obtained from natural F1 hybrids between a naturally grown a female and a male carob tree. DNA was extracted from the leaves of 12 carob plants. Using BSA, the female and male bulks were formed from five female and five male plants, respectively, using equal amounts of DNA from each plant. In this study, 130 RAPD primers were tested. That of 21 primers tested showed polymorphisms between male and female bulks. While the fragment of 750 bp from the OPA17 RAPD primer was not detected in the female parent, female bulk, and female F1 hybrids; it was observed in the male parent and four out of five male F1 hybrids. This is the first report in the literature that one RAPD marker, namely OPA17-750, related to 80% reliability to male sex in carob was determined.

Keragaman Genetik Bawang Merah (Allium cepa var. aggregatum) Berdasarkan Marka Morfologi dan Molekuler

Bawang merah di Indonesia pada umumnya diperbanyak secara vegetatif, tetapi bawang merah yang tersebar tersebut beragam bentuknya sehingga diduga memiliki keragaman genetik yang tinggi. Penelitian ini bertujuan mengidentifikasi keragaman morfologi menggunakan 21 karakter bawang merah serta keragaman molekuler menggunakan penanda RAPD. Hasil penelitian menunjukkan bahwa karakter morfologi membagi 40 genotipe bawang merah menjadi 2 kelompok utama pada koefisien ketidakmiripan 0.59 yaitu kelompok A (38 genotipe) dan B (2 genotipe), sedangkan analisis morfologi berdasarkan 14 karakter morfologi umbi menunjukkan adanya dua kelompok utama pada koefisien ketidakmiripan 0.50 yaitu kelompok A dan B, masing-masing terdiri atas 36 dan 4 genotipe. Analisis berdasarkan penanda molekuler menunjukkan dua kelompok utama pada koefisien ketidakmiripan 0.41 yaitu kelompok A (3 genotipe) dan B (37 genotipe). Marka RAPD menghasilkan 229 pita polimorfik DNA dengan total 100% dan primer informatif adalah SBN2, OPE11 dan SBN9. Pengelompokkan dari 40 genotipe dalam penelitian ini tidak berhubungan dengan asal geografis. Genotipe BM12, BM19, BM78 dan BM01 memiliki ukuran daun yang panjang, ukuran dan diameter umbi besar dan memiliki intensitas warna dasar kulit umbi kering yang gelap. BM63 dan BM24 memiliki perilaku daun yang tegak, panjang daun sedang, ukuran dan diameter umbi sedang. Genotipe potensial ini dapat dikembangkan untuk meningkatkan varietas bawang merah di Indonesia. Kata kunci: genotipe, koefisien ketidakmiripan, RAPD, primer informatif

Identification of interspecific hybrid between Jatropha curcas × J. integerrima using morphological and molecular markers

Saptadi D, Asbani N, Heliyanto B, Setiawan A, Sudarsono. 2020. Identification of interspecific hybrid between Jatropha curcas x J. integerrima using morphological and molecular markers. Biodiversitas 21: 814-823. Eight F1 progenies derived from Jatropha curcas × J. integerrima hybridizations were evaluated for their morphological characters and using RAPD, ISSR and SSR markers. Morphological variations among the hybrids were limited and they were intermediate between the Jatropha parents. The eight F1 progenies derived from J. curcas × J. integerrima hybridizations were most probably the interspecific F1 hybrids. The confirmed identity of the progenies as interspecific hybrids between J. curcas × J. integerrima was based on the presence of several phenotypic characters from both parents in the F1 progenies and by similarity of the molecular marker banding patterns among the parents and the F1 progenies. Among the evaluated molecular markers, the ISSR primers and the majority of either RAPD and SSR primers were not able to generate marker for confirming the identity of F1 progenies as interspecific hybrids between J. curcas × J. integerrima. However, the RAPD primer OPC 10 and the SSR primers AF469003, EU099522 and EU586348 were able to generate polymorphic markers in the Jatropha parents and their F1 progenies. Therefore, these four primers were able to generate usable markers for confirming the identity of F1 progenies as interspecific hybrids between J. curcas × J. integerrima. The evaluated interspecific F1 progenies are potentially useful to increase genetic diversity of J. curcas and support its breeding program.

Isolation and Identification of Molecular Markers for Fingerprinting of Chilli Hybrids & its Parental Lines

Chilli (Capsicum annum) is the predominant sp., which is cultivated in both hot and sweet papers. The maintenance of the genetic purity of chilli plant is a matter of great concern for the breeders. For genetic purity analysis, between true hybrids and off-types, breeders find out morphological differences between them, but this technique is cannot be recognized easily and also costly, tedious to score, and environmentally sensitive. Alternatively, molecular markers based genetic purity analysis can be employed. The molecular marker-based technique was thus used to overcome the conventional method drawbacks. The main objective of the study is to identify informative molecular markers (ISSR and RAPD) capable of distinguishing Chilli hybrids and their parental lines and their utilization in seed purity assessment. Five parental lines of Chilli (i.eCH10, CH12, CH530, CH709, CH734) were used for the production of 3 hybrids. Total 30 ISSR and 8 RAPD primers were selected for the study of 5 parental lines, among them 2ISSR and 1 RAPD primers produced unique fingerprinting across the hybrids. The ISSR marker UBC815 amplified alleles specific to different parental lines(CH10 & CH12) for hybrids (ACH112), The ISSR marker UBC 827, amplified alleles specific to different parental lines(CH709 & CH12) for hybrids (ACH179). Likewise, RAPD primer B20 for hybrid ACH 753 and their parental lines(CH734 & CH530). Thus, the above study showed that the aid of molecular markers is more reliable, highly efficient, and reproducible for assessing fingerprinting of Chilli commercial hybrid seeds with more accuracy.