Pengelolaan Plasma Nutfah Tanaman Terintegrasi dengan Program Pemuliaan

<p>Plant breeding, as an applied of plant genetics, is based and is supported by various subdisciplines of genetic sciences, includeing plant germplasm, classical genetics, molecular genetics, cytogenetics, gene-transformation techniques, etc. Linkage and team work system between plant germplasm management and plant breeding program is most required, since the success of plant breeding maybe obtained from the contribution of gene donor parents, derived from the germplasm management. Without the flow of genes from the germplasm collection, varieties produced by the plant breeder would suffer a narrow genetical based or a bottle-necking genetic based. Plant germplasm research is an integral part of the germplasm management, aimed to (1) evaluate the genetic variation of the germplasm collection, to be readily available for the breeding program and to be used for scientific publications, (2) tracing the origin of plant species, and (3) officially release a selected germplasm, containing new economic gene (s). The linkage between germplasm management and plant breeding research program could be facilitated through the following activities (1) identifying an elit germplasm for varietal release, (2) selection and stabilization of a promising germplasm accession for possible varietal releases, (3) use of germplasm accession as a gene donor parent to incorporate adaptive genes into improved variety, (4) use of germplasm accession for a specific donor gene, (5) use of germplasm to broaden the genetical base of varieties through an introgression and nobilization, (6) use of germplasm to improve the genetic value of the breeding population, and (7) to develop multiple crossess involving many parents to broaden the genetical base of the breeding population. Another important function of the germplasm management is to conserve accessions carrying genes which may be useful in the future, to anticipate the dynamic changing of biological and environmental stresses on crop. Germplasm management is considered successfully conducted when it is continously supplying donor gene parents to breeders for parental crosses on their breeding program, conversely, breeding program in considered successfully managed, when it uses the rich genetic variability available on the germplasm collection. Separating the organizational units among the breeding program, germplasm management and molecular genetic research, is only for enhancing the intensity of the research, but should not separate the linkage program of the research.</p><p> </p><p><strong>Abstrak</strong></p><p>Pemuliaan tanaman merupakan ilmu genetika terapan yang didukung oleh berbagai cabang ilmu kegenetikaan, termasuk plasma nutfah, genetika klasik, genetika molekuler, sitogenetika, dan genetika transformasi. Keterpaduan antara pengelolaan plasma nutfah dengan pemuliaan tanaman tidak dapat ditawar, karena keberhasilan pemuliaan sangat tergantung dari ketersediaan sumber gen yang disediakan oleh pengelola plasma nutfah. Tanpa kontribusi sumber gen dari pengelola plasma nutfah, hasil pemuliaan tanaman mengalami penyempitan kandungan genetik, atau terjadi gejala leher botol genetik. Penelitian plasma nutfah merupakan bagian integral dari pengelolaan materi plasma nutfah, bertujuan untuk (1) menggali kekayaan sifat genetik plasma nutfah guna penyediaan tetua persilangan dan bahan publikasi ilmiah, (2) menelusuri asal-usul spesies tanaman, (3) melepas secara resmi plasma nutfah sebagai sumber gen yang diakui kepemilikannya. Keterkaitan pengelolaan plasma nutfah dengan program pemuliaan dapat dilaksanakan melalui (1) pemanfaatan langsung aksesi plasma nutfah elit untuk dilepas sebagai varietas unggul, (2) pemurnian dan pemantapan populasi aksesi plasma nutfah sebagai calon varietas, (3) pemanfaatan aksesi plasma nutfah sebagai donor gen untuk rekombinasi gen-gen unggul adaptif, (4) plasma nutfah sebagai donor gen spesifik, (5) plasma nutfah sebagai bahan perluasan latar belakang genetik varietas melalui proses introgresi dan nobilisasi, (6) pemanfaatan plasma nutfah untuk perbaikan genetik populasi seleksi, dan (7) pembentukan populasi dasar yang mengandung keragaman genetik luas melalui persilangan banyak tetua. Fungsi pengelolaan plasma nutfah lainnya adalah melestarikan sumber daya genetik untuk kebutuhan gen di masa depan, agar dapat menyediakan gen-gen untuk mengantisipasi perubahan ras patogen dan tipe baru serangga hama yang bersifat dinamis, serta penyediaan gen guna mengatasi cekaman abiotik alamiah. Pengelolaan plasma nutfah dinilai berhasil apabila telah mampu menyediakan aksesi plasma nutfah sebagai sumber gen donor dalam program pemuliaan. Pemuliaan tanaman berhasil secara optimal apabila telah memanfaatkan keragaman genetik sifat yang diinginkan, yang tersedia dalam koleksi plasma nutfah. Keterpisahan kelembagaan antara unit kerja pengelolaan plasma nutfah dengan program pemuliaan tidak boleh membatasi keterpaduan program penelitian antara kedua cabang disiplin keilmuan tersebut.</p>

