PUTRANTO, Riza Arief
INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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Kloning dan karakterisasi daerah promoter gen penyandi ADP glucose pyrophosphorylase dari Metroxylon sagu rendemen pati-tinggi dan -rendah [Cloning and characterization of promoter region of ADP glucose pyrophosphorylase-encoding gene from Metroxylon sagu with high- and low-starch content] BUDIANI, Asmini; PUTRANTO, Riza Arief; MINARSIH, Hayati; RIYADI, Imron; SUMARYONO, .; ABBAS, Barahima
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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ADP-glucose pyrophosphorylase (AGPase) is one of the key enzymes in the starch biosynthesis. In many plants, the activity of this enzyme was reported to affect the yield and composition of the produced starch. This research is a part of an effort to develop molecular markers for early selection of high starch-yielding of sago palm. The purpose of the research was to isolate promoters of AGP gene and to analyze the differences in their DNA sequences between sago palm with high starch content (MsHS) and low starch content (MsLS). DNA was isolated and purified from the leaves of the two sago palm. The promoter region of AGP was amplified by Genome Walking technique. The specific primers were designed by Primer3 program based on the information of DNA sequence of AGP genes of sago palm from previous studies. Selected DNA fragments resulted from Genome Walking were isolated from the gel, cloned into E. coli, and analyzed its DNA sequence. DNA sequence analysis showed that one DNA fragment from MsHS  (± 1500 bp) and one DNA fragment from MsLS (> 2000 bp) were confirmed as a 5’ upstream of the AGP gene.  Further in silico analysis using MEME program identified various DNA motifs of cis-acting elements, which confirmed that those DNA fragment were promoter region of the gene. Preliminary analysis showed the differences in DNA sequences and motives of cis-acting elements in the promoter region of the two samples which might influence or indirectly associated with the character of the starch yield in sago palm.
Genetic mapping studies in Coffea sp using molecular marker methods Studi peta genetik pada Coffea sp. menggunakan metode penanda molekuler PRIYONO, .; PUTRANTO, Riza Arief
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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AbstrakAnalisis genetik telah  menjadi alat yang penting  dalam  pemuliaan  tanaman untuk perbaikan sifat penting tanaman. Salah satu potensi terbesar dari analisis tersebut adalah identifikasi penanda molekuler yang berguna untuk pemetaan genetik. Pemetaan genetik  merupakan  salah satu langkah penting dari analisis  genetik.  Intisari  dari   semua pemetaan genetik adalah  menempatkan  koleksi  pe- nanda molekuler pada posisi tertentu dalam genom. Hal tersebut dapat kemudian digunakan untuk meng- identifikasi lokus sifat kuantitatif (QTLs) dengan memanfaatan keragaman genetik alami yang tersedia dan meningkatkan sifat-sifat penting serta berharga. Sampai saat ini, tiga belas peta genetik telah dipublikasi dan tersedia pada Coffea sp. yang menciptakan database besar untuk kerangka genetik. Sebuah peta genetik terbaru dengan akses terbuka dan berfungsi sebagai referensi telah dibangun oleh International Coffee Genomics Network (ICGN). Peta tersebut tediri dari 3230 lokus, dengan panjang peta 1471 cM (1cm ~ 500 Kb) serta kepadatan satu penanda setiap 220 Kb. Peta-peta genetik pada tanaman kopi telah digunakan dari karakterisasi gen hingga analisis komparatif genom dengan spesies tanaman yang berbeda. Saat ini, pesatnya kemajuan teknologi New Genome Sequencing (NGS) untuk sekuensing DNA dan RNA memungkinkan validasi dari peta-peta genetik untuk prediksi QTLs serta gen-gen yang membawa sifat penting Coffea sp.AbstractGenetic analysis has become an important tool in plant breeding for crop improvement. One of their greatest potential appears to be the identification of molecular markers useful for genetic mapping. Genetic mapping is one of important steps in genetic analysis. The essence of all genetic mapping is to place a collection of molecular markers onto their respective positions on the genome. Thus, it leads to identification of new quantitative trait loci (QTLs) by making benefits of natural available genetic diversity.and to improve important and valuable traits. Until present, thirteen genetic maps were published and available in Coffea sp. creating a huge database for genetic framework. One most recent and open reference genetic map for robusta coffee has been generated by the International Coffee Genomics Network (ICGN) comprising 3230 loci, genetic size 1471 cM (1cM ~500 Kb), with an average density close to one marker every 220 Kb. The Coffea genetic maps have been utilized from gene characterization to genomic comparative analysis with different plant species. Nowadays, the feasibility of NGS for DNA and RNA sequencing allow the validation of genetic map related to the prediction of QTLs and adjacent genes related to important traits for Coffea sp. 