Cyclin D1 Overexpression In Clonal Plasma Cells In Systemic AL Amyloidosis Is Associated with Differential Expression of Protein Quality Control Genes and Bias In Clonal Germline IgVL donor Gene Use

Abstract 4043 Cyclin D1 (CCND1) overexpression in AL plasma cells (PC) is associated with patient characteristics such as production of free immunoglobulin (Ig) light chains (FLC) without an intact M-protein (that is, without partner Ig heavy chains), increased cardiac biomarkers and shorter survival (Amyloid 2010;17(S1):61a; Blood 2008;111:4700; Haematologica 2009;94:380). The molecular ramifications of CCND1 overexpression within AL PC clones have not been described. To study these associations, we used CD138+ AL PC from 69 untreated AL patients at diagnosis for (1) gene expression profiling (GEP, Affymetrix U133A 2.0) (n=16), (2) qRT-PCR to validate GEP findings (n=53), and (3) clonal IgVL germline donor gene identification (n=69) by established methods (Blood 2008;111:549; Blood 2001;98:714). By GEP, all cases displayed significant overexpression of the appropriate isotypic IgVL constant region gene, confirming the preponderance of clonal AL PC. Five cases were CCND1hi and 11 CCND1lo, and a supervised analysis of CCND1hi vs CCND1lo transcriptomes showed that in CCND1hi PC among the most down-regulated genes were IGHG1, IGHG3 and CCND2 while among the most up-regulated ones (after CCND1) were FAM129A, WARS, SEC63, PDIA6 and SEL1L. By RT-PCR all 53 cases used for qRT-PCR displayed prominent amplification of spliced and unspliced XBP1, confirming PC derivation. By qRT-PCR, median CCND1 expression was 1.51 (range, 0–19.36) with 27 cases above (CCND1hi) and 26 below the median (CCND1lo) with clear-cut quartile differences (25% 0.02, 75% 4.78). We examined PDIA6 and SEL1L expression by qRT-PCR, and found that both correlated with CCND1 expression (PDIA6, P=0.018, r=0.452; SEL1L, P=0.038, r=0.395). In addition, PDIA6 and SEL1L values above and below the CCND1 median differed significantly (P=0.01, P=0.04). The genes up-regulated in CCND1hi cases are involved in endoplasmic reticulum (ER) and protein control processes: WARS in protein production, FAM129A in autophagy, SEC63 in ER protein transport, PDIA6 in catalysis of disulfide bonds and SEL1L in modifying misfolded proteins and channeling them to cytosolic proteasomes. We then identified the clonal IgVL germline donor genes in the CCND1hi (n=32) and CCND1lo (n=37) AL PC clones. We knew that CCND1hi clones displayed biased Ig light chain restriction with 10/12 κ and 22/57 λ cases being CCND1hi (p=0.009, Fisher's exact). Surprisingly, we also identified biased λ family use as only 6/27 λ1 and λ2 cases were CCND1hi compared to 16/30 λ3 and λ6 cases (P=0.03). Overall these results confirm that CCND1hi AL PC clones express significantly higher levels of important ER protein quality control genes than CCND1lo clones, possibly due to CCND1hi AL PC clones adapting to the production of FLC without partner Ig heavy chains. Moreover, CCND1hi AL PC clones display a biased clonal IgVL germline donor gene repertoire, raising questions about the origin of CCND1hi clones since germline gene selection is an early and CCND1 overexpression likely a late event in malignant clonal PC emergence. Disclosures: No relevant conflicts of interest to declare.

Donor T Cell Gene Expression and GVHD.

GVHD is initiated by donor T cell responses to host alloantigens. However, the occurrence and severity of GVHD are not determined solely by the level of histoincompatibility between donor and recipient. Two MHC-identical subjects will display over 50 minor histocompatibility antigen differences. If histoincompatibility is sufficient for triggering GVHD, the rate of GVHD in MHC-matched recipients of allogeneic hematopoietic cell transplantation (HCT) that receive no immunosuppressive agents should be 100%. Under these conditions, however, GVHD is found in only 50% and 73% of mouse and human recipients, respectively. Histoincompatibility is thus necessary but not sufficient to elicit GVHD. We tested the hypothesis that some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. To this end, we studied the gene expression profiles of CD4 and CD8 T cells from 50 HCT donors using microarrays and qRT-PCR. We found that gene expression profiling before HCT was able to distinguish those donors whose cells caused GVHD from those whose cells did not. The “dangerous donor” trait (GVHD+ recipient) is under polygenic control and is shaped by the activity of genes that regulate TGF-β signaling and cell proliferation. The donor gene profile defined on day 0 shows strong correlation with that of recipient CD4 and CD8 T cells harvested one year post-AHCT. The latter correlation provides compelling evidence that a significant portion of the differential gene profiles between GVHD+ and GVHD– donors is imprinted at the hematopoietic stem cell level. Moreover, stability of the gene expression profiles over a one-year period suggests that the profiles result from inherited genetic traits as opposed to environmental factors. The gene with the best GVHD-predictive accuracy was SMAD3, a key component of the TGF-β pathway. By testing a cohort of 450 subjects using qRT-PCR, we found that amounts of SMAD3 transcripts varied over a 6-fold range. In mice and humans, SMAD3 is constitutively activated (as evidenced by phosphorylation and accumulation in the nucleus) in many leukocyte subsets. We found in mice that induction of TGF-β signaling in donor T cells is an early event following AHCT and that Smad3-deficient donors trigger more severe GVHD than wild-type littermates. These findings strongly suggest that the donor gene expression profile has a dominant influence on the occurrence of GVHD. In allogeneic HCT, the ability to discriminate strong and weak alloresponders using gene expression profiling could help select low-risk donors and permit tailoring GVHD prophylaxis regimens according to the probability of GVHD occurrence.

Transgene Deletions in Long-Term Circulating Donor Gene-Modified T Lymphocytes Infused at Time of Hematopoietic Transplantation.

A phase I/II trial of Herpes-Simplex Virus-thymidine kinase (HSV-tk)-expressing gene modified T cells (GMTC) administration at time of HLA-identical sibling bone marrow transplantation (BMT) demonstrated that such an approach could modulate deleterious alloreactivity after BMT (Tiberghien et al., Blood 2001). The Neomycin resistance gene (NeoR) was transferred together with the HSV-tk gene in a retroviral vector to allow for in vitro GMTC selection. More than 6 years after BMT, 4 patients are alive in complete remission, off immunosuppression, free of chronic graft-versus-host disease (GvHD). Long-term circulating GMTC are continuously found in all 4 patients, as assessed by quantitative PCR (QPCR) for NeoR gene (6 years post-BMT: 0.031 +/- 0.017% of PBMC). Unexpectedly, presence of NeoR gene was readily detected by single run PCR whereas a nested PCR was necessary for HSV-tk gene detection. As PCR assays for both genes have a similar sensitivity, this result suggested that deletions may be present in GMTC. For each patient, PBMC were polyclonally activated and expanded before a one week G418-mediated selection. The % of GMTC in the post-selection samples, as assessed of NeoR QPCR, was > 80% in 3 patients while of only 7% in the last patient, thus demonstrating long-term NeoR transgene expression in at least a fraction of circulating GMTC more than 6 years after BMT. Successfully re-selected GMTC were cloned. As expected, most clones (25 out of 26) were found to be NeoR by PCR. In contrast, only 2/26 were HSV-tk+: one clone contained the full length HSV-tk gene while the second contained the ganciclovir (GCV)-resistant truncated form of the HSV-tk gene (Garin et al., Blood 2001). Clones generated from freshly produced-GMTC were found to be both NeoR+ and HSV-tk+ thus demonstrating that such gene deletions did not occur with a significant frequency during the GMTC production nor during the cloning process. We have previously established that an immune response against GMTC occurred in most patients (Mercier et al., ASH 2004). Immune responses were found against both transgenes and predominantly so against HSV-tk. Immuno-selection of initially rare GMTC containing a deleted transgene without expression of immunogenic peptides might significantly contribute to the high proportion of long-term circulating GMTC containing mutated HSV-tk genes. Such deletions within the transgenes most likely explain the coexistence of an immune response against the transgenes and persistent circulating GMTC. Nevertheless, GCV-sensitivity of circulating GMTC and more importantly so, of GvHD during the first 4 months after BMT suggests that the high frequency of the mutated GMTC occurred late enough after BMT to not interfere with GCV-mediated control of GvHD.