Evaluasi 18 primer SSR untuk pengembangan sidikjari DNA tanaman karet (Hevea brasiliensis Muell. Arg.) Evaluation of 18 SSR primers to develop DNA fingerprint of rubber tree (Hevea brasiliensis Muell. Arg.) BUDIANI, Asmini; WOELAN, Sekar; MINARSIH, Hayati; NUHAIMI-HARIS, .; PUTRANTO, Riza Arief
E-Journal Menara Perkebunan Vol 82, No 2: Desember 2014
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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Abstract Breeding program of rubber tree to produce elite clones is hampered by the length of selection cycles. On the other hand, attempts to increase production by extensification of the plantation area is also facing a problem from the availability of the rootstock, causing the occurence of fake clones without any information of their origin. Therefore, the availability of molecular markers to be used as DNA fingerprint of rubber tree clones is needed. This will help the breeder to shorten the length of selection program and to identify the purity of the clone. This research was aimed to evaluate 18 SSR primer pairs that had been published to identify 17 rubber clones. Pure genomic DNAs were isolated from 17 clones, followed by experiment to optimize annealing tempe-rature for each primer to obtain the best amplification product. Initially, the PCR product was run in both the agarose and polyacrylamide gels. However, the analysis of all PCR products were then conducted on SDS polyacrylamide gel, since this gel can separate DNA fragments with only a few bases differences. The results showed that 14 clones have been identified specifically using 11 primers. Four out of 18 primer pairs used could identify 12 rubber tree clones, which are PR 107, PR 261, SP 217, PB 330, PB 340, IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 and RRIM 712. Each clone can be distinguished from each other using only one primer pair. Identification of the other tree clones (PB 5/51, PB 260, and RRIC 110) has to be conducted by combining the several PCR products using different primer pairs. Although these results showed that SSR markers had high potential to be used as DNA fingerprint on rubber tree clones, the set of the primer pairs should be tested among other clones, as well as other SSR primers should be tested to identify the clones which could not be identified using the 18 primer pairs in this experiment.Abstrak Pemuliaan tanaman karet untuk menghasilkan klon-klon unggul baru menghadapi masalah lamanya siklus seleksi. Di sisi lain, upaya peningkatan produksi melalui pembukaan lahan baru, juga terkendala oleh ketersediaan bibit, yang memicu beredarnya bibit palsu, yang umumnya tidak jelas asal usulnya. Oleh karena itu, diperlukan ketersediaan marka yang dapat digunakan sebagai sidikjari DNA bagi klon-klon tanaman karet yang ada, sehingga dapat membantu mempercepat proses seleksi dan mengetahui kemurnian bibit. Penelitian ini bertujuan untuk mengevaluasi 18 primer SSR yang telah dipublikasikan untuk mengidentifikasi 17 klon karet. DNA yang murni diisolasi dari 17 klon, kemudian dilakukan optimasi suhu annealing untuk setiap jenis primer agar diperoleh hasil amplifikasi terbaik.  Pada awal percobaan hasil PCR dicek pada gel agarosa dan gel poliakrilamida, namun analisis untuk seluruh hasil PCR dilakukan pada gel SDS poliakrilamid, karena gel ini secara nyata dapat memisahkan fragmen DNA yang hanya berbeda beberapa basa. Hasil percobaan menunjukkan bahwa 14 klon dapat diidentifikasi secara spesifik menggunakan 11 primer. Empat dari 18 pasang primer yang diuji dapat mengidentifikasi 12 klon yang dianalisis, yaitu PR 107, PR 261, SP 217, PB 330, PB 340,. IRR 5, IRR 112, IRR 118, IRR 220, GT 1, BPM 101 dan RRIM 712. Masing-maing klon tersebut dapat dibedakan dari klon lainnya hanya dengan menggunakan satu jenis primer. Sedangkan identifikasi tiga klon lainnya (PB 5/51, PB 260, dan RRIC110) harus dilakukan dengan menggabungkan hasil PCR menggunakan beberapa primer. Meskipun hasil percobaan ini menunjukkan bahwa marka SSR sangat berpotensi untuk digunakan sebagai sidikjari DNA klon-klon karet, namun primer yang sama perlu diuji untuk klon-klon lainnya. Demikian pula primer lain perlu diuji untuk mengidentifikasi klon-klon yang belum teridentifikasi menggunakan 18 primer dalam penelitian ini.
Ekspresi dan kloning gen penyandi ADP-Glucose Phyrophosphorylase dari tanaman sagu (Metroxylon sagu Rottb.) Expression and cloning of gene encoding ADP-Glucose Phyrophosphorylase from sago palm (Metroxylon sagu Rottb.) BUDIANI1, Asmini; PUTRANTO, Riza Arief; MINARSIH, Hayati; RIYADI, Imron; SUMARYONO, .; ABBAS, Barahima
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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AbstractSago palm (Metroxylon sagu Rottb.) is a potential food and energy resources becouse it is the highest starch producing plant.  Breeding of sago palm should be directed to produce elite genotype with superior characters such as high starch content, wider pith diameter, without spine and high starch quality. However, research on sago palm in Indonesia so far is limited espescially in the field of cultivation and breeding, and attempt to produce such elite would take long time. Availability of molecular marker for starch content would be beneficial to shorten the length period of breeding. ADP-Glucose Phyrophosphorylase is one of the important enzymes in starch biosynthesis. Therefore its gene is an interesting subject in order to develope molecular marker of high starch content.  This research was aimed to study the expression of gene encoding AGP in the sago palm with high starch content versus low starch content, and to clone the full cds of the gene. RNA was isolated from leaf and pith of both palms. Exspression analysis and amplify-cation of full cds were conducted by Reverse Transcryptase-Polymerase Chain Reaction (RT-PCR) using specific primers. The results showed that sago palm with higher starch content expressed AGP higher than that of sago palm with lower  starch content. Expression of AGP in the full developing leaf was higher than in the young leaf, and there was no expression detected in the pith. The full cds of AGP was successfully amplified and cloned. Even though the DNA sequence showed high homology with DNA sequence of the same gene that has been deposited in GenBank, there were differences in severall nucleotide including that in the active domain of the enzyme.AbstrakTanaman sagu merupakan sumber pangan dan energi yang sangat potensial untuk dikembangkan karena merupakan tanaman penghasil karbihidrat tertinggi. Pemuliaan tanaman sagu mestinya diarah-kan untuk menghasilkan bibit sagu yang selain memiliki rendemen pati tinggi, juga memiliki diameter empulur besar, tidak berduri dan memiliki cita rasa pati yang enak. Namun, sampai saat ini riset mengenai sagu di Indonesia masih sangat terbatas, sehingga pemuliaan sagu untuk menghasilkan bibit unggul demikian akan memerlukan waktu lama. Ketersediaan penanda rendemen pati akan sangat membantu mempercepat pemuliaan tanaman tersebut. ADP-Glucose Pyrophosphorylase adalah salah satu enzim yang berperan penting dalam biosintesis pati, sehingga gene penyandinya merupakan subjek yang menarik dalam pengembangan marka kandungan pati tinggi.  Sebagai bagian dari upaya untuk mendapat-kan penanda rendemen pati tinggi pada tanaman sagu, penelitian ini bertujuan untuk mempelajari ekspresi gen penyandi AGP. RNA diisolasi dari daun tanaman sagu rendemen pati rendah dan tanaman sagu rendemen pati tinggi. Perbedaan tingkat ekspresi gen penyandi AGP dari tanaman sagu rendemen pati tinggi vs rendemen pati rendah, dianalisis dengan teknik Reverse-Transcryptase PCR menggunakan primer spesifik. Hasil penelitian menunjukkan bahwa tanaman sagu rendemen pati tinggi mengekspresikan AGP lebih tinggi dibandingkan dengan tanaman sagu rendemen pati rendah. Ekspresi gen tersebut pada daun tua (full developing leaf) lebih tinggi di-bandingkan dengan pada daun muda, dan pada empulur tidak dideteksi ekspresi gen tersebut. Daerah penyandi lengkap AGP subunit kecil telah diklon. Meskipun memiliki homologi yang tinggi dengan sekuen DNA gen yang sama yang telah dideposit pada  GenBank,  namun terdapat perbedaan beberapa nukleotida termasuk pada daerah domain aktif dari enzim tersebut. 
Identifikasi famili gen putatif penyandi protease inhibitor dengan pendekatan in silico komparatif pada genom Hevea brasiliensis Muell. Arg (Identification of putative gene family encoding protease inhibitors by in silico comparative analysis in Hevea brasiliensis Muell. Arg genome) MARTIANSYAH, Irfan; PUTRANTO, Riza Arief; KHUMAIDA, Nurul
E-Journal Menara Perkebunan Vol 85, No 2 (2017): Oktober 2017
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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AbstractProtease inhibitors (PIs) are small proteins that form complexes with proteases and inhibits their proteolytic activity. Its potential application as an antimicrobial agent has been studied. Most of PIs molecule size is around 8-22 kDa depending on their protein families.To date, on the basis of sequence homologies of inhibitor domains, PIs have been classified into 48 families in all organisms. In plant, more than 13 families of PIs have been identified but they were not widely identified in the rubber tree (Hevea brasiliensis Muell.Arg). In the present study, 40 putative HbPI genes, designated as HbPI01 to HbPI36, were identified from whole-genome sequence of rubber tree clone Reyan 7-33-97 using 7453 scaffolds available online in NCBI with the accession code: LVXX01000000. Multiple sequence alignment using MUSCLE algorithm discovered seven conserved motifs (Motifs I-VII) among HbPIs. Phylogenetic analysis of 50 and 36 PI amino acid residues of 32 scaffolds containing putative PI genes from Arabidopsis thaliana and H. brasiliensis showed three clusters (families): LTP-I, SERPIN and LTP-II. LTP-I has 23 putative HbPI genes (HbPI05 to HbPI27) and 12 AtPI genes. SERPIN, a family member of serine protease inhibitor group, has 11 putative HbPI genes (HbPI01 to HbPI04 and HbPI28 to HbPI34) and 22 AtPI genes. LTP-II has 2 putative HbPI genes (HbPI35 to HbPI36) and 16 AtPI genes. In conclusion, this work provides valuable information for further functional characterization of HbPI genes in H. brasiliensis.[Key words: protease inhibitor, genome-wide, scaffold, in silico, Hevea brasiliensis]. AbstrakProtease inhibitor (PI) merupakan protein yang membentuk kompleks dengan protease dan menghambat aktivitas proteolitik dari enzim tersebut. Potensi penggunaan protease inhibitor sebagai agensia antimikroba telah diketahui. Kebanyakan PI memiliki ukuran molekul sekitar 8-22 kDa bergantung pada familinya. Saat ini, PI dapat diklasifikasikan menjadi 48 famili di seluruh organisme berdasarkan kemiripan sekuen dari domain inhibitornya. Pada tanaman, lebih dari 13 famili PI telah diketahui tetapi pada tanaman karet (Hevea brasiliensis Muell.Arg) belum diidentifikasi. Pada penelitian ini, sebanyak 40 gen putatif penyandi PI (HbPI01 hingga HbPI36) telah berhasil diidentifikasi dari 7453 scaffold genom utuh tanaman karet klon Reyan 7-33-97 yang tersedia secara daring dengan kode aksesi LVXX01000000. Penjajaran sekuen menggunakan algoritma MUSCLE memper-lihatkan tujuh konservasi motif (Motif I-VIII) pada famili gen putatif HbPIs. Analisis pohon filogenetik dari tanaman Arabidopsis thaliana dan H. brasiliensis sebanyak 50 dan 36 sekuen residu asam amino dari 32 scaffold yang mengandung gen putatif PI menunjukkan adanya tiga klaster besar, yaitu famili LTP-I, SERPIN dan LTP-II. LTP-I terdiri dari 23 gen putatif HbPI (HbPI05 hingga HbPI27) dan 12 gen AtPI. SERPIN yang merupakan anggota kelas protease inhibitor serin terdiri dari 11 gen putatif HbPI (HbPI01hingga HbPI04 dan HbPI28 hingga HbPI34) dan 22 gen AtPI. LTP-II terdiri dari 2 gen putatif HbPI (HbPI35 hingga HbPI36) dan 16 gen AtPIs. Penelitian ini menghasilkan informasi penting untuk melakukan karakterisasi fungsional lebih mendalam pada gen HbPI tanaman karet ke depannya.[Kata kunci: protease inhibitor, genome-wide,scaffold, in silico, Hevea brasiliensis].
Evaluation of eleven reference genes for Reverse Transcriptase Quantitative PCR of rubber tree under water deficit Evaluasi sebelas gen referensi untuk Reverse Transcriptase Quantitative PCR pada tanaman karet tercekam kekeringan PUTRANTO, Riza Arief; LECLERCQ, Julie; MONTORO, Pascal
E-Journal Menara Perkebunan Vol 83, No 2: Desember 2015
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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AbstrakReverse Transcriptase Quantitative PCR (RT-qPCR) merupakan teknik yang sangat ampuh untuk mendeteksi jumlah mRNA yang rendah dalam sel tanaman. Pengukuran akumulasi transkrip tersebut relatif terhadap kontrol ekspresi seperti gen-gen housekeeping. Keandalan teknik RT-qPCR ber-gantung pada pemilihan kontrol internal yang disebut pula gen referensi. Hal tersebut menjadi alasan kenapa validasi gen referensi disarankan untuk setiap set sampel cDNAs yang akan diguna-kan pada eksperimen RT-qPCR baru. Penelitian ini bertujuan untuk menganalisis stabilitas sebelas gen-gen housekeeping terpilih pada tiga organ Hevea brasiliensis (daun, kulit batang dan akar) tercekam kekeringan moderat selama 15 hari. RNA total diisolasi dari 18 sampel yang terdiri dari tanaman kontrol dan tercekam kekeringan pada hari ke-0 (D0), ke-5 (D5) dan ke-15 (D15). Kualitas cDNA yang disintesis divalidasi dengan amplifikasi PCR menggunakan primer HbActin. Kesebelas pasangan primer penyandi gen-gen housekeeping pada Hevea (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S dan HbUBI) divalidasi dengan amplifikasi PCR. Nilai Crossing-point (Cp) yang diukur dengan metode derivatif kedua pasca analisis RT-qPCR mengungkapkan nilai rerata Cp yang lebih tinggi secara signifikan untuk kesebelas gen housekeeping pada titik sampling D5 dibanding D0 dan D15. Studi ini menyarankan bahwa metode perhitungan koefisien keragaman (CV) sederhana dapat digunakan untuk menentu-kan peringkat gen referensi pada tanaman karet berdasarkan ekspresinya yang stabil. Lima gen housekeeping (HbRH2b, HbRH8, HbUBC4, HbαTUB dan HbActin) dapat digunakan sebagai gen referensi untuk analisis RT-qPCR pada Hevea brasiliensis yang tercekam kekeringan moderat. Gen HbRH2b memiliki ekspresi paling stabil dibanding yang lain.AbstractReverse Transcriptase Quantitative PCR (RT-qPCR) is a powerful technique in order to detect low abundance of mRNA in the plant cell. The measurement of transcript abundance is relative to the control of expression such as housekeeping genes. Therefore, the reliability of RT-qPCR depends essentially to the choice of these internal controls also called reference genes. That is the reason why a prior validation of reference genes is suggested for every set of cDNA samples used in a new RT-qPCR experiment. This study aimed to analyze the stability of eleven selected house-keeping genes in three Hevea brasiliensis tissues (leaf, bark and root) under15 days of moderate water deficit. Total RNA was isolated from 18 samples consisting of control and stressed-plants collected at day-0 (D0), day-5 (D5) and day-15 (D15).The quality of cDNA synthesized was examined by PCR using HbActin primer. The eleventh primers encoding Hevea housekeeping genes (HbActin, HbelF1Aa, HbUBC4, HbUBC2b, HbYLS8, HbRH2b, HbRH8, HbUBC2a, HbαTub, Hb40S and HbUBI) were validated using PCR amplification. The Crossing-point (Cp) values were measured using a second derivative method after RT-qPCR analysis revealing a significantly higher Cp mean values for 11 housekeeping genes at D5 compared to D0 and D15 sampling points. This study suggests that a simple coefficient of variation (CV) method can be used to rank Hevea reference genes based on its stable expression. Five housekeeping genes (HbRH2b, HbRH8, HbUBC4, HbαTUB and HbActin) can be used for RT-qPCR analysis in Hevea brasiliensis under moderate water deficit. The HbRH2b gene was the most stable among others.
The Hevea brasiliensis AP2/ERF superfamily: from ethylene signalling to latex harvesting and physiological disease response PUTRANTO, Riza Arief; MONTORO, Pascal
E-Journal Menara Perkebunan Vol 84, No 1: Oktober 2016
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

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Ethylene is a hormone known for its involvement in the process of latex harvesting in Hevea brasiliensis. It facilitates latex flow by activation of endogenous metabolism in the anastomosed latex cells called laticifers. In regard to its ambivalent role, ethylene is both favourable to the latex production and unfavourable, to a certain level, to the apparition of a physiological disease termed as tapping panel dryness (TPD). Comprehensive researches have been carried out to reveal the molecular actors in ethylene biosynthesis and signalling pathways in Hevea brasiliensis. One of the most important superfamily implicated as the last transcription factor known in plant ethylene signalling is the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF). Currently, 114 unique sequences related to the Hevea AP2/ERF gene superfamily have been identified and characterized. Specific characterizations under the condition of harvesting stress and the occurrence of TPD have identified 36 gene expression markers (GEMs). Eighteen of these GEMs were predicted as ortholog with 19 Arabidopsis AP2/ERF genes. The characterization was mainly focused on transcriptional regulation, whilst potential post-transcriptional and post-translational regulations of HbAP2/ERF genes were formerly predicted. Three HbERF groups (HbERF-VII, HbERF-VIII and HbERF-IX) were hypothesized to have an important role in Hevea tolerance during latex production as they highly accumulated in laticifers and in response to multiple abiotic stresses. Further functional analysis of several key genes is suggested in order to fully understand the regulation of HbAP2/ERFs. Finally, the molecular markers for future Hevea breeding could be possibly developed from this superfamily